g. xatisaSvili, m. gordeziani, T. varazi, m. yuraSvili, m. fruiZe

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(fiziologia da bioqimia)

Tbilisi

2009

G. Khatisashvili, M. Gordeziani, T. Varazi, M. Kurashvili, M. Pruidze

Oxigenases of Biomembranes (Physiology and bBiochemistry)

Tbilisi, 2009

durmiSiZis bioqimiisa da bioteqnologiis instituti

Durmishidze Institute of Biochemistry and Biotechnology

g. xatisaSvili, m. gordeziani, T. varazi, m. yuraSvili, m. fruiZe biomembranebis oqsigenazebi (fiziologia da bioqimia)

monografiaSi ganxilulia monooqsigenazuri fermentuli sistema,

romelic qsenobiotikebis metabolur gardaqmnebzea orientirebuli. es

rTuli multifermentuli kompleqsia da farTodaa gavrcelebuli

cocxal samyaroSi. warmodgenili masala “evoluciuri qronologiiT”

asaxavs, rogor itvirTes P450-Semcvelma monooqsigenazebma detoqsi-

kaciis funqcia.

naSromSi detaluradaa gaaanalizebuli literaturuli da sakuTari

monacemebi monooqsigenazebis fiziologiuri da bioqimiuri rolis

Sesaxeb. calke Tavi aqvs daTmobili citoqrom P450-is fermentuli

aqtivobis regulaciis funqciur da struqturul aspeqtebs.

wigni gankuTvnilia gansxvavebuli dargebis specialistebisaTvis:

qimikosebisaTvis, bioqimikosebisaTvis, biofizikosebisaTvis farmakologe-

bisaTvis, toqsikologebisaTvis, ekologebisaTvis, fiziologebisaTvis,

agreTve biofakultetebis pedagogebisa da zeda kursebis studen-

tebisaTvis. igi SeiZleba sasargeblo aRmoCndes membranul biologiaSi

da toqsikologiaSi momuSave medikosebisaTvis.

redaqtori: saqarTvelos mecnierebaTa erovnuli akademiis

wevr-korespondenti n. aleqsiZe

recenzenti: biologiis mecnierebaTa kandidati e. imediZe

ISBN 978-9941-0-1699-8

oqsigenazebi. fiziologia da bioqimia (Sesavali) ............................................. 6 Tavi 1. monooqsigenazebi. ................................................................................. 9 moqmedebis funqcia, struqturuli organizacia, da katalizuri cikli ......... 9 monooqsigenazebis kvlevis mokle istoriuli mimoxilva ................................. 9 1.1 mikrosomuli hidroqsilirebis biologiuri azri da misi meqanizmi .......... 14 1.2 oqsigenazebi ............................................................................................. 19 1.3 mikrosomuli monooqsigenazebis fermentuli Semadgenloba ..................... 22 1.3.1 NADPH-citoqrom P450-reduqtaza (NADPH-specifikuri

flavoproteini EC 1.6.2.4) ............................................................................ 23 1.3.2 NADH-citoqrom b5-reduqtaza (EC 1.6.2.1) ............................................ 28 1.3.3 citoqromi b5 ........................................................................................ 32 1.3.4 citoqrom P450 _ mikrosomebis eleqtronTa satransporto sistemis

terminaluri komponenti ............................................................................... 41 1.3.4.1 mikrosomuli hidroqsilirebis calkeuli stadiebi (monooqsigenazuri

cikli) .......................................................................................................... 55 1.4 citoqrom P450-is mravlobiTi formebi da maTi Tanamedrove

nomenklatura ................................................................................................ 77 1.4.1 citoqrom P450-is mravlobiTi formebis induqcia ................................. 88 1.5 mikrosomuli fosfolipidi _ mahidroqsilirebeli kompleqsis

aucilebeli komponenti ................................................................................. 92 literatura ................................................................................................ 102

Tavi 2. oqsigenazebi Sidaujredul metabolur cvlaSi .......................................124 2.1 citoqrom P450 mcenaris azotfiqsaciaSi ................................................ 124 2.2 oqsigenazebi cximovan mJavaTa zeJangur JangvaSi ..................................... 125 2.2.1 jaWvis gagrZelebis stadia ................................................................... 131 2.2.2 citoqrom P450-is monawileoba cximovani mJavebis hidroqsilirebasa

da epoqsidirebaSi ........................................................................................ 142 2.3 citoqrom P450 fenolur naerTTa biogenezSi ......................................... 148 2.4 citoqrom P450 alkaloidebis biosinTezSi ............................................ 152 2.5 citoqrom P450-is monawileoba hiberelinebis biosinTezSi ................... 155 2.6 steroidTa mahidroqsilirebeli oqsigenazebi ......................................... 158 2.6.1 Tirkmelzeda jirkvlis qerqis mitoqondriebis citoqrom P450 .......... 160 2.6.2 NADPH-adrenodoqsinreduqtaza (EC 1.6.7.1) ........................................ 164 2.6.3 adrenodoqsini ..................................................................................... 165 2.6.4 steroidmahidroqsilirebeli sistemis moqmedebis meqanizmi ................ 168 2.6.4.1 qolesterinis gverdiTi alifaturi jaWvis moxleCvis meqanizmis

Sesaxeb ......................................................................................................... 176

2.6.5 qolesterini mcenareSi da misi metaboluri gardaqmnebi ...................... 181 2.6.5.1 qolesterinis eTerifikacia mcenareSi ................................................ 181 2.6.5.2 qolesterinis Jangva mikroorganizmebsa da mcenareSi (SedarebiTi

analizi) ...................................................................................................... 184 2.7 urTierTkavSiri oqsigenazur da imunur sistemebs Soris ....................... 191 2.7.1 molekuluri receptorologiis zogadi principebi ............................... 198 2.7.2 oqsidazebi da oqsigenazebi mcenaris mdgradobasa da

fitoimunitetSi ........................................................................................... 201 literatura ................................................................................................ 207

Tavi 3. oqsigenazebi qsenobiotikebis detoqsikaciaSi .................................... 219 3.1 citoqrom P450-is roli qsenobiotikebis metabolizmSi .......................... 219 3.2 citoqrom P450-is mier katalizebuli reaqciebi .................................... 222 3.2.1 hidroqsilireba .................................................................................... 223 3.2.2 demeTilireba ....................................................................................... 237 3.2.3 N- da S-Jangva ..................................................................................... 240 literatura ................................................................................................ 245 Tavi 4. monooqsigenazuri sistemis regulacia ............................................ 249 4.1 regulacia aRmdgeneli eqvivalentebis doneze ......................................... 249 4.1.1 urTierTqmedeba eleqtron-satransporto gzebs Soris .......................... 249 4.1.2 eleqtronTa transmembranuli cvla mikrosomasa da

mitoqondrias Soris ......................................................................................................... 254

4.1.2.1 mitoqondriuli kontroli ................................................................. 255 4.1.2.2 NADPH-is Tavisufali mikrosomuli Jangva ....................................... 259 4.2 monooqsigenazuri sistemis aqtivobis substratuli regulacia ............. 263 4.3 citoqrom P450-is regulacia induqciis doneze ..................................... 267 4.4 monooqsigenazuri sistemis aqtivobis regulacia membranul doneze ....... 271 4.4.1 membranuli fosfolipidebis gavlena citoqrom P450-is meoreul

struqturaze da aqtivobaze ......................................................................... 271 4.4.2 membranuli fosfolipidebis roli NADPH-citoqrom P450-reduqtazisa da citoqrom P450-is urTierTqmedebaSi ....................................................... 273 4.4.3 NADH-citoqrom b5-reduqtazisa da citoqrom b5-is damokidebuleba

membranul fosfolipidebze .......................................................................... 279 4.4.4 monooqsigenazebis da lipidebis erTiani sistema .................................. 283 4.4.5 lipidebis fermentuli peroqsidacia da qsenobiotikebis Jangva

endoplazmur membranebSi erTi da igive eleqtronis satransporto

sistemiT katalizdeba .................................................................................. 288 4.5 regulacia citoqrom P450-is qimiuri modifikaciis doneze ................... 294

4.5.1 NADPH-citoqrom P450-reduqtazas SesaZlo funqciebi

citoqrom P450-is peroqsidazad transformaciis pirobebSi ......................... 319 literatura ................................................................................................ 326

mecnierebi, romlebmac mniSvnelovani wvlili Seitanes biologiuri Jangvis

oqsigenazuri meqanizmebis gamokvlevaSi......................................................... 336

wignis gamocema dafinansebulia saqarTvelos erovnuli samecniero

fondis (GNSF) da ukrainis mecnierebisa da teqnologiebis centris

(STCU) erToblivi grantiT #4674

6

oqsigenazebi

fiziologia da bioqimia

Sesavali

citoqrom P450-Semcveli monooqsigenazebi endogenuri da egzo-

genuri warmoSobis lipofiluri naerTebis metabolizmSi monawileoben.

am nivTierebaTa CamonaTvali sakmaod grZelia. esenia sinTezuri da

bunebrivi samkurnalo saSualebebi, samrewvelo narCenebi, pesticidebi,

saRebavebi, navTobproduqtebi, plastmasebi, sayofacxovrebo moxmarebis

produqtebi, kosmetikuri saSualebebi da sxv. sakveb produqtebSic ki,

romelTac yoveldRiurad viyenebT, sayuaTo nivTierebebi “sufTa” saxiT

iSviaTad gvxvdeba da yovelTvisaa organizmisTvis usargeblo balasturi

minarevebi. aRarafers vambobT sakveb danamatebze da konservantebze. es

nivTierebebi sawyisi formiT organizmSi arc energiis da arc plas-

tikuri cvlis miznebisaTvis ar gamoiyeneba da “ucxos” (Zveli gansaz-

RvriT) warmoadgenen. amJamad isini “qsenobiotikebis” (berZn: “qsenos” –

ucxo, “bios” – sicocxle) saerTo saxelwodebiT arian cnobili, Tumca

bioqimiaSi, farmakologiaSi da toqsikologiaSi orive termini dResac

TanasworuflebianobiT sargeblobs.

garemodan organizmSi qsenobiotikebi SemTxveviT an sxvadasxva

mizeziT, organizmis nebisagan damoukideblad xvdebian. aq isini sponta-

nurad an katalizurad ganicdian gardaqmnebs. metaboluri aqtivaciis

Sedegad eleqtrofiluri reaqciisunariani Sualedi produqtebi war-

moiqmnebian, romlebic rTuli JangviTi gardaqmnebis gziT an sabolood

detoqsicirdebian (uvnebeldebian), an urTierTqmedeben Sidaujredul

sasicocxlod aucilebel naerTebTan (gansakuTrebiT makromoleku-

lebTan) da es procesi mutagenur, toqsikur, teratogenul da kance-

rogenul efeqtebs ukavSirdeba.

sadReisod qsenobioqimia biologiuri mecnierebis damoukidebel dar-

gadaa Camoyalibebuli. mas Tavisi Teoriuli safuZvlebi da teqnikur-

meToduri midgomebi gaaCnia. principSi qsenobioqimia dargTaSorisi dis-

ciplinaa, ramdenadac bioqimiis, organuli qimiis, analizuri qimiis,

farmakologiisa da toqsikologiis garkveul sferoebs moicavs. misi

7

interesebi qsenobiotikebis katalizisa da organizmze maTi moqmedebis

meqanizmebis dadgenaSi mdgomareobs.

fermentuli sistema, romelic qsenobiotikebis “metabolur aqtiva-

ciaze” da gardaqmnebzea orientirebuli, monooqsigenazebs miekuTvneba.

es rTuli multifermentuli kompleqsia da farTodaa gavrcelebuli

cocxal samyaroSi. misi fiziologiuri da bioqimiuri rolis deta-

luri Seswavla Tanamedrove qsenobioqimiis erT-erTi yvelaze “cxeli”

mimarTulebaa. gamomdinare iqidan, rom monooqsigenaza membranadakavSi-

rebul (integralur) cilas warmoadgens, fermentuli aqtivobis regu-

laciis TvalsazrisiT did yuradRebas imsaxurebs misi aramarto

kvlevis funqciuri, aramed struqturuli aspeqtebic.

hidrolizisa da dehidrogenirebis sxvadasxva reaqciebis zogadi

meqanizmi didi xania cnobilia. miuxedavad amisa, endoplazmuri retiku-

lumis (mikrosomebis) fermentebiT ucxo naerTTa Jangva-aRdgenis reaq-

ciebis meqanizmebis kvleva amJamadac intensiurad grZeldeba. dadgenilia,

rom aRniSnuli procesebis makatalizebel fermentul sistemaSi cent-

raluri pozicia citoqrom P450-s ukavia. qsenobiotikis metaboluri

gardaqmnis siCqare am hemoproteinis maqsimaluri aqtivobiT da substra-

tisadmi misi swrafviT limitirdeba.

yoveli cocxali qsovili maRali specifikurobis mqone “oqsi-

genazebis anakrebs” Seicavs. mravali maTgani wyalSi xsnadi fermentia

da amdenad hidrofiluri Tvisebebis mqone endogenuri substratebis

Jangvas emsaxureba. es, upirveles yovlisa, aminomJavebis (fenilalaninis,

triftofanis, lizinis, prolinis) oqsigenazebia. farTodaa gavrcele-

buli poliujeri cximovani mJavebis oqsigenaza (lipoqsigenaza),

laqtatoqsigenaza, indolaminooqsigenaza da a.S. maTi kvlevis “mSvidi”

msvleloba mas Semdeg dairRva, rac aRmoCenil iqna universaluri

oqsigenazebi citoqrom P450-is SemcvelobiT. isini monawileoben

rogorc endogenuri warmoSobis substratebis (steroluri hormonebis,

cximovani mJavebis, prostaglandinebis, alkaloidebis, hiberelinebis da

mravali sxv.) biosinTezebSi da JangvaSi, aseve mravali ucxo naerTis

biodegradaciaSi.

cxadia, Soreul warsulSi, rodesac garemoSi qsenobiotikebis done

nulovan niSnuls utoldeboda, citoqrom P450-Semcveli oqsigenazebi

8

aqtiur monawileobas endogenur plastikur cvlaSi iRebdnen. mxolod

sinTezuri samkurnalo saSualebebis gamoyenebis da garemos qimiuri

dabinZurebis Semdeg itvirTes maT detoqsikaciis funqcia da tipiur

“garegan” monooqsigenazebad iqcnen. ase rom, damcvelobiTi funqcia am

fermentuli kompleqsis gacilebiT gvian SeZenili Tvisebaa. amitom

winamdebare monografiaSi warmodgenili masalac aseTi “evoluciuri

qronologiiTaa” dalagebuli.

9

Tavi 1

monooqsigenazebi.

moqmedebis funqcia, struqturuli organizacia,

da katalizuri cikli

monooqsigenazebis kvlevis mokle istoriuli mimoxilva

monooqsigenazebis kvleva Tavis saTaves gasuli saukunis 50-iani

wlebidan iRebs. jer kidev brodisa da Tanaavtorebis [1], agreTve

aqselrodis [2] samuSaoebiT dadgenil iqna RviZlis endoplazmur

retikulumSi fermentuli sistemis arseboba, romelsac gansxvavebuli

tipis wamlebisa da aromatuli naerTebis ufro polarul produqtebad

gardaqmnis unari gaaCnda. gairkva, rom am procesSi CarTuli arian

Mg2+-is ionebi da NADPH-generirebadi sistema (NADP + glukozo-6-

fosfatdehidrogenaza – pentozuri ciklis sawyisi fermenti). agreTve

gamovlinda, rom aRniSnuli gardaqmnebi mxolod molekuluri Jangbadis

uSualo TanamyofobiT xorcieldeba. amasTan dakavSirebiT masonma da

Tanaavtorebma [3] warmoadgines monacemebi substratze Jangbadis gadam-

tani fermentuli sistemis funqcionirebis Sesaxeb da igi miakuTvnes

reaqciis fenolazur tips, romelic moqmedebis Semdegi meqanizmiT

xasiaTdeba:

AH + NADPH + H+ + O2 → AOH + NADP + H2O

SemdgomSi es gardaqmna monooqsigenazuri reaqciis zogad gantole-

bad iqna aRiarebuli.

momdevno wlebSi garfinkelma [4] da klingenbergma [5] RviZlis mi-

krosomebSi daadastures CO-damkavSirebeli pigmentis arseboba, romel-

ic NADPH-iTa da diTionitiT ganicdida aRdgenas. maT pirvelebma aC-

venes, rom es pigmenti da misi CO-kompleqsi STanTqmis maqsimums 450

nm-ze amJRavneben. 1964 wels iaponelma mkvlevarebma omuram da satom

[6, 7] am pigmentis hemoproteinuli buneba gamoavlines da mas pirobi-

Tad citoqrom P450 uwodes (P – pigmentis aRmniSvneli asoa).

Semdgomi gamokvlevebiT eqsperimentulad dadasturda, rom mikroso-

muli monooqsigenazuri kompleqsi Seicavs eleqtronis Tavisufal

10

(energiis generirebasTan araSeuRlebuli) satransporto jaWvs, romlis

sawyis eleqtronul donors NADPH-damokidebul reduqtaza warmoad-

gens, xolo citoqrom P450 aq namdvilad eleqtronTa terminaluri aq-

ceptoris rols asrulebs [8, 9].

ganxiluli monacemebi ZiriTadad mikrosomebze da citoqrom P450-is nawilobriv gasufTavebul preparatebze tardeboda. 1968 wels ki kata-

hiris [10] jgufma Pseudomonas putidas baqteriuli mutantidan warmate-

biT gamoyo am fermentis xsnadi forma, romelic qafuris 5-

epoqsihidroqsiqafurad hidroqsilirebas axorcielebda.

metad sagulisxmoa is faqti, rom monooqsigenazebis kvlevis dawye-

bis Tanadroulad saTanado yuradReba mieqca ucxo naerTTa biodegrada-

ciaSi TviT endoplazmuri retikulumis membranuli sistemis mniSvnelo-

bis garkvevas. paladisa da sikevicis [11] mier nanaxi iqna, rom diferen-

cialuri centrifugirebiT miRebuli mikrosomuli fraqcia sferulad

Sekrul am membranul struqturebs warmoadgens da mravali cilis bio-

sinTezSi aramxolod matriqsis (“Stativis” an “samagri blokis”),

aramed fermentuli aqtivobis gamomJRavnebisaTvis kofaqtorul funq-

ciasac asrulebs.

mcenareSi mikrosomuli citoqromebis kvleva ukavSirdeba cianidre-

zistentuli sunTqvis (“narCeni sunTqvis”) fenomenis gamovlenas [12, 13]

da imis dadgenas, Tu konkretulad romeli oqsidazebi ganapirobeben am

movlenas. amTaviTve SevniSnavT, rom es sakiTxi principulad dResac

araa gadawyvetili. safiqrebeli iyo, rom narCen sunTqvaSi CarTuli

unda yofiliyvnen citoqromebisagan gansxvavebuli cianidisa da azidi-

sadmi rezistentuli oqsidazebi da amis realur kandidatebad flavi-

nur fermentebs miiCnevdnen. am jgufis pirveli warmomadgeneli rogorc

cnobilia 1932 wels varburgma da kristianma [14] aRmoaCines. qimiurma

gamokvlevebma aCvenes, rom igi izoaleqsazinur mononukleotids (fla-

vinmononukleotids – FMN-s) warmoadgens. aRdgenil mdgomareobaSi mas

intensiuri yviTeli Seferiloba aqvs da Jangvisas uferuldeba. TviT

aRmomCenebma mas yviTeli fermenti (literaturaSi “Zveli yviTeli fer-

mentis” saxelwodebiTaa cnobili) uwodes. sadReisod mas riboflavinad

vicnobT. dadgenil iqna, rom NADPH-is Tanamyofobisas es fermenti

Jangavs glukozofosfats da am substrats wyalbadi uSualod Jang-

11

badze gadaaqvs. amdenad igi oqsidazas warmoadgens. amave mkvlevarebma

1938 wels “axali yviTeli fermenti” aRmoaCines, romelic flavinade-

nindinukleotids (FAD-s) warmoadgens.

yviTeli fermentis aRmoCenam saTave daudo flavinuri oqsidazebis

fiziologiuri rolis kvlevas. aRmoCnda, rom zogierTi adre cnobili

oqsidaza prostetul jgufad FAD-s an ufro iSviaTad FMN-s (mag.,

qsantinoqsidaza, D- da L-aminomJavaTa oqsidazebi, aldehidoqsidaza da

sxv.) Seicavs. maT substratebTan uSualo reagirebis unari aqvT da

maTgan mocilebuli wyalbadis Jangbadze gadatanas axorcieleben. amis

Sedegad reaqciis produqtad zeJangi warmoiqmneba, romelsac SemdgomSi

katalaza an peroqsidaza gardaqmnis. FMN da FAD SedarebiT gvian aR-

moCenil ferment-dehidrogenazebis aqtiur jgufebs warmoadgenen da

NAD(P)H-is eleqtronebs sxvadasxva aqceptorebs gadascemen.

narCen sunTqvaSi flavinuri fermentebis monawileobis sasargeblod

arsebuli faqtebi miuTiTeben garkveul paralelizmze am fermentTa

raodenobriv Semcvelobasa da narCeni sunTqvis sidides Soris da, rac

mTavaria, maT rezistentobaze cianidisa da azidis mainhibirebeli moq-

medebis mimarT.

saerTod, gasuli saukunis 50-iani wlebi, rogorc flavinuri fer-

mentebis, aseve citoqromuli sistemis intensiuri kvleviT aRiniSneba.

SesaZlebeli Seiqmna citoqromoqsidaza iseT mcenareul obieqtebSic

gamovleniliyo, romlebSic adre misi identifikacia ar xerxdeboda.

amis Sedegad hipoTezac ki Camoyalibda, romlis Tanaxmadac citoqro-

muli sistema sunTqvas mcenaris zrdis adreul etapebze axorcielebs,

xolo Semdeg mas flavinuri fermentebi cvlian, anu adgili aqvs

sunTqvis sistemaTa cvlas. SemdgomSi es hipoTeza praqtikulad uaryo-

fil iqna. amis sapirispirod Camoyalibda e.w. “oqsidazebis siWarbis

Teoria”, romlis Tanaxmadac oqsidazebis nawili sunTqvis siCqares ar

gansazRvraven da isini ujredSi siWarbiT imyofebian. amitom Sxamis dam-

kavSirebeli ubnebis Semdegac ki rCeba sunTqvisaTvis sakmarisi oqsida-

zebis nawili. speqtrofotometruli gamokvlevebis safuZvelze am Sex-

edulebam garkveuli dasabuTeba moipova. aRniSnuli meTodis gamoyenebiT

SesaZlebeli gaxda cianidis Tanamyofobisas citoqromoqsidazas rogorc

aRdgenili, aseve daJanguli formebis erTdroulad arsebobis demon-

12

strireba. jer kidev 1945 wels lundegardma [15] sunTqvis mxolod

12%-iT daTrgunva miiRo, maSin rodesac citoqromoqsidazas 82% cia-

nidTan iyo dakavSirebuli. Cansis da haketis [16] cdebSi egzogenuri

NADH-is Jangvisas, romelic Zlier intensiurad mimdinareobs, cianidi-

sa da azidis Tanamyofobisas c da a3 citoqromebis ZiriTadi (99%) na-

wilis aRdgenili formiT arseboba ar vlindeboda. amgvarad, adre Tu

miRebuli iyo, rom narCeni sunTqva arametalSemcveli flavinuri oqsi-

dazebiT xorcieldeba, amJamad am fermentTa roli mainc ukana planzea

gadaweuli da Tvlian, rom sunTqvaSi wamyvani roli upiratesad cito-

qromul sistemas ekuTvnis.

mcenareSi monooqsigenazebis kvlevas kerZod rac Seexeba, 1955 wels

martinisa da mortonis [17] mier Saqris Warxlis foTlis mikrosomebSi

pirvelad iqna aRmoCenili hemSemcveli proteini, romelic cianidis mi-

marT inertulobas amJRavnebda. cota mogvianebiT amave avtorebma [18]

SeZles daedginaT, rom es mikrosomuli pigmenti “b“ jgufis citoqro-mebs miekuTvneba da mas rkina-Jangbadiani kompleqsis warmoqmnis unari

aqvs. mcenare iukas (Arum maculatum) mikrosomebSi karotinoidebTan er-Tad ori citoqromuli komponentis arseboba daadgines bendalma da

hilma [19], xolo kasinskisa da Tanaavtorebis [20] mier naCvenebi iqna

lobios eTiolirebuli aRmonacenebis gvelis SxamiT damuSavebuli mi-

krosomebidan CO-damkavSirebeli pigmentis miRebis SesaZlebloba da am

fraqciaSi b-tipis ori citoqromis arseboba. analogiuri Sedegebi mi-

Rebul iqna ambikesa da baqsteris [21] mier. maT gamoavlines Claviceps purpurea-s miceliumSi CO-damkavSirebeli citoqromi, romelic gvelis

SxamiT an natriumis qolatiT solubilizdeboda. hemoproteinis inaqti-

vacias Tan axlda speqtrul TvisebaTa cvlilebebi.

Sereuli funqciis oqsidaza, romelic bambis aRmonacenebis hipoko-

tilebidan miRebul mikrosomebSi Sardovanas warmoebulebis demeTili-

rebas axorcielebda, gamoyves da daaxasiaTes frearma da Tanaavtorebma

[22]. fermentuli preparatis aqtivirebisaTvis aucilebeli aRmoCnda

molekuluri Jangbadi da kofaqtorad NAD(P)H-is Tanamyofoba. ionuri

detergentebiTa da ionuri aqceptorebiT, agreTve naxSirbadis monooq-

sidiT procesis inhibirebam am avtorebs saSualeba misca gaekeTebinaT

daskvna mcenareuli da cxoveluri mikrosomuli eleqtronis satrans-

13

porto sistemebis struqturuli organizaciis msgavsebis Sesaxeb.

250 nm-ze STanTqmis maqsimumiT CO-damkavSirebeli pigmentis speq-

trulma kvlevam misi arseboba daadastura bardis (Pisum sativum) aRmo-nacenebSi da simindis (Zea mays) lebnebSi [23]. specifikuri inhibitore-

bis (SKF-525A-isa da meTiraponis) da hidroqsilirebis substratebis

(aminopirinisa da dimeTilanilinis) daxmarebiT gordonma da Tanaavto-

rebma [24] xorblis aRmonacenebis fesvebSi gamoavlines mikrosomuli

redoqs-sistemisa da citoqrom P450-is funqcionireba. sayuradReboa,

rom eqsperimentebSi isini iyenebdnen ara mikrosomul fraqcias, aramed

mTlian mcenareul qsovils.

2,4-diqlorfenoqsiZmarmJavas (2,4-D) arilhidroqsilireba kitrisa

da bardis foTlebSi Seiswavleboda Ckanikovisa da Tanaavtorebis [25,

26] mier. naCvenebi iqna, rom es procesi 2,4-D-s metabolizmSi erT-erTi

umniSvnelovanesi stadiaa da Sereuli funqciebis mqone mikrosomuli

oqsidaziT katalizdeba. TvisebebiT igi Zlier waagavs meore mikroso-

mul oqsidazas _ dariCinmJava-4-hidroqsilazas, ramdenadac orive fer-

menti aqtivobisaTvis saWiroeben O2-s, NADPH-s da mTlianad iTrgune-

bian naxSirbadis monooqsidiT. hidroqsilirebis produqtis maqsimaluri

gamosavali miiReboda NADPH-Tan erTad 2-merkaptoeTanolis Tanamyo-

fobisas, rac am avtorTa mier axsnili iqna membranaze misi mastimuli-

rebeli moqmedebiT, radgan NADPH-is gareSe es nivTiereba fermentis

aqtivobaze araviTar zemoqmedebas ar axdenda. agreTve dadgenil iqna,

rom arilhidroqsilirebis reaqciis kinetika eqvemdebareba mixaelis-

menTenis gantolebas da uCvenebs, rom dasaJangi substratebis ferment-

Tan dakavSireba konkurentuli bunebisaa.

gordezianisa da mis TanaavtorTa [27] mier naCvenebia, rom aerobul

pirobebSi soiasa da simindis gajirjvebuli lebnebi axorcieleben ami-

dopirinis, dimeTilanilinisa da monuronis (Sardovanas warmoebuli N-meTilis jgufebis Semcveli herbicidis) N-demeTilirebas (reaqciis

msvleloba fasdeboda gamoyofili formaldehidiT) da anilinis p-hidroqsilirebas. orive reaqcia damokidebuli aRmoCnda NADPH-ze,

ramdenadac stimulaciis xarisxs am kosubstratis koncentracia gan-

sazRvravda. garda amisa, dadgenil iqna, rom benzidinisa da naftalinis

SemTxvevaSic aromatuli birTvis Jangvis sawyis stadias NADPH- da

14

O2-damokidebuli hidroqsilireba warmoadgens. procesi inhibirdeboda

naxSirbadis monooqsidiT da SemdgomSi fotoreaqtivacias ganicdida

[28]. es monacemebi pirdapir miuTiTeben, rom mcenareSi mimdinare aRniS-

nul procesebSi uSualod citoqrom P450 monawileobs.

mcenaris mikrosomebi citoqromuli komponentebis mravalferovnebiT

xasiaTdebian. maTi Semcveloba farTo sazRvrebSi meryeobs. aqedan gamom-

dinare Tvlian, rom aq eleqtronis ramdenime satransporto sistema

unda funqcionirebdes, romelTa aqtivobebic calkeuli qsovilis indi-

vidualur metabolur moTxovnebzea damokidebuli [29, 30].

citoqrom P450 identificirebulia eukariotul organizmebSic. gan-

sakuTrebiT mniSvnelovania am hemoproteinis roli zogierT safuar so-

koSi, romlebic zrdisaTvis aerobul pirobebs iTxoven da naxSirbadis

erTaderT wyarod alkanebs moixmaren [31]. safuvris (Candida tropicalis) tetradekanze gamozrdisas inducirdeba fermenti, romelic cximovani

mJavebis, alkanebisa da wamlebis hidroqsilirebas axorcielebs [32].

fermenti afinuri qromatografiis meTodiT iqna gawmendili da misi

cxovelur citoqrom P450-Tan identuroba dadasturda.

1.1 mikrosomuli hidroqsilirebis biologiuri azri

da misi meqanizmi

qsenobiotikTa umravlesoba (∼98%) wyalSi uxsnad (an mcired

xsnad) arapolarul hidrofobul nivTierebebs miekuTvneba. ujredSi

SeRwevis Semdeg isini, rogorc cximSi xsnadi nivTierebebi, swrafad

idevnebian citoplazmis wylovani fazidan da biomembranebis fosfoli-

pidebis an membranis hidrofobuli cilebis fazaSi koncentrirdebian. es

garemoeba mniSvnelovnad unda zRudavdes oqsidazuri sistemiT maT de-

gradacias, radgan es fermentebi Tavis funqcias warmatebiT mxolod

wylovan fazaSi axorcieleben. aqedan gamomdinare, cxadi xdeba, rom uc-

xo naerTTa detoqsikaciis rTuli safexurebrivi gardaqmnebisaTvis au-

cilebel winapirobas warmoadgens maTTvis wyalSi xsnadobis unaris

(hidrofobulidan hidrofilurad gardaqmnis) miniWeba. swored am mizans

emsaxureba membranaSi mimdinare JangviTi reaqciebi. maTi saSualebiT qse-

15

nobiotikis molekulaSi axali funqciuri jgufis (hidroqsilis, kar-

boqsilis, epoqsidis an sxv.) Seyvana xdeba. amitom am stadias funqcio-

nalizacias uwodeben. am dros mcirdeba nivTierebis qimiuri inertulo-

ba da miRebuli produqtebis polaruli jgufebi fermentuli gardaqm-

nebisaTvis gacilebiT xelmisawvdomi xdeba. gardasaqmneli nivTierebis

molekulaSi ramdenime funqciuri jgufis Seyvana kidev ufro efeqturs

xdis masze fermentTa erTobliv moqmedebas da molekulis gansxvavebul

ubnebze fermentTa swraf “Setevas”. sxvagvarad rom iTqvas, funqciona-

lizaciis Sedegad ucxo naerTi iseT pirvelad gardaqmnebs ganicdis,

rom oqsidazebisadmi igi advilad “Secnobadi” xdeba. am procesSi oqsi-

dazebis garda SeiZleba CaerTvnen reduqtazebi, esTerazebi, dehalogena-

zebi da sxv. arcTu iSviaTad funqcionizacia SeiZleba am fermentTa

urTierTSenacvlebiT realizdes.

funqcionizaciis yvelaze gavrcelebul formas hidroqsilireba war-

moadgens. cxovelSi es procesi RviZlSi arsebul citoqrom P450 Semc-veli monooqsigenazebiT xorcieldeba. am mxriv mcenarem ufro farTo

arCevani gaakeTa da hidroqsilirebisaTvis am hemoproteinis garda pe-

roqsidazebi da fenoloqsidazebic gamoiyena. reaqcia aqtivaciis maRal

energias saWiroebs da amis gamo Senelebulad mimdinareobs. meore

mxriv, ujredisaTvis sasicocxlod aucilebelia is, rom rac ufro

swrafad ganxorcieldeba ucxo naerTis polarobis gazrda, miT ufro

swrafad warimarTeba misi Semdgomi JangviTi gardaqmnebi. aqedan gamomdi-

nareobs daskvna, rom ucxo naerTis mravalsafexurebrivi JangviTi de-

gradaciisaTvis funqcionalizacia procesis siCqaremalimitirebel sta-

dias warmoadgens da sruli detoqsikaciis regulaciac swored am sta-

diaze unda xorcieldebodes.

mikrosomuli monooqsigenazebiT katalizebuli bioqimiuri Jangvis

reaqciebi metad farTo wres moicavs, magram Zneli araa imis SemCneva,

rom qvemoTmoyvanil reaqciebs:

− aromatul hidroqsilirebas

OC6H5X HO_C6H4X

− aciklur Jangvas

16

RCH3 RCH2OHO

− O-dealkilirebas

ROCH3 ROCH2OH ROH+HCHOO

− N-dealkilirebas

NHCH3

R

R'

ONH2

R

R'NHCH2OH

R

R'

− dezaminirebas

NHCH3

R

R'

ONH2

R

R'NHCH2OH

R

R'

− sulfoJangvas

RSR'O

RSOR' + H(OH)R'++RS

erTi da igive, kerZod, hidroqsilirebis saerTo meqanizmi udevs safuZ-

vlad. yvela am reaqcias aRdgenili NADPH da atmosferuli Jangbadi

esaWiroeba, radgan 18O2-isa da H2

18O-is gamoyenebiT Catarebulma cdebma

aCvenes, rom ucxo naerTis molekulaSi Canergili hidroqsilis Jangbadi

atmosferul Jangbads, da ara wylis molekulas ekuTvnis. amitom ucxo

naerTebis Jangva NADPH-is oqsidaciasTan pirdapir damokidebulebaSia

da igi SeiZleba konkurentulad daiTrgunos mikrosomuli Jangvis sxva

substratiT [33]. am Sedegidan gamomdinareobs, rom mikrosomuli hi-

droqsilireba SeuRlebuli Jangva-aRdgeniT xorcieldeba: garkveuli

kofermenti NADPH-iT ganicdis aRdgenas da Semdgom JangbadTan mier-

TebiT e.w. “aqtiur Jangbads” generirebs. es ukanaskneli ki sxvadasxva

hidroqsilazebis monawileobiT ucxo naerTTan urTierTqmedebs da hi-

droqsilirebuli produqti, an misi winamorbedi aqtiuri intermediati

warmoiqmneba.

17

garkveuli drois ganmavlobaSi iTvleboda, rom mTeli rigi qseno-

biotikebis hidroqsilirebaSi araspecifikuri fermentis erTaderTi

forma monawileobs, magram saxeobrivma da fermentuli inhibitorebisa

da induqtorebisadmi gansxvavebulma mgrZnobiarobam mkafiod gamoavlina

sxvadasxva fermentis arseboba [34, 35]. rogorc gamoirkva, hidroqsi-

lirebis yvela reaqciisaTvis saerToa mxolod Sualedi produqtis

warmoqmna NADPH-iT da O2-iT generirebuli “aqtiuri JangbadiT”.

zustad aseve, “aqtiuri Jangbadi” saerTos warmoadgens sterolebis ma-

hidroqsilirebeli fermentebisa [36] da cximebis zeJanguri Jangvis ma-

katalizebeli fermentebisaTvis [37].

aucilebelia aRiniSnos, rom N-dealkilireba, romlis drosac nax-

Sirbad–azotis kavSiri wydeba da karboniluri naerTi da dealkilire-

buli amini warmoiqmeba, bioqimiaSi sakmaod cnobili reaqciaa. misi gan-

xorcielebis mizania azotze damatebiTi aqtiuri wyalbadis gaCena,

romlis saSualebiTac N-koniugatebis warmoqmna da organizmidan maTi

swrafad gamoZeveba xdeba. maSasadame, dealkilirebis Sedegad sawyisi

nivTierebis polaroba izrdeba, rasac Tan sdevs ganawilebis moculobis

Semcireba [38]. N-dealkilireba da dezaminireba identuri procesebia da

ucxo naerTTa metabolizmSi maT erT-erTi centraluri pozicia ukavia.

RviZlis mikrosomebSi N-dealkilirebis reaqcia SeiZleba N-JangviT

daiwyos. amitom C_N-kavSiris gawyveta SesaZloa aminooqsigenazam gana-

xorcielos. es imas niSnavs, rom citoqrom P450-is garda N-oqsi-

dazebis funqcias gansazRvruli flavinuri fermentebi inawileben [39].

amis sasargeblod metyveleben arCakovisa da Tanaavtorebis [40, 41]

monacemebic. oqsidazuri reaqciebis gaaqtivebis mizniT maT gamoiyenes

Mg2+-is sxvadasxva koncentraciebi da Seiswavles dimeTilanilinis N-

demeTilirebaze da p-hidroqsilirebaze am metalis ionebis gavlena.

aRmoCnda, rom orive reaqciisaTvis aqtivaciis koeficienti Sesabamisad

1.5-s da 2.2-s Seadgenda, maSin rodesac N-Jangvaze Mg2+ araviTar

zemoqmedebas ar avlenda. am Sedegebis safuZvelze avtorebi im daskv-

namde mividnen, rom demeTilirebisa da hidroqsilirebis reaqciebze

Mg2+-is mastimulirebeli moqmedeba an NADPH-specifikuri flavopro-

teiniT ar unda iyos ganpirobebuli, an N-JangvaSi sxva, Mg2+-isadmi

aramgrZnobiare flavoproteini unda iyos CarTuli. aqve SevniSnavT, rom

18

jer kidev gasuli saukunis 60-ian wlebSi ciglerma da petitma [42]

aCvenes, rom N,N-dimeTilanilinis oqsidi intermediats warmoadgens

N,N-dimeTilanilinis N-demeTilirebis gzaze. substrati iJangeboda

erT-erTi flavin-damokidebuli oqsidaziT, xolo Semdgom citoqrom

P450-iT demeTilirdeboda, Tumca mogvianebiT vilim da bikelma [43]

daaskvnes, rom N-oqsiduri dealkilireba, rogorc α-naxSirbadis Jang-vis damatebiTi gza, zogad wesad ar unda miiCneodes.

aromatul naerTebSi hidroqsilirebis kvlevisa da miRebuli pro-

duqtebis analizis safuZvelze sxvadasxva mosazrebebia warmodgenili,

romlebic mikrosomuli Jangvis am ZiriTadi meqanizmis garkvevas exebian.

amasTan dakavSirebiT intensiurad mimdinareobda biologiuri hidro-

qsilirebisa da modeluri sistemis mier ganxorcielebuli nacnobi

meqanizmis mqone hidroqsilirebis SedarebiTi Seswavla.

modeluri sistemebidan yvelaze popularulia fentonis reaqtivi

(H2O2+Fe2+) da iudenfrendis [44] sistema (askorbati_Fe2+

_EDTA_O2 an

H2O2), romlebic Sesabamisad hidroqsilisa (•HO ) da perhidroqsilis

(•

2HO ) radikalebs warmoqmnian. iudenfrendis sistema, romelSic EDTA

(eTilendiamintetraZmarmJava) Setanilia, rogorc metalebTan kompleqs-

warmomqmneli maxelatirebeli agenti, Jangvis ramodenime reaqcias

akatalizebs. isini principSi mikrosomul fermentul Jangvas, maT

Soris aromatul hidroqsilirebas, aciklur Jangvas, O-dealkilirebas,

N-dealkilirebas da aminoJangvas Seesabamebian. mikrosomul da modelur

sistemebze Catarebulma gamokvlevebma SesaZlebeli gaxada daSvebuliyo,

rom Jangvis biologiuri meqanizmis safuZvels Tavisufal (SesaZloa

perhidroqsilis) radikalebs Soris urTierTqmedebis reaqcia

warmoadgens.

aromatuli naerTebis hidroqsilirebisa da warmoqmnil produqtTa

gamosavalSi mikrosomul da modelur sistemaTa Soris msgavseba im

pirobiT xdeba ufro TvalsaCino, Tu davuSvebT, rom ucxo naerTi

Jangbadis gaaqtivebul formasTan, rogorc mahidroqsilirebel agentTan,

SeSfoTebul molekulur (aRgznebul mdgomareobaSi myofi uaryofiTi

ioni an molekula) mdgomareobaSi urTierTqmedebs [45]. aman SeiZleba

epoqsidis an N-oqsidis, xolo Semdgom maTi gadajgufebiT, Sesabamisad,

fenolis an N-hidroqsiluri naerTis warmoqmna gamoiwvios. isic unda

19

iTqvas, rom fermentulad da modeluri sistemebiT ganxorcielebul

hidroqsilirebas Soris msgavseba xSir SemTxvevaSi arasrulia da mTeli

rigi ucxo naerTebis mimarT SesamCnevi gansxvavebebi arsebobs. miuxe-

davad amisa, orive mimarTulebiT Catarebuli gamokvlevebis safuZvelze

SesaZlebeli Seiqmna gakeTebuliyo mniSvnelovani zogadi daskvna, rom

mikrosomuli mahidroqsilirebeli sistema fosfolipid_protohem_sul-

fidproteinul kompleqss unda warmoadgendes, romelSic citoqrom

P450 Jangbadis gamaaqtivebeli komponentis funqcias asrulebs [46-48].

1.2 oqsigenazebi

biologiuri Jangvis reaqciaTa meqanizmebis kvleva mdidaria

winaaRmdegobrivi da xSirad erTmaneTis gamomricxavi TeoriebiT. ori

diametralurad gansxvavebuli Tvalsazrisi, romelic aRiarebda an

uaryofda molekuluri Jangbadis gaaqtiurebisa da organul naerTebSi

misi Semdgomi CarTvis SesaZleblobas, ”mSvidobiani TanaarsebobiT”

dasrulda. baxis da engler-vildis [49] mier 1897 wels Seqmnilma

Senelebuli Jangvis zeJangurma Teoriam warmatebiT daasabuTa

biologiur Jangvaze mecnieruli Sexeduleba, romelic molekuluri

Jangbadis dasaJang substratSi (R) uSualo CarTvas iTvaliswinebs:

/1 2 O2+RH2 R + H2O RH2 R + H2O2+ O2

gamomdinare im faqtidan, rom O=O bmis sruli daSlisaTvis ufro

meti energiaa saWiro, vidre _O_O_-ad gardasaqmnelad, am mkvlevarebma

miiCnies, rom Senelebuli Jangvisas Jangbadis molekula swored aseT

cvlilebas unda ganicdides. igi xorcieldeba aramdgrad wonasworobaSi

myofi autooqsidabeluri (TviTJangvadi) nivTierebis xarjze, romelic

JangbadTan urTierTqmedebisas zeJangs warmoqmnis:

AO

OA + O2

zeJangi advilad iSleba atomuri Jangbadis gamoTavisuflebiT, ro-

20

melic Zlieri damJangvelia:

AO

O+ B AO + BO

baxi-engleris Teorias mikrosomuli Jangvis dRes arsebul cnobil

faqtebTan Tu SevajerebT, Semdegi daskvnis gakeTeba SeiZleba: autooq-

sidabeluri cila-fermenti, romelsac zeJanguri dajgufebis mierTeba

SeuZlia da Semdgom aqtiuri Jangbadi Znelad dasaJang nivTierebaze ga-

daaqvs, sxva araferia, Tu ara mikrosomuli monooqsigenazas ZiriTadi

komponenti citoqrom P450. saerTod, meoce saukunis dasawyisi biologiur Jangvaze warmodgene-

bis mkveTri cvlilebebiT aRiniSna. am periodSi molekuluri Jangbadi

kerZo SemTxvevad iqna CaTvlili, sadac igi eleqtronuli aqceptoris

erT-erT SesaZlo pretendentadaa aRiarebuli. wina planze wamoiwia

fermentuli dehidrirebis reaqciam, romlis Tanaxmadac biologiuri

Jangva dasaJangi substratidan (protonTa R-donoridan) wyalbadis

atomebis (an eleqtronebis) A-aqceptorze gadatanas gulisxmobs.

RH2 + A R + AH2

Jangva

hipoTezis erT-erT avtorad vielandi [50] iTvleba. reaqciis maka-

talizebeli fermentebi _ dehidrogenazebi gansakuTrebul klass war-

moadgenen. isini wyalbadis aqceptorad O2-s iyeneben. “dehidrogenazulma

Teoriam” gasuli saukunis 60-ian wlebamde “ibatona” da pirveli “bza-

ri” mas Semdeg gauCnda, rac dadgenil iqna, rom Jangvis produqtebSi

CarTuli 18O rogorc zeviT iyo aRniSnuli, ekuTvnis molekulur Jang-

bads (bax-engleris Tanaxmad) da ara wyals (vielandis Tanaxmad).

1960-1965 wlebidan dawyebuli oqsigenazuri Teoria kvlav iZens

Tavis aRiarebas. vlindebian fermentTa sxvadasxva mono- da dioqsigena-

zuri tipebi. pirvelebi iseTi moqmedebiT xasiaTdebian, romliTac Jang-

badis mxolod erTi atomis CarTva xdeba dasaJang substratSi, xolo

meore wylis warmoqmnaze ixarjeba. dioqsigenazebs rac Seexeba, isini

Jangbadis orive atoms axmaren substratis Jangvas [51].

oqsigenazebis Tanamedrove klasifikacia ase gamoiyureba:

21

R + O2 + DH2 RO + D + H2O

RH2 + O2 RO + H2O

R + O2 RO2

R1 + O2 + R2 R1O + R2O

monooqsigenazebi dioqsigenazebi

garegani

Sinagani

Sidamolekuluri

molekulaTSorisi

maSasadame, monooqsigenazebis ori klasidan erTni funqcionirebisaT-

vis molekulur Jangbadze gadasatanad eleqtronTa gare wyaroebs (do-

norebs) saWiroeben, xolo meoreni gare redoqs-eqvivalentebs ar iTxo-

ven da eleqtronebs TviT dasaJangi molekulebidan iReben. aseTi kla-

sifikaciis garda, monooqsigenazebi imis mixedviTac SeiZleba dayofi-

liyo, Seicaven Tu ara maTi molekulebi metalis ionebs, an maT kom-

pleqsebs. yvelaze xSirad es fermentebi spilenZis an rkinis ionebs

Seicaven. swored maT miekuTvneba hemoproteini citoqrom P450 [52]. ka-talizur reaqciebSi, romlebic monooqsigenazuri kompleqsiT xor-

cieldebian, citoqrom P450 Taviseburi “aqtiuri centris” rols

asrulebs [53]. igi urTierTqmedebs substratTan, molekulur Jangbad-

Tan da Sesabamisi donorebidan iRebs eleqtronebs:

e_

H2O

citoqrom P450

SH2O2

SOH

specialur literaturaSi monoqsigenazebi xSirad “hidroqsilaze-

bad” moixseneba. es gansazRvra arazustia, ramdenadac arseboben fermen-

tebi, romlebic hidroqsilirebas akatalizeben, magram es procesi mo-

nooqsigenazuri meqanizmiT ar mimdinareobs.

dioqsigenazebis ori klasi erTmaneTisagan imiT gansxvavdeba, rom

erT SemTxvevaSi Jangbadis ori atomi erT molekulas uerTdeba (Sida-

molekuluri), xolo meoreSi _ isini dasaJangi substratis or mole-

kulas Soris nawildeba (molekulaTaSorisi). cnobilia oqsigenazebi

22

(mag., indolamindioqsigenaza), romlebic substratad ara molekulur

Jangbads, aramed misi erTi eleqtroniT aRdgenil produqts _ supe-

roqsid-radikals moixmaren.

sadReisod oqsigenazur sistemaTa polifunqciuroba eWvs aRar iw-

vevs. baqteriul da safuvris ujredebSi isini ZiriTadad plastikur

miznebs emsaxurebian. naxSirbadSemcvel nivTierebaTa Jangvis Sedegad

isini naxSirbadis sarezervo fonds qmnian. mcenareebSi monooqsigenazuri

reaqciebiT sinTezdeba: bioregulatorebi, alkaloidebi, steroidebi da

sxv. cxovelisa da adamianis ujredebSi Semonaxul plastikur funq-

ciebTan erTad am fermentul sistemas kidev erTi, damcvelobiTi funq-

ciac gauCnda. monooqsigenazebis ZiriTadi fiziologiuri rolis _ mo-

lekulis polarobis gazrdis zogadi sqema erTnairad optimalurad

esadageba rogorc qsenobiotikebs, aseve mraval endogenur hidrofobul

nivTierebebs. evoluciuri kibis yvelaze maRal safexurze dgas RviZlis

citoqrom P450-Semcveli monooqsigenaza. aqedan gamomdinare, gasakviri

araa, rom am organos mikrosomuli Jangvis sistema sawyisi da univer-

saluri modelia yvela eukariotuli organizmis membranasTan dakavSi-

rebuli oqsigenazebis dasaxasiaTeblad.

1.3 mikrosomuli monooqsigenazebis fermentuli

Semadgenloba

membranuli monooqsigenaza fermentuli kompleqsia, romelic fos-

folipidur matriqsSia CaSenebuli. reaqciisaTvis saWiro eleqtronebis

wyaros aq ZiriTadad NADPH warmoadgens, saidanac aRmdgeneli eqviva-

lentebi flavoproteinis saSualebiT citoqrom P450-ze gadaitanebian. am fermentis saxelwodebaa NADPH-citoqrom P450-reduqtaza. mikro-somul membranaSi kidev ori komponenti imyofeba, romlebic aseve mona-

wileoben ucxo naerTTa metabolizmSi da eleqtronTa satransporto

jaWvs qmnian. esenia NADPH-citoqrom b5-reduqtaza da citoqrom b5.

xangrZlivi periodis manZilze iTvleboda, rom aRniSnuli fermentuli

sistema erTaderTia, romelic RviZlis endoplazmur membranaSi NADPH-

s Jangavs. SemdgomSi mikrosomebidan gamoyofil iqna kidev erTi

23

NADPH-specifikuri flavoproteini, romelic Tavisi molekuluri

TvisebebiT gansxvavdeba citoqrom P450-Semcveli monooqsigenazas fla-

voproteinisagan. igi umTavresad mesameuli meTilCanacvlebuli aminebis

N-JangvaSi monawileobs. amitom amJamad SeiZleba iTqvas, rom mikrosoma-

Si NADPH-is Jangvis ori sistema funqcionirebs [54-57]. rogorc

gairkva, monooqsigenazur kompleqsSi flavoproteinis erT molekulaze

citoqrom P450-is ramdenime molekula modis [58].

1.3.1 NADPH-citoqrom P450-reduqtaza

(NADPH-specifikuri flavoproteini EC 1.6.2.4)

fermenti endoplazmuri membranis ZiriTadi redoqs-jaWvis sawyisi

komponentia. xSirad mas aRniSnaven rogorc FP1-s (flavoprotein l-s). misi saSualebiT citoqrom P450 eleqtronebs jer ferment-substratis

kompleqsis, xolo Semdgom sammagi kompleqsis (fermenti_substra-

ti_O2) Tanmimdevruli aRdgenisaTvis iyenebs.

fermenti pirvelad mikrosomebidan gamoyofil iqna 1962 wels,

mkvlevarTa ori damoukidebeli jgufis mier [59, 60]. citoqrom с-sadmi maRali swrafvis gamo mas Tavdapirvelad NADPH-citoqrom с-reduqtaza ewoda. aqve SevniSnavT, rom citoqromi с mikrosomebSi Za-lian mcire raodenobiTaa. eleqtronTa WeSmariti bunebrivi aqceptori

sadReisod jerac ucnobia. sayuradReboa is faqtic, rom flavoprotei-

nis swrafva citoqrom b5-is mimarT izrdeba da citoqrom с-s mimarT swrafvas utoldeba garemos ionuri Zalis gaormagebisas [61]. fermenti

membranasTan mtkiced dakavSirebuli cilaa. natiuri saxiT igi 81 kD

molekuluri masis mqone dimers warmoadgens da ori 40.5 kD moleku-

luri masis mqone suberTeulisagan Sedgeba. gamoyofis meTodis miuxeda-

vad, reduqtaza erT polipeptidur jaWvze TiTo molekula FMN-s da

FAD-s Seicavs [62]. rogorc irkveva, isini cilasTan kovalenturad ar

arian dakavSirebulebi, amitom fermentisagan maTi mocileba pH-is daq-

veiTebiT, an xsnaris maRali ionuri Zalisa da dabali temperaturis

pirobebSi xerxdeba. reduqtazas molekulis stoqsis radiusi 4 nm-s

aRwevs, xolo standartuli redoqs-potenciali -310 mV-s utoldeba.

24

aminomJavuri Semadgenlobis analizma aCvena, rom igi ∼31%-mde hidro-

fobul aminomJavur naSTebs, agreTve cisteinis 6 da triftofanis 1

naSTs Seicavs. Tiolur jgufebs, rogorc Cans, garkveuli roli unda

gaaCndeT reduqtazisa da NADPH-is urTierTqmedebaSi. daJangul mdgo-

mareobaSi flavoproteinis STanTqmis speqtri sami maqsimumiT (277, 380

da 455 nm-ebze) da erTi mxariT (485 nm-ze) xasiaTdeba. gansazRvrulia

maRali sisufTavis fermentis eqstinqciis koeficienti, romelic 455

nm-ze 10.7 mM-1.sm-1-s Seadgens.

reduqtaza mkacrad specifiuria NADPH-is mimarT da TiTqmis Tana-

bari siCqariT SeuZlia aRadginos erT- da oreleqtroniani aqceptorebi:

citoqromi c, K3[Fe(CN)6], diqlorfenolindolfenoli, magram ara ci-

toqromi b5, koenzimi Q10, neotetrazoliumi da citoqromebi P420 an P450 [63-66]. izolirebul ferments neotetrazoliumis aRdgenis unari

mxolod fosfatidilqolinis Tanamyofobisas uCndeba. lecitinis iseTi

uaxloesi analogi, rogoric fosfatidileTanolaminia, am SemTxvevaSi

araefeqturia. izolirebuli flavoproteini molekuluri JangbadiT

Zlier dabali siCqariT iJangeba. reduqtazuli reaqciis SesaZlo meqa-

nizmi mocemulia nax. 1.1-ze.

NADPH

NADP

2FAD

2FADH

2FADH2

aRdgenili aqceptori

daJanguli aqceptori

NADPH

NADP

+

+

+

nax. 1.1. FAD-is aRdgenis reaqciis meqanizmi [64].

mocemuli meqanizmis Tavisebureba imaSi mdgomareobs, rom am dros

fermentis naxevradaRdgenili stabiluri forma warmoiqmneba [65, 66].

amis sasargeblod arsebuli eqsperimentuli monacemebis Tanaxmad, aero-

bul pirobebSi NADPH-specifikur redoqs-jaWvSi kofermentis Tana-

myofobisas erTdroulad SeiZleba aRdgenili flavinis ori forma

25

(FADH da FADH2) arsebobdes, romelTa Sorisac dinamikuri wonaswo-

roba myardeba [61]. flavinis naxevradaRdgenil formas citoqrom c-sa da eleqtronTa sxva aqceptorebis aRdgenis unari ar gaaCnia. am reaq-

ciebSi aqtiur aRmdgenels mxolod FADH2 warmoadgens. reaqciaTa ste-

qiometriis Seswavlam aCvena, rom 1 moli NADPH 2 mol citoqrom c-s aRadgens da NADP+

-is garda, am dros produqtad FADH-ic miiReba.

aRniSnulidan gamomdinare, daaskvnes, rom substratis Jangvisa da aq-

ceptoris aRdgenis procesSi flavini naxevradaRdgenilidan srulad

aRdgenil formad ciklur gardaqmnebs ganicdis. am TvalTaxedvis mixed-

viT reduqtazas sruli aRdgenis reaqciis Tanmimdevroba SeiZleba ase

warmovidginoT:

NADPHFAD_FMN FADH2_FMN FADH_FMNH

FAD_FMNH2NADPH FADH2_FMNH2

. . .

principulad gansxvavebuli Sedegebia miRebuli, da Sesabamisad, sxva

Tvalsazrisia Camoyalibebuli inagisa da Tanaavt. [67, 68] mier, rom-

lebmac daadgines, rom reduqtazas molekula yovel 69 kD molekulu-

ri masis mqone polipeptidze 2 mol flavins Seicavs. maTgan erTi FAD,

xolo meore FMN-ia. aq centraluri problema NADPH-dan citoqrom

P450-ze eleqtronebis gadatanaSi mdgomareobs. piridinnukleotidebi

ori eleqtronis donorebi arian, magram erTi hemis Semcvel hemoprote-

ins erTi eleqtronis miReba SeuZlia. amdenad cila, romelic NADPH-

dan citoqrom P450-ze eleqtronTa gadatanas emsaxureba, erTdroulad

ori eleqtronis aqceptorisa da erTi eleqtronis donoris Tvisebebs

unda flobdes. es meqanizmi TviT NADPH-flavoproteinuli reduqta-

zis moqmedebiT SeiZleba aixsnas. igi erTaderTi sadReisod cnobili

fermentia, romelic kofaqtorebad FAD-s da FMN-s Seicavs. reduqtaza

NADPH-dan myisierad Rebulobs or eleqtrons, magram maT cal-calke

gadascems an rkina-gogirdovan cilas (mitoqondriaSi), an pirdapir ci-

toqrom P450-s (mikrosomaSi). meore SemTxvevaSi eleqtronTa trans-

portisaTvis rkina-gogirdovani cilebis monawileoba saWiro aRaraa.

aerobul pirobebSi redoqs-jaWvis funqcionirebisas flavinis erTi

molekula stabilur semiqinonis formaSi gadadis. meore flavini ki

26

srulad daJangul mdgomareobaSi rCeba. warmoqmnili semiqinoni epr-

speqtris damaxasiaTebel signals g=2-is olqSi iZleva, romelic mi-

krosomuli preparatebisTvisac daregistrirda. avtorebma ver gamoav-

lines am fermentisaTvis damaxasiaTebeli zeviTaRniSnuli naxevradaRd-

genili da srulad aRdgenili formebis Seqcevadi gadasvlebi. amitom,

maTi azriT, flavoproteinis aRdgena oTx-eleqtronian stadias unda

moicavdes da srulad aRdgenil ferments citoqrom P450-Tan uSualo

urTierTqmedeba unda SeeZlos; anu, rogorc iTqva, cila, romelic

NADPH-dan hemoproteinze eleqtronTa gadamtanis rols asrulebs,

marTlac unda avlendes ori eleqtronis aqceptorisa da erTeleqtro-

niani donoris Tvisebebs.

FAD da FMN, vitamin riboflavinis warmoebulebia da izoaloqsa-

zinis birTvs Seicaven. maTi qimiuri buneba savsebiT Seesatyviseba nax.

1.1-ze aRwerili procesebis specifikas: rodesac srulad daJanguli

flavonukleotidi mxolod erT eleqtrons (wyalbadis erT atoms)

ierTebs, izoaloqsazinis birTvis naxevrad aRdgenili (semiqinonuri)

forma warmoiqmneba. garda amisa, igi SeiZleba imyofebodes an srulad

daJangul, an erTi da ori eleqtroniT aRdgenil mdgomareobebSi:

CNHC

N

N

NCH3

CH3

O

O

R

H

e + H+_e + H+_

CNHC

N

N

NCH3

CH3

O

O

R

CNHC

N

N

NCH3

CH3

O

O

R

H

H

_

.

(daJanguli) (semiqinoni)

(aRdgenili)

FAD an FMN FAD an FMN

FADH2 an FMNH2

. .

27

aRniSnuli garemoeba imas ganapirobebs, rom am klasis flavoproteinebi

ufro mravalricxovan reaqciebs akatalizeben, vidre piridinnukleoti-

duri dehidrogenazebi.

dehidrogenazul reaqciaTa ZiriTadi Sualeduri produqtebis Semad-

genlobaSi, rogorc wesi, ar identificirdeba flavinis radikalebi,

amdenad, substratis flavindamokidebuli dehidrogenizacia oreleq-

tronian gadatanasTan aris dakavSirebuli [69]. O_H-kavSiris oreleq-

troniani aqtivaciis meqanizmi Seesabameba dabali potencialis ubans anu

“Sesasvlels” biologiuri Jangvisas da maRali potencialis ubans anu

“gamosasvlels” fotosinTezisas. Tumca unda aRiniSnos, rom metalTa

kompleqsebTan flavini erTeleqtroniani meqanizmiT reagirebs, romelic

sunTqviTi jaWvis flavinuri ubnidan “gamosasvlels” da fotosinTezur

jaWvSi “Sesasvlels” Seesabameba. es imasTanaa dakavSirebuli, rom cile-

bi zrdian flavinuri radikalis mdgradobas [70]. amgvarad, reduqtazas

SeuZlia miiRos eleqtronebi NADPH-dan da isini gaanawilos flavinis

or kofaqtors Soris, vidre maT hemoproteins gadascems.

miuxedavad imisa, rom gasufTavebuli, homogenuri flavoproteini

JangbadTan Zalian nela reagirebs, membranaSi igi mniSvnelovan autooq-

sidabelobas amJRavnebs. amitom savsebiT SesaZlebelia, redoqs-jaWvis

muSaobisas H2O2-is warmoqmna am ubanze iyos lokalizebuli. NADPH-

oqsidazuri aqtivoba advilad rekonstruirdeba maRali ionuri Zalis

mqone areSi NADPH-specifikuri flavoproteinis, citoqrom b5-isa da

Fe_EDTA-s kompleqsis gamoyenebisas [63, 71]. naCvenebia mocemuli fer-

mentis monawileoba azoreduqtazul reaqciaSi. gasufTavebuli saxiT

mas unari aqvs aRadginos azosaRebavebi, kerZod dimeTilaminoazobenzo-

li. am reaqciis kinetika da inhibitorebisadmi mgrZnobiaroba srul Se-

sabamisobaSia NADPH-citoqrom-c reduqtazul reaqciasTan [72, 73].

trifsiniT solubilizebul reduqtazas citoqrom b5-is aRdgenis

unari garemos maRali ionuri Zalis pirobebSi gaaCnia; detergentiT

gamoyofils ki marilTa dabali koncentraciis xsnarebSic SeuZlia he-

moproteinis aRdgena. flavinze warmoqmnili superoqsiduli anioni ci-

toqromis aRdgenaSi ar monawileobs [74].

28

1.3.2 NADH-citoqrom b5-reduqtaza (EC 1.6.2.1)

endoplazmuri membranis eleqtronTa satransporto meore sistema

NADH-damokidebuli citoqrom b5-reduqtaziTa da citoqrom b5-iTaa

warmodgenili. orive cila amfifiluri bunebisaa, anu rogorc

hidrofobul, ise hidrofilur ubnebs Seicavs. amTaviTve SevniSnavT,

rom eleqtronTa gadatanis es gza ujredis yvelaze naklebad

Seswavlil fermentul sistemas warmoadgens da misi WeSmariti

funqciuri roli kvlavac daudgeneli rCeba.

NADH-damokidebuli redoqs jaWvis wyoba SeiZleba ase warmovidgi-

noT (nax. 1.2).

FP1 citoqrom b5

askorbati

cianidmgrZnobiare faqtori

fericianidi

semidehidroaskorbati,

neotetrazoliumi

citoqrom c,

NADH

nax. 1.2. NADH-damokidebuli redoqs jaWvi. uwyveti xazebiT gamosax-

ulia citoqrom b5-is aRdgenis, xolo punqtiriT misi Jangvis

gzebi [75, 76].

gamoyofili da gasufTavebuli NADH-citoqrom b5-reduqtazas

(Cveulebriv, mas aRniSnaven, rogorc FP2-s), molekuluri masa 38-41 kD-

ia. igi ar Seicavs rkinas da an sxva romelime cvalebadi valentobis

metalis ions. erT mol apofermentze reduqtaza erT mol FAD-s Sei-

cavs. koferment-cilis urTierTqmedebas, rogorc irkveva, Tirozinis

naSTi ganapirobebs, ramdenadac erTi fenoluri jgufis iodireba am

urTierTqmedebas absoluturad ablokirebs [77-79]. safiqrebelia, rom

apofermentTan FAD-is dakavSirebaSi sulfhidriluri jgufebi ar mona-

wileoben, radgan maTi inhibitorebi _ p-qlormerkurbenzoati da mer-

29

salili flavinis mierTebaze ar moqmedeben, Tumca erT-erTi Tavisufali

SH-jgufi mainc pasuxismgebeli unda iyos cilasTan NADH-is dakavSi-

rebaze. NADH-flavoproteinis kompleqsi apofermentis SH-jgufs icavs

p-qlormerkurbenzoatisa da N-eTilmaleinidis moqmedebisagan. kompleq-

sis warmoqmnaSi NADH-is fosforibozuli naSTi monawileobs. steqi-

ometriulad fermentis erTi molis aRdgenaze NADH-is 1 moli ixar-

jeba [77-80]. FAD-is aRdgena stereospecifikurad, piridinuli birTvis

4A-mdgomareobidan flavinze wyalbadis pirdapiri gadataniT xorciel-

deba. eleqtronul aqceptorad fericianidis gamoyenebisas swored C_H-

bmis gaxleCva warmoadgens reaqciis siCqaris malimitirebel stadias.

Setutianebisas fermentis inaqtivacias Tan axlavs polipeptiduri

jaWvis gafaSareba, agreTve triftofanis naSTisa da flavinis gadasvla

membranis polarul fazaSi. cvlilebebs eqvemdebareba cisteinisa da Ti-

rozinis naSTebic, magram daSlas mxolod NADH-is dakavSirebuli

centri ganicdis [81, 82].

Jangbadisa da citoqrom c-s (rogorc eleqtronebis aqceptorebis)

mimarT da NADPH-is (rogorc eleqtronebis donoris) mimarT, fermen-

tis molekuluri aqtivoba uaRresad dabalia. amis sapirispirod, Zalian

maRalia reduqtazas citoqrom b5-isadmi da K3[Fe(CN)6]-isadmi swrafva [79]. gasufTavebul ferments neotetrazoliumis aRdgenis unari ar

gaaCnia, magram citoqrom b5-is damatebis Semdeg vlindeba NADH-

neotetrazolium-reduqtazuli aqtivoba. citoqrom b5-is gavleniT

diqlorfenolindofenolis aRdgenis siCqare TiTqmis ormagdeba da mi-

krosomebSi NADH-diqlorfenolindofenol-reduqtazuli aqtivobis

50%-mde flavoproteinis wilad modis [83].

eleqtronebis aqceptorad O2-is gamoyenebisas flavoproteiniT

NADH-is Jangvis siCqare Zalze dabalia da fericianiduli Jangvis siC-

qaris 0.01%-s utoldeba. Zalian dabalia agreTve (0.3%-mde) fermentiT

NADPH-is daJangvis siCqare fericianidisa da Jangbadis Tanamyofobisas.

maSasadame, ferments TviTdaJangvis unari ar gaaCnia. eleqtronebis do-

norebis _ NADPH-isa da NADH-is mimarT flavoproteinis swrafva

imdenad gansxvavebulia (molekulur aqtivobaTa Soris sxvaoba NADH-

isaTvis 3-rigiT maRalia), rom fermenti NADH-isadmi absoluturad

specifikurad SeiZleba CaiTvalos [81].

30

NADH-citoqrom b5-reduqtazuli reaqciis meqanizmi safuZvlianad

jerac araa Seswavlili. safiqrebelia, rom citoqromis aRdgena erTe-

leqtroniani gadatanis Sedegs warmoadgens [84, 85]. Tavdapirvelad xde-

ba fermentisa da NADH-is kompleqsis formireba. reduqtazas piridi-

nuli birTvi apofermentis SH-jgufTan urTierTqmedebs, xolo adeno-

zindifosforibozis fosfati garkveul eleqtrofilur jgufs ukav-

Sirdeba. procesi imdenad swrafad xorcieldeba, rom mTliani reaqciis

siCqaris limitireba aq ar unda xdebodes. momdevno stadiaze xdeba

C_H bmis daSla da piridinuli birTvidan flavinze wyalbadis gadata-

na. es reaqcia gacilebiT Senelebulad xorcieldeba da amdenad, SeiZle-

ba mTliani procesis siCqaris limitirebas axdendes. am mosazrebas isic

adasturebs, rom substratad NADH-is gamoyenebisas reaqciis siCqaris

konstanta (68 mM.wm-1) korelaciaSia am piridinnukleotidis daJangvis

jamuri reaqciis siCqaresTan [86]. yvelaze naklebadaa Seswavlili fla-

voproteinidan citoqrom b5-ze eleqtronis gadatanis stadia, maSin

rodesac swored mas Tvlian reduqtazuli reaqciis siCqaris malimiti-

rebel etapad [87, 88].

membranis fosfolipidi aadvilebs NADPH-reduqtazul reaqcias da

mis swraf msvlelobas uzrunvelyofs, xolo NADH-fericianid-

reduqtazul aqtivobaze fosfolipidi gavlenas ar axdens.

triton X-100-iT damuSavebuli RviZlis mikrosomebidan gamoyofili

reduqtaza tipiuri amfifiluri cilaa (molekuluri masiT 43 kD) da

381 aminomJaur naSTs (detergentul fragments) Seicavs [89, 90]. pro-

teinazuli (katefsinazuri) damuSavebis Semdeg ferments 33 kD mole-

kuluri masa gaaCnia da 292 aminomJauri naSTisagan Sedgeba. detergen-

tul fermentSi arapolaruli aminomJavebis Semcveloba gacilebiT me-

tia, vidre proteazulSi. detergentuli fermentis qimotrifsiniT da-

muSaveba 10 kD molekuluri masis mqone polipeptidis mocilebas iw-

vevs, romelic upiratesad hidrofobuli aminomJavebiTaa warmodgenili.

detergentuli fermenti wyalxsnarSi 600 kD molekuluri masis agre-

gatebs warmoqmnis. NADH-isa da citoqrom b5-isadmi orive fermentis

swrafva erTnairia: NADH-is SemTxvevaSi KM Sesabamisad 6.0 µM da 2.5

µM-ia, xolo citoqrom b5-isaTvis KM Sesabamisad 45 µM-isa da 10

µM-s Seadgens. am monacemebis safuZvelze gakeTebulia daskvna, rom ka-

31

talizurad aqtiuri centri molekulis hidrofobul nawilSi imyofeba,

romelic fermentis mTliani molekulis 25%-s ar aRemateba. flavo-

proteini membranaSi CaSenebis gasaocar unars amJRavnebs; kerZod, igi

100-jer aRemateba misi sawyisi Semcvelobis raodenobas. membranaSi Ca-

Senebuli reduqtaza funqciurad uaRresad aqtiuria da membranul

struqturas hidrofobuli urTierTqmedebis ZalebiT mtkiced ukavSir-

deba. garda amisa, ferments membranis sibrtyeSi advilad difundirebisa

da citoqrom b5-is ramdenime molekulis dakavSirebis unari aqvs.

xsnarSi da membranaSi reduqtazasa da citoqrom b5-s moqmedebis erT-

nairi kinetika aqvT, rac saSualebas gvaZlevs misaRebad CavTvaloT mo-

sazreba imis Sesaxeb, rom membranaSi ar arsebobs flavoproteinisa da

citoqrom b5-is specializirebuli kompleqsi da mikrosomebis Txevad

fazaSi redoqs-jaWvis funqcionireba gadamtanTa SemTxveviT Sejaxebebs

unda emyarebodes [89, 90].

arsebobs monacemebi, romlebic mikrosomebSi NADH-citoqrom b5-

reduqtazas garda, NADH-is Jangvis sxva gzis arsebobaze miuTiTeben

[58, 75, 91, 92]. varaudoben kidev erTi flavoproteinis funqcionire-

bas, romelic prostetul jgufad FMN-s Seicavs da maRali swrafva

gaaCnia citoqrom c-s mimarT [93]. am reaqciiT citoqrom c-s aRdgena sulfhidriluri Sxamebisa da detergentebis mimarT mdgradobas amJRav-

nebs [75, 91] da amiT gansxvavdeba NADH-citoqrom b5-reduqtazuli

aqtivobisagan.

aRvniSnavT agreTve, rom gamoyofilia NADH-reduqtazas polimeru-

li forma molekuluri masiT 200-400 kD. dodecilsulfatis moqmede-

biT es polimeri 35-39 kD molekuluri masis mqone suberTeulebad

iSleba. flavoproteinis monomeruli formisagan gansxvavebiT, polimeri

citoqrom c-s mimarT ufro moWarbebul swrafvas amJRavnebs, vidre fe-

ricianidis mimarT, anu monomerTan SedarebiT reduqtazas polimeruli

forma ufro “fiziologiuria” [94, 95].

32

1.3.3 citoqromi b5

cila-fermentebis mniSvnelovan klass warmoadgenen hemi- an hemo-proteinebi, romlebic prostetul jgufad rkina-porfirinebs Seicaven.

mraval sxva funqciebTan (hemoglobiniT Jangbadis transporti, mioglo-biniT Jangbadis Senaxva, citoqrom c-oqsidaziT Jangbadis wylamde

aRdgena, citoqrom P450-iT sxvadasxva funqciuri jgufebis daJangva

Jangbadis saSualebiT da sxv.) erTad, isini monawileoben eleqtronTa

gadatanaSi (citoqromebi b da c). winsarTebi hemi- da hemo- warmodgeba rkinaporfirinis dajgufebis saxelwodebidan: rkina (II) porfirinis

kompleqss uwodeben hems, xolo rkina (III) porfirins _ hemins. sami yvelaze gavrcelebuli hemi (a, b da c) dakavSirebulia a, b da c cito-qromebTan. b-hemis porfirinul ligands warmoadgens protoporfirini

IX. amitom xSirad b-hems uwodeben: rkina (II) protoporfirin IX-s, an

protohems, an ubralod hems [96]:

N

N

N

N

CH3 CH CH2

CH3

CH CH2

CH3CH2

CH2

CO2H

CH3

CH2

CH2

CO2H

Fe...... ......

......

......

1 2

3

4

56

7

8

NH

N

NH

N

CH3 CH CH2

CH3

CH CH2

CH3CH2

CH2

CO2H

CH3

CH2

CH2

CO2H

1 2

3

4

56

7

8

protoforfirin IX

(2,4-difenildeiteroporfirini)hemi b _ anu protohemi

romauli cifri IX porfirinul ciklSi Canacvlebulebis specifikur mdeba-

reobas aRniSnavs. porfirinis birTvis 8 naxSirbadatomTan SeiZleba sxvadasxva

sigrZis da funqciuri jgufebis mqone Camnacvlebeli iyos dakavSirebuli. erT-

erTi maTganis specifikuri ganlageba (rigiT IX) b-hemis warmomqmnel ligands

_ protoporfirin IX-s axasiaTebs (detalurad ix. 1.6).

33

mikrosomul membranebSi arsebuli b-tipis citoqromuli cilebi sa-

dReisod safuZvlianadaa Seswavlili. isini farTod arian gavrcelebu-

li sruliad gansxvavebul obieqtebSi. am jgufis warmomadgeneli cito-

qromi b5 identificirebulia RviZlisa da tvinis qsovilebSi, cximovan

qsovilSi, umaRles da umdables mcenareTa organoebSi, mikroorganiz-

mebSi, anaerobulad mzard safuvrebSi da sxv. igi mtkiced ukavSirdeba

ujredis organelebs da amitom xsnadi formiT misi miReba mxolod

fermentuli hidrolizis Sedegadaa SesaZlebeli [97-100]. citoqromi b5

gamoyofilia monomeris saxiT, romelsac 11-13 kD molekuluri masa

gaaCnia. 1 mol apofermentze misi molekula 1 mol rkina-protopor-

firin IX-s Seicavs. igi amfifaturi cilaa, romelSic hidrofobuli

ubnis sidide ufro metia (mTeli molekulis 25%), vidre flavopro-

teinSi. hidrofobuli nawili membranaSia CaZiruli da gareT myofi ci-

lis didi nawilisaTvis damWeri “Ruzis” rols asrulebs. daJanguli

formiT es hemoproteini kargad gamosaxul STanTqmis maqsimums 413 nm-

ze avlens, xolo aRdgenili forma aseve kargad gamosaxul STanTqmis

maqsimumebs 424, 525 da 556 nm-ebze amJRavnebs [101, 102].

gasufTavebuli hemoproteini aRdgeba diTionitiT, cisteiniT, askor-

batiT da fermentulad – NADH-citoqrom b5-reduqtaziT. igi iJangeba

citoqrom c-s, FeCl2-is da saTanado redoqs-potencialis mqone saRebave-

bis moqmedebiT. JangbadTan igi Zalian nela urTierTqmedebs [103-105].

naxSirbadis monooqsidTan, azidTan da cianidTan es hemoproteini ar

reagirebs, xolo mJavebisa da tuteebis zemoqmedebisadmi sakmaod

mdgradia [106, 108]. naxSirbadis monooqsidi da cianidi, rogorc li-

gandebi, cila-hematohemis kompleqsSi rkinas koordinaciulad ukavSir-

debian. es procesi rkinasTan cilis jgufebis CanacvlebiT mimdinareobs.

sadReisod sruladaa gaSifruli citoqrom b5-is aminomJavuri Tan-

mimdevroba [103, 104, 109-111]. sxvadasxva wyaroebidan proteolizuri

damuSavebis Sedegad gamoyofili hemoproteini TiToeul polipeptidur

jaWvSi Tirozinis sam da triftofanis erT naSTs Seicavs. molekulaSi

gamovlenili araa gogirdSemcveli aminomJavebis arseboba. isini gansx-

vavdebian Tavisi N-kidura aminomJavebiT. savsebiT SesaZlebelia, rom

erTi obieqtidan miRebuli heterogenurobiTac xasiaTdebodes. amis ma-

34

galiTia xbos RviZlidan miRebuli cilis preparati, romelic Seicavs

eleqtroforezuli Zvradobisa da DEAE-celulozaze qromatografire-

bis unariT erTmaneTisagan gansxvavebul citoqromi b5-is qvefraqciebs

[112]. pirvelad struqturaSi gansxvaveba imaSi mdgomareobs, rom erTi

maTgani _ citoqrom b5(1) C-kidura dipeptidad glutamin-serins Sei-

cavs, maSin rodesac meore citoqrom b5(2)-s es dipeptidi saerTod ar

gaaCnia. citoqrom b5-is ori qvefraqciis arseboba erTmniSvnelovnad ar

wyvets sakiTxs imis Sesaxeb, namdvilad arsebobs Tu ara es ori cito-

qromi, an gansxvaveba maT struqturaSi xom araa artefaqti, anu solu-

bilizaciis procedurisas proteolizuri dazianebis Sedegi.

RviZlidan gamoyofil hemoproteinis preparatebSi ori citoqrom

b5-is arseboba sxva avtorebis mierac aris naCvenebi. aRmoCnda, rom

trifsiniT damuSavebisas DEAE-celulozaze swrafad da Senelebulad

Zvradi citoqrom b5-ebis fardoba Sesabamisad 7:1-s, xolo baqteriuli

proteaziT damuSavebul preparatebSi 1:4-s Seadgens [101]. citoqrom b5-

is mesame tipi, romelic wina orisagan arsebiTad gansxvavdeba, gamoyo-

filia mitoqondriis gare membranidan [113], xolo TviT mitoqondriaSi

identificirebulia sami citoqrom b5, romlebic membranul struqtu-

rebTan kavSiris simtkiciTa da imunologiuri TvisebebiT gansxvavdebian

[114, 115].

jerjerobiT araa gacemuli pasuxi kiTxvaze _ warmoadgenen Tu ara

citoqrom b5-is preparatebi heterogenul formebs? am problemis kvle-

vaSi mniSvnelovani roli Seasrules iaponelma mkvlevarebma itom da

satom [116, 117]. swored maT mier iqna naCvenebi, rom citoqrom b5-s Tu

miviRebT ara proteazuli damuSavebiT, aramed detergentebiT solubi-

lizaciis Sedegad, SesaZlebeli gaxdeba gamoiyos citoqromis monomeri,

romelsac Cveulebrivze orjer meti molekuluri masa (24-26 kD)

gaaCnia. am gziT izolirebuli citoqromi b5-is monomeri uaRresad hi-

drofobulia da swrafva aqvs warmoqmnas polimeri molekuluri masiT

120 kD. am mkvlevarTa azriT, proteazuli damuSavebisas solubiliza-

cias ganicdis citoqrom b5-is mxolod fermentulad aqtiuri nawili.

molekulis meore nawili, romelic membranaSi rCeba da mis cila-

lipidur kompleqsTan dakavSirebazea pasuxismgebeli, proteazuli ze-

moqmedebisas xsnarSi ar gadadis. amis sapirispirod detergenti hemo-

35

proteinis mTliani molekulis solubilizacias iwvevs.

miuxedavad imisa, rom arsebuli monacemebi gadaWriT ver asabuTeben

RviZlis mikrosomebSi ori gansxvavebuli citoqrom b5-is funqcionire-

bas, aseve maTi arsebobis ignorireba mainc ar SeiZleba. amis saTqmelad

safuZvels gvaZlevs arCakovisa da misi TanamS. [118-123] mier miRebuli

faqtebi, romlebic mocemuli hemoproteinis funqciur heterogenulo-

baze miuTiTeben. maT mier sakmaod sarwmunod iqna naCvenebi, rom cito-

qrom b5-s SeuZlia aRdges rogorc NADH-iT, aseve NADPH-iT. NADH-

is gamoyenebisas mixaelis-menTenis tipiuri mrudi isaxeba, xolo anae-

robul pirobebSi mikrosomuli suspenziis NADPH-iT aRdgenisas adgi-

li aqvs citoqrom b5-is aRdgenis xarisxis Senelebul zrdas da es

procesi piridinnukleotidis koncentraciazea damokidebuli. NADPH-is

eleqtronebiT citoqrom b5-is aRdgena NADPH-specifikuri jaWvidan

NADH-is Jangvis jaWvze reducirebuli eqvivalentebis gadasvlas anu

mikrosomebis mahidroqsilirebel kompleqsSi citoqrom b5-is monawi-

leobas mowmobs [124, 125].

NADPH-is koncentraciis gazrdiT (10-dan 100 µM-mde) citoqrom

b5-is aRdgenilobis xarisxi izrdeba da es SeiZleba aixsnas misi CarT-

viT mZlavri autooqsidabeluri terminaluri aqceptoris mqone eleq-

tronTa satransporto jaWvSi. NADPH-specifikur jaWvSi aseT aqcep-

torad citoqrom P450 funqcionirebs. garda amisa, sainkubacio narevSi hidroqsilirebis substratis-dimeTilanilinis Seyvanisas citoqrom b5-

is aRdgenilobis xarisxi klebulobs, rac hidroqsilirebis reaqciaSi

eleqtronul gadamtanad citoqrom b5-is uSualo monawileobis pirda-

piri maCvenebelia.

NADPH-is Jangvis jaWvSi citoqrom b5-is erT-erTi qvefraqciis

funqcionirebis mniSvnelovan dadasturebad SeiZleba CaiTvalos im eqs-

perimentebis Sedegebi, romlebSic NADH-isa da NADPH-is Tanamyofo-

bis pirobebSi izomeboda citoqrom b5-is Jangva-aRdgenis siCqareebi da

igi edreboda am substratTa Tanamyofobisas citoqrom b5-is staciona-

ruli mdgomareobis doneebs. amis safuZvelze diferencirebul iqna

principulad ori gansxvavebuli sqema [126]:

36

NADPH

NADH

O2

NADPH

NADH

O2

O2

FP1

FP2

FP1

FP2

citoqrom P450

citoqrom P450

citoqrom b5

citoqrom b5

citoqrom b5

a)

b)

sqema 1.1. (ganmarteba teqstSi).

pirveli sqema (a), romelsac mkvlevarTa umetesoba iziarebs, cito-

qrom b5-is mxolod erTi fraqciis arsebobas iTvaliswinebs. igi Car-

Tulia NADH-is Jangvis jaWvSi, magram NADPH-specifikuri flavo-

proteiniTac SeuZlia aRdgena. sxva SesaZleblobaa naCvenebi (b) sqemaze,

romelic sakmaod originaluria da citoqrom b5-is ori qvefraqciis

arsebobas iTvaliswinebs. Tu gaizomeba citoqrom b5-is Jangva-aRdgeniTi

reaqciis sawyisi siCqareebi, SeiZleba gamoiTvalos aerobul pirobebSi

misi aRmdgenelobis done da eqsperimenturad dadgenils Sedardes. am

sidideTa damTxveva eleqtronTa gadatanis ganxiluli sqemis sisworeze,

xolo sawinaaRmdego SemTxvevaSi mis araadeqvaturobaze miuTiTebs [121].

mikrosomul membranebSi NADH-isa da NADPH-is Tanmyofobisas cito-

qrom b5-is aRmdgenelobiTi doneebis eqsperimentulad miRebuli da

Teoriulad gamoTvlili sidideebis monacemebi ase gamoiyureba:

ADH ADPH

eqsperimentuli sidideebi 70 57

Teoriulad gamoTvlili sidideebi

sqema a 100 99

sqema b 74 60

kargad Cans, rom aseT damTxvevas mxolod (b) sqemis SemTxvevaSi

aqvs adgili.

NADPH-is Jangvis reaqciebSi citoqrom b5-is monawileoba SesaZle-

beli gaxda gamovleniliyo cdebiT, romlebSic Seiswavleboda NaCl-is

37

Tanamyofobisas NADPH-citoqrom b5- da NADPH-citoqrom P450-reduqtazul aqtivobaTa stimulireba. aRmoCnda, rom sainkubacio areSi

NaCl-is SetaniT izrdeba NADPH-iT da ara NADH-iT citoqromebis _

b5-isa da P450-is aRdgenis reaqciaTa siCqareebi [119, 112]. aRniSnulidan gamomdinare, gamoiTqva mosazreba endoplazmur re-

tikulumSi citoqrom b5-is ori qvefraqciis arsebobis Sesaxeb, ro-

melTa Jangva-aRdgena gansxvavebuli gzebiT xorcieldeba. amasTan, erTi

qvefraqcia aRdgeba NADPH-iT da iJangeba citoqrom P450-iT, xolo

meore NADH-iT ganicdis aRdgenas da SemdgomSi SeiZleba sxva gziT

daiJangos.

endoplazmur membranebSi ori eleqtrongadamtani kompleqsuri sis-

temis arseboba, romelTagan TiToeuli Tavis citoqrom b5-s Seicavs,

gamokvleul iqna triton X-100-iT damuSavebul mikrosomuli membrane-

bis preparatebSi. varaudobdnen, rom detergenti daarRvevda ra fermen-

tul kompleqsTa mkacr sivrcobriv orientacias, mospobda citoqrome-

bis kompartmentalizacias da imavdroulad gaaqrobda gansxvavebas hemo-

proteinebis Jangvis gzebs Soris. naCvenebi iqna, rom detergentis dax-

marebiT SesaZlebelia citoqrom b5-is or fraqcias Soris gansxvavebis

nivelireba da miiReba citoqromebis erTiani fondi, romelic Tanabari

doniT aRdgeba rogorc NADH-iT, aseve NADPH-iT. amisaTvis aucile-

beli aRmoCnda, rom piridinnukleotidebis koncentraciebi TiTqmis er-

Ti rigiT gansxvavdebodnen erTmaneTisagan. aerobul pirobebSi NADPH-

is sxvadasxva koncentraciebiT citoqrom b5-is aRdgenis mrudi proce-

sis orstadiurobaze miuTiTebs (nax. 1.3): pirvelisaTvis damaxasiaTebe-

lia aerobul (a) da anaerobul (b) pirobebSi citoqrom b5-is aRdgenis

donis damokidebuleba NADPH-isa (1) da NADH-is (2) koncentraciebze

[126].

NADPH-is mimarT aRmdgeneli sistemis maRali swrafva (KM=0.3 µM

NADPH), da gajereba, NADPH-is maRali koncentraciebis (100 mkm)

gamoyenebisasac ki ar miiRweva.

anaerobul pirobebSi citoqrom b5-is Senelebuli aRdgenis meore

faza qreba da mxolod am hemoproteinis swrafi gajerebis faza (KM ∼2.5 µM) rCeba. es garemoeba SeiZleba NADPH-specifikur jaWvSi ci-

toqrom P450-is _ mZlavri eleqtronuli aqceptoris arsebobiT

38

Na2S2O4Na2S2O4

_

_

_

0.15

0.10

0.05

a b 1

2

1 2 3 4 12 36 60 84 108 4 12 20 28 NADPHNADH0.4 1.2 2.0 2.8 µΜµΜ

∆A408_484 ∆A408_484

2

1

_

_

_

0.15

0.10

0.05

nax. 1.3. aerobul (a) da anaerobul (b) pirobebSi citoqrom b5-is aRd-

genilobis donis damokidebuleba NADPH-isa (1) da NADH-is

(2) koncentraciebze [126].

aixsnas. swored citoqrom b5-is swrafi daJangvis reaqcia iwvevs imas,

rom stacionaruli mdgomareobis miRwevis Semdeg misi aRmdgenelobis

done NADPH-is koncentraciis gazrdisas umniSvnelod matulobs.

NADH-is Tanamyofobisas citoqrom b5-is maqsimaluri aRdgenilobis

done erTnairad advilad miiRweva rogorc aerobul, aseve anaerobul

pirobebSi (KM=0.4 µM). substratad NADH-is gamoyenebisas misi aRd-

genis Senelebuli fazis ararseboba saSualebas iZleva misaRebad CaiT-

valos, rom RviZlis endoplazmuri retikulumis membranebSi funqcio-

nireben citoqrom b5-is gansxvavebuli qvefraqciebi, romelTaganac erTi

_ swrafad Jangvadi _ uSualodaa CarTuli NADPH-specifikur redoqs-

jaWvSi da aq terminalur eleqtronul aqceptors citoqromi b5 war-

moadgens, xolo meore _ Senelebulad Jangvadi _ CarTulia NADH-

specifikur eleqtronis satransporto sistemaSi. am or redoqs-

sistemas Soris eleqtronebis cvla unda xorcieldebodes [126]. anae-

39

robul pirobebSi wyalbadis zeJangis damatebisas NADH-iT aRdgenili

citoqrom b5-is araumetes 20%-is daJangva xdeba, maSin roca NADPH-

iT aRdgenili citoqromi srulad iJangeba. es Sedegi imaze metyvelebs,

rom NADH-is Jangvis jaWvSi CarTul citoqrom b5-s sakuTari termi-

naluri autooqsidabeluri eleqtronis aqceptori ar gaaCnia.

anaerobul pirobebSi NADPH-iT aRdgenili citoqrom b5-is nawi-

lobrivi daJangva SeiZleba aixsnas citoqrom b5-is im qvefraqciis da-

JangviT, romelic NADPH-is Jangvis jaWvSia CarTuli da romelic

anaerobiozisas NADH-iT aRdgenas ganicdis. aerobul pirobebSi cito-

qrom b5-is es qvefraqcia NADH-iT ar aRdgeba, radgan NADH-is Jang-

vis jaWvidan NADPH-is Jangvis jaWvze eleqtronebis gadatana Zlier

umniSvneloa.

kidev erTi Znelad asaxsneli faqtis Sesaxeb: absoluturad gauge-

baria Tu +20 mV redoqs-potencialis mqone citoqrom b5-idan Tu ro-

gor transportirdebian aRmdgeneli eqvivalentebi citoqrom P450-ze, romlis redoqs-potenciali -200-idan -300 mV-mde Seadgens da Tan

NADPH-redoqs-jaWvSi es orive gadamtani TiTqmis srulad daJangul

mdgomareobaSi imyofeba. amasTan dakavSirebiT arsebobs metad sayuradRe-

bo Tvalsazrisi imis Sesaxeb, rom sunTqviTi jaWvis am ubanze eleq-

tronTa gadatana eleqtrul velSi xorcieldeba, romelic citoqrom

P450-sa (“pliusi” hemis rkinaze) da Jangbads (“minusi”) Soris war-

moiqmna. es ukanaskneli rkinisagan mniSvnelovan manZilze imyofeba. mem-

branis ganivad ase ganwyobil eleqtrul velSi eleqtronebi SeiZleba

efeqturad gadaadgildnen citoqrom b5-idan citoqrom P450-ze [126]. citoqrom b5-is meoradi da mesameuli struqtura sadReisod deta-

luradaa Seswavlili. cnobilia, rom molekulaSi hemis rkina dakavSi-

rebulia apofermentis histidinis or imidazolTan. apofermentis nuk-

leofiluri dajgufebebi da lizinis ε-aminojgufebi apofermentTan he-mis mierTebaSi ar monawileoben [104, 105, 112, 127]. apofermentTan hemis

rkinis liganduri urTierTqmedebiT citoqromi b5 Zlier waagavs he-

moglobinis molekulas, magram am cilaTa funqciebi mniSvnelovnad

gansxvavdebian erTmaneTisagan [127]. citoqromis molekula elifsoiduri

formisaa 25×25×32 nm zomebiT. mTeli hemi molekulis hidrofobuli

ubnis SigniTaa ganlagebuli da mxolod propionis mJavas naSTi gamodis

40

apofermentis zedapirze [128, 129].

citoqrom b5-is sxva maxasiaTeblebidan saWiroa aRiniSnos misi mag-

nituri Tvisebebi. mikrosomul preparatSi hemoproteinis epr-signali

Zalian sustia da amitom misi magnituri Tvisebebis kvleva mxolod

izolirebul fermentzea SesaZlebeli. am dros Semdegi signalebi vlin-

debian: dabalspinuri (g-faqtoriT 3.03, 2.23 da 1.43) da maRalspinuri

komponenti (g-faqtori 6.2). tute areSi fermentis denaturaciisas ma-Ralspinuri signalis amplituda mkveTrad izrdeba [130, 131].

citoqrom b5-is WeSmariti funqcia jerac idumalebiT moculi rCe-

ba. am mimarTulebiT kvlevas is aferxebs, rom gamovlenili araa moce-

muli hemoproteinis Semcveli NADH-is mJangveli sistemis saboloo

eleqtronuli aqceptori. amasTan dakavSirebiT ramdenime Tvalsazrisi

arsebobs. maTgan upirveles yovlisa unda aRiniSnos varaudi membranaSi

CaSenebis Semdeg am hemoproteinis mier TviTdaJangvis SeZenis unaris

Sesaxeb. es Tvalsazrisi eqsperimentulad ar dasabuTebula. amasTan

dadgenil iqna, rom izolirebul da membranaSi CaSenebul citoqroms

erTnairi redoqs-potenciali gaaCnia da amdenad nakleb mosalodnelia

membranaSi CaSenebisas hemoproteinis fizikuri Tvisebebis aseTi mkveTri

cvlilebebi, ramac misi TviTdaJangva unda daaCqaros.

sadavo rCeba meore alternativac imis Sesaxeb, rom mikrosomebSi

arsebobs gansakuTrebuli cianidmgrZnobiare faqtori, romelic NADH-

is Jangvis jaWvis terminalur aqceptors SeiZleba warmoadgendes da

najer cximovan mJavaTa denaturaciis reaqciebSi monawileobs [132-134].

aRmoCnda, rom flavoproteinisa da citoqrom b5-is garda, am reaqciebSi

monawileobs komponenti, romelic NaCN-isadmi Tavisi swrafviT Zlier

waagavs citoqromoqsidazisadmi NaN3-is swrafvas. citoqromoqsidazisa-

gan gansxvavebiT, cianidmgrZnobiare faqtori ar inhibirdeba Na2S-iT. swored am faqtoris xorklian membranebSi swrafi sinTezi da Semdgom

misi gadasvla gluv membranebSi pasuxismgebeli aRmoCnda naxSirwylebiT

mdidar dietaze myofi virTxebis RviZlis endoplazmur membranebSi de-

saturazuli aqtivobis induqciaze [134].

cianidmgrZnobiare faqtori izolirebulia maRali sisufTavis mdgo-

mareobaSi. es preparati ar Seicavs arc flavo- da hemoproteins. ci-

la : fosfolipidis Tanafardoba masSi 1 : 1-zea. am monacemebis safuZ-

41

velze aRniSnuli faqtori unda ganixilebodes ara rogorc eleqtrone-

bis gadamtani, aramed lipoproteini, romelic aucilebelia desatura-

ciis reaqciisaTvis.

NADH-is Jangvis jaWvis inhibitorulma analizma aCvena, rom

NADH-is oqsidazur reaqciaSi monawileobs orvalentian metalTa io-

nebi. es reaqcia inhibirdeba EDTA-iT da aqtiurdeba Ca2+-iT. safiqrebe-

lia, rom Ca2+-is gamaaqtivebeli moqmedeba gamowveuli iyos NADH-isaT-

vis endoplazmuri membranis ganvladobis gazrdiT. NADH-is Jangva ara-

mgrZnobiarea mitoqondriuli Sxamebis _ rotenonisa da antimicin A-s

mimarT. igi mdgradia cianidis, azidis da sulfhidriluri Sxamebis mi-

marTac. mikrosomebSi NADH-is Jangvis siCqareze damTrgunvelad moqme-

deben citoqrom P450 hidroqsilazas substratebi da inhibitorebi

[126]. arsebuli monacemebi saSualebas iZleva NADH-is Jangva CavTvaloT

mikrosomuli Jangvis gansakuTrebul saxed, romelic aramgrZnobiarea

tipiuri mitoqondriuli inhibitorebis mimarT. amave dros, ueWvelia am

reaqciebSi cianidmgrZnobiare eleqtronTa terminaluri aqceptoris mo-

nawileoba. savsebiT SesaZlebelia, rom citoqrom P450 garkveul rols

asrulebdes NADH-oqsidazur reaqciebSi. yvela nivTiereba, romelic am

hemoproteinTan urTierTqmedebs, NADH-is Jangvis inhibirebas iwvevs.

daTrgunvis meqanizmi kvlavac ucnobia.

1.3.4 citoqrom P450 _ mikrosomebis eleqtronTa satransporto

sistemis terminaluri komponenti

mikrosomebis suspenziaSi aRmdgeni agentis damatebis da naxSirbadis

monooqsidis gatarebis Sedegad warmoqmnil produqts speqtris 450 nm-

ze STanTqmis maqsimumi axasiaTebs. hemSemcveli proteinebis warmomad-

genlebs, romelTac zolebi STanTqmis speqtris am olqSi (sores olq-

Si) gaaCniaT, citoqrom P450-ebs akuTvneben [6]. eleqtronTa satransporto sistemebSi citoqrom P450-is sxvadasxva

formebi terminaluri aqceptoris rols asruleben. es sistemebi Ziri-

Tadad endoplazmur da mitoqondriul membranebSi lokalizdebian. iseve

42

rogorc yvela sxva citoqromi, P450-ic rkina-porfirinis prostetul

jgufs Seicavs. igi tetrapiroluri naerTis _ porfirinis sawyisi war-

moebulia. misi molekulebi oTxi meTilis, ori vinilis jgufebisa da

propionmJavas naSTisagan Sedgeba. ramdenadac protoporfirinebi sami

gansxvavebuli tipis Camnacvlelebs Seicaven, maT gverdiTi jaWvis rva

SesaZlo mdgomareobaSi Canacvlebulebis ganlagebis rigis mixedviT

TxuTmeti izomeruli formiT arseboba SeuZliaT. am izomerebidan mniSv-

nelovania protoporfirini IX (ix. 1.5), romelsac rogorc citoqrom

b5, citoqrom P450-ic Seicavs. igi rkinis, magniumis, TuTiis, kobaltisa

da spilenZis ionebTan “oTxbirTvian” kompleqsebs warmoqmnis. hemSi

porfirinis oTxi liganduri jgufi rkinasTan kompleqss iZleva, ro-

melsac brtyeli aRnagoba aqvs, xolo rkinis mexuTe da meeqvse koordi-

naciuli kavSirebi porfirinis birTvis sibrtyis perpendikularulia.

yvela cnobili citoqromis hemis rkina dakavSirebulia porfirinis

birTvis pirolis azotis oTx atomTan. mexuTe aqsialuri (perpendiku-

larul mdgomareobaSi arsebuli) ligandi masTan cisteinis naSTis mer-

kaptiluri jgufiTaa dakavSirebuli, xolo meeqvse SeiZleba sxva li-

gandiT (mag., JangbadiT, naxSirbadis monooqsidiT, cianidiT da a.S.) iyos

Canacvlebuli. bunebrivia, rom hemoproteinis koordinaciuli kompleq-

sis ZiriTadi fizikuri maxasiaTeblebi damokidebuli unda iyos mexuTe

da meeqvse ligandebze, romlebic hemis prostetul jgufs ukavSirde-

bian [96].

modeluri sistemebi, romlebSic mexuTe ligands gogirdis atomi,

xolo meeqvses imidazolis azoti warmoadgenen, yvelaze optimalurad

imeoreben hemoproteinis speqtrul Tvisebebs. swored es ligandebi in-

acvlebian sulfhidriluri SxamebiTa da citoqrom P450-is specifikuri inhibitoriT _ meTiraponiT. diTionitiT aRdgenisas citoqrom P450-s eleqtronis miReba SeuZlia, rac imaze miuTiTebs, rom hemoproteini,

hemuri rkinis garda, eleqtronis kidev erT aqceptors unda Seicavdes

[96, 126].

xangrZlivi periodis ganmavlobaSi ar xerxdeboda membranidan cito-

qrom P450-is natiuri formiT solubilizacia. amis mizezs misi uaRre-

sad maRali labiluroba warmoadgens, ris gamoc igi advilad gardaiqm-

neba (konversirdeba) araaqtiur citoqrom P420-ad. am ukanasknels b-

43

tipis citoqromebisaTvis damaxasiaTebeli speqtri gaaCnia. unda aRiniS-

nos, rom P450-is konversia marto solubilizaciisas ar SeiniSneba. mas

adgili aqvs membranaze neitralur marilTa maRali koncentraciebiT,

trifsiniT, lizolecitiniT, p-qlormerkuribenzoatiT, organuli gamxs-

nelebiTa da sxva naerTebiT zemoqmedebisas. arc erT CamoTvlil SemTx-

vevaSi membranidan fermentis solubilizacia ar xdeba. amis gamo kvle-

vaTa umravlesoba ZiriTadad suspenziur preparatebze tardeboda [135-

137]. 1974 wels iaponeli mkvlevarebis imaisa da satos mier fenobar-

bitaliT inducirebuli kurdRlis RviZlis mikrosomebidan gamoyofil

iqna citoqrom P450 homogenur mdgomareobaSi (hemoproteinis Semcvelo-

ba 17 nmoli/mg cilaze). am mizniT maT gamoiyenes afinuri qromatogra-

fia ω-aminooqtilsefarozaze, radgan am droisaTvis ukve cnobili iyo,

rom n-oqtilamini TviTon warmoadgens gansakuTrebiT Zlier ligands

daJanguli formis citoqrom P450-isaTvis [140]. amJamad sayovelTaodaa

miRebuli, rom sefarozas rogorc alkiluri, aseve ionuri jgufebi

erTnairad mniSvnelovania mikrosomul fermentTa gamoyofis procesSi

[141]. TiTqmis imavdroulad, 1974 wels kunma da misma TanamS. [142]

warmoadgines homogenuri citoqrom P450-is miRebis meTodi, romelSic

fraqcionirebisaTvis polieTilenglikoli iqna gamoyenebuli. aqac Ca-

tarda fenobarbitaliT winaswari induqcia, oRond kurdRlis RviZlis

mikrosomebi damuSavda pirofosfatiT, solubilizacia moxda natriumis

qolatiT, xolo fraqcionireba _ polieTilenglikol 6000-iT. fer-

mentis Semdgomi gasufTaveba ganxorcielda qromatografiiT ramdenime

safexurad: DEAE-celulozaze, hidroqsiapatitze da kalciumis fos-

fatian gelze. damamTavrebel stadiaze gamoyenebul iqna araionuri de-

tergenti reneqs-690. am gziT aRniSnulma mkvlevarebma miiRes cito-

qrom P450-is formebi, romlebic erTmaneTisgan gansxvavdebodnen eleq-

troforezuli ZvradobiT da aRdgenili karboniluri (CO-dakavSi-rebuli) kompleqsebis speqtrebiT. eleqtroforezuli Zvradobis Semci-

rebis rigisa da TiToeuli suberTeulis molekuluri masis zrdis mix-

edviT es formebi pirobiTad Caiwera, rogorc LM1, LM2 da a.S. fenobar-

bitaliT inducirebuli citoqrom P450 aRiniSna, rogorc P450LM2

[143]. fermenti 90%-iani sisufTavisaa da eleqtroforezulad homoge-

nuria. igi 49 kD molekuluri masis mqone polipeptids Seicavs da

44

409 aminomJavuri naSTisagan Sedgeba. hemoproteinis polimerul formas

gaaCnia 280 kD molekuluri masa da, rogorc Cans, 6 suberTeulisagan

Sedgeba. yovel polipeptidze 6 cisteini da ∼50%-mde arapolaruli

aminomJava modis. am cilis tipiuri absoluturi STanTqmis speqtrebi

mocemulia suraTze (nax. 1.4) [144].

.

.

..

.

.

.

.

.

.

.

.

.

.

.

.

.

.

0.08

0.07

0.06

0.05

0.04

0.03

0.02

0.01

λ, nm

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

1

2

3

350 400 450 500 550 600 650

3

1

2

nax. 1.4. RviZlis mikrosomebis citoqrom P450-is STanTqmis speqtrebi.

1. daJanguli forma;

2. aRdgenili forma;

3. aRdgenili da CO-sTan kompleqsirebuli forma.

citoqrom P450-is mier katalizuri aqtis gansaxorcieleblad

NAD(P)H-is aRmdgeneli eqvivalentebiT misi aRdgenis siCqares ZiriTa-

dad aqtiur centrSi myofi rkinis ionis spinuri mdgomareoba gan-

sazRvravs. am ions 5 sxvadasxva sivrculi orientaciis mqone 3d orbi-talze 6 eleqtroni aqvs (nax. 1.5). 3dxy orbitali urTierTsapirispiro

spinis mqone or eleqtrons Seicavs, xolo danarCeni orbitalebi erT-

nairi spinis mqone TiTo eleqtroniTaa dakavebuli. rodesac oqtaedrul

struqturaSi Fe2+ eqvsi ligandiTaa garemoculi, maSin ligandebis

45

+ 6H2O

3dxy 3dyz 3dxz

+ 6H2O

t2g t2geg eg

3dxy 3dyz 3dxz3dxy 3dyz 3dxz

[Fe(H2O)6]2+

H2O

H2

H2O H2O

H2O

Fe2+

O

H2O

NC

C

CN NC

NC

Fe2+

N

NC

_

_

_

_

_

_

[Fe(CN)6]4+

Fe2+

3dz2

3dz2 3dx2_y2

maRalspinuri Fe2+ dabalspinuri Fe2+

3dx2_y23dz2

3dx2_y2

nax. 1.5. rkinis (II) ionis spinuri mdgomareobebi (ganmarteba ix.

teqstSi).

oqtaedruli velis gavleniT 3d orbitali gansxvavebuli energiis mqone

eg da t2g qvedoneebad ixliCeba; amasTan, eleqtronebis aRniSnul qvedo-

neebze gadanawileba damokidebulia oqtaedruli velis siZliereze.

SesaZlebelia ori zRvruli SemTxveva:

1. susti oqtaedruli velis SemTxvevaSi, romelsac, mag., [Fe(H2O)6]2+-

kompleqsSi Fe2+-is garSemo ganlagebuli wylis molekulebi qmnian,

maRali energiis eg-qvedoneze ganlagdebian 3dz2 da 3dx2-y2 orbitale-

bis eleqtronebi, romlebic ligandebTan σ-bmebs warmoqmnian; 3dxy

3dyz da 3dxz orbitalebidan dabali energiis t2g qvedoneze gadasuli

eleqtronebi ki π bmebis warmoqmnaSi iReben monawileobas; amasTan

orive qvedoneze eleqtronebi ise arian orientirebuli, rom maTi

spinuri qvanturi ricxvebis jami maqsimaluria da +2-s aRwevs. ase-

Ti garemocvisas Fe2+ maRalspinur mdgomareobaSi imyofeba.

2. Zlieri oqtaedruli velis SemTxvevaSi mag., [Fe(CN)6]4--kompleqsSi

eqvsive eleqtroni dabali energiis t2g-qvedonezea gadasuli. isini

urTierTsapirispiro mimarTulebiT Tavsdebian. Sesabamisad am eleq-

tronebis spinuri qvanturi ricxvebis jami nulis toli iqneba. ami-

tom am kompleqsSi Fe2+ dabalspinur mdgomareobaSia.

zogierTi hemoproteinisaTvis dadgenilia maRal- (S=5/2) da dabal-

46

(S=1/2) spinur formebs Soris wonasworobis arseboba, anu sxvadasxva obieqtebidan miRebul mikrosomebSi citoqrom P450 faqtiurad orive spinuri formis narevis saxiT aris warmodgenili [145, 146]. es mdgoma-

reobebi erTmaneTisagan energetikulad didad ar gansxvavdeba da gare

faqtorTa gavleniT SesaZlebelia maTi urTierTgadasvla. ligandebis

velis Teoriis Tanaxmad gansazRvruli ligandebis zemoqmedebas e.w. spi-

nebis Sewyvilebis energia Seesabameba.

rkinis ionTa daJangulobis xarisxis Seqcevadi cvlileba FeFe citoqrom P450-s saSualebas aZlevs Tavisi WeSmariti, eleq-

tronTa gadatanis funqcia Seasrulos. substratis mierTeba hemis rki-

nis eleqtronuli Rrublis SeSfoTebas iwvevs. amis gamo misi redoqs-

potenciali ufro dadebiTi xdeba, vidre substratis gareSea. es gare-

moeba SesaZlebels xdis, rom es hemoproteini aRdges eleqtronebiT,

romelic mas NADPH-dan flavoproteinis gavliT, NADPH-citoqrom

P450-reduqtazas saSualebiT gadaecema. hidroqsilirebis aqtis gansax-

orcieleblad ori eleqtronia saWiro da maT citoqromi erTmaneTisa-

gan damoukideblad Rebulobs. hemis rkina daJangulobis +2-xarisxSi

unda imyofebodes, raTa man Jangbadis mierTeba SeZlos. amitom pirveli

eleqtroni hemTan Jangbadis mierTebas, xolo meore _ Jangbadis gaaqti-

vebas xmardeba [135].

sxvadasxva warmoSobis citoqrom P450-ebi speqtruli TvisebebiT

identurebia. oTaxis temperaturaze da pH 7.0-ze am hemoproteinis da-

Jangul da aRdgenil formebs sores zolebi Sesabamisad 415 da 412 nm-

ebze gaaCniaT. speqtrSi 424 nm-ze mxaric registrirdeba. sxva cito-

qromebisagan gansxvavebiT, daJanguli citoqrom P450 ramdenadme ati-

piur absolutur STanTqmis speqtrs iZleva, rac imaSi mdgomareobs,

rom STanTqmis Cveulebrivi zolebis garda, rac rkinis dabalspinuri

mdgomareobisTvisaa damaxasiaTebeli, 650 nm-is olqSi Cndeba axali

farTo zoli, es ki imis maCvenebelia, rom CO-s gareSe aRdgenisas igi

maRalspinur mdgomareobaSi imyofeba. garda amisa, TvalSi sacemia aRdge-

nili citoqrom P450-is STanTqmis speqtrSi sores olqis anomaliuri

ganlageba 448 nm-ze CO-sTan kompleqsirebisas da 450 nm-ze hemopro-

teinis formaze damokidebulebiT. amis sapirispirod sxva hemoproteine-

bi CO-kompleqsTan STanTqmis maqsimums 420 nm-ze amJRavneben. aRdgenili

47

citoqrom P450-is karboniluri kompleqsis eqstinqciis koeficienti

91 000 moli-1.sm

-1-is tolia da am sidides fermentis koncentraciis

gansasazRvrad iyeneben [7].

citoqrom P450 naxSirbadis monooqsids mxolod aRdgenili formiT

ikavSirebs. amasTan Cndeba STanTqmis erTi maqsimumi 450 nm-ze. CO-sadmi

fermentis swrafva Zlier maRalia: KCO=1.3 µM-s da meore rigis reaq-

ciis siCqaris konstanta 25°C-ze da 2.4.105 moli.wm-1-is tolia. alki-

lizocianidebi erTnairad ukavSirdebian rogorc aRdgenil, aseve daJan-

gul citoqrom P450-s. amasTan, daJanguli hemoproteini STanTqmis

mxolod erT maqsimums warmoqmnis 434 nm-ze, daJanguli ki or maqsimums

amJRavnebs 430 da 455 nm-ebze. zolebis intensivobis Tanafardoba bu-

feris ionur Zalasa da garemos pH-zea damokidebuli [126].

solubilizebuli citoqrom P420 b-tipis hemoproteinebisaTvis da-maxasiaTebel Tvisebebs avlens. misi daJanguli forma sores zols 414

nm-ze flobs, xolo aRdgenili _ maqsimumebs 427-is, 530-isa da 560 nm-

is olqSi iZleva. fermentis redoqs-potenciali pH 7.0-ze -20

mV-is tolia. pH 9.5-ze misi monomeris molekuluri masa 150 kD-is

rigisaa [126].

apofermentTan prosTetuli jgufis specifikuri urTierTqmedeba

gansazRvravs citoqrom P450-is speqtrul, magnitur, qimiur da funq-

ciur maxasiaTeblebs. did magnitur velebSi mikrosomebis epr-

speqtrebi pirvelad meisonisa da misi TanamS. [147] mieraa daregistri-

rebuli da dadgenilia g-faqtorebis mniSvnelobebi. aRmoCnda, rom am

hemoproteins dabalspinuri formis damaxasiaTebel epr-speqtrs Seda-

rebiT viwro sami zoli aqvs da g-faqtori 1.91-is, 2.24-isa da 2.42-is tolia. maRalspinur formas g-faqtorTa 1.7-is, 3.7-isa da 8-is toli

mniSvnelobebi Seesabameba. citoqrom P450-isaTvis dasaxelebul sidide-

Ta dadgena safuZvels qmnis hemoproteinis JangbadTan, aRmdgenel agen-

tebTan, substratTan da inhibitorebTan urTierTqmedebis meqanizmis

asaxsnelad. gansxvavebuli spinuri mdgomareobis narevze zogierTi sub-

stratis damatebisas SeiniSneba hemoproteinis maRalspinur mdgomareo-

baSi srulad gadasvla. citoqrom P450-is solubilizacias da araaq-

tiur P420-ad konversias Tan axlavs maRalspinuri signalis aRZvra

6.0-is toli g-faqtoriT. amasTan, citoqrom P420-isaTvis damaxasiaTe-

48

beli 2.25 g-faqtoris signali SenarCunebulia. dadginda, rom citoqrom

P420 SeiZleba arsebobdes sam (β, γ da σ) mdgomareobaSi erTnairi

speqtrebiT da gansxvavebuli spinuri mdomareobebiT. citoqrom P450-is inaqtivacias Tan axlavs misi natiuri α-mdgomareobidan β-formebis gavliT maRalspinur γ-formaSi gadasvla. gamoirkva agreTve, rom arse-

bobs nivTierebebi (mag., eTilenglikoli, propilenglikoli, ribitoli,

glukoza, saqaroza, sorbitali, dimeTilsulfoqsidi da a.S.), romleb-

sac citoqrom P420-is citoqrom P450-ad rekonversia SeuZliaT.

citoqrom P450-is epr-speqtris Taviseburebas warmoadgens hemuri

rkinis simetriis Zlieri darRveva (anizotropia), romelic ligandebis

velis Teoriis CarCoebSi SeiZleba im parametrebiT daxasiaTdes, rom-

lebic rombisebur da aqsialur gadaxrebs asaxaven [148]. msgavsi sime-

triis darRveva sxva hemoproteinebis mimarT ar vlindeba. adreul ga-

mokvlevebSi anizotropuli dabalspinuri epr-signalis gaCenas mikro-

somebSi Fex komponentis arsebobas ukavSireben [149]. SemdgomSi aRmoCn-

da, rom es signali citoqrom P450-s ganekuTvneba. amitom Fex-

komponentis Semcvelobis gansazRvra orive hemoproteinis jamur raode-

nobas ufro asaxavs [150-153].

ar SeiZleba ar aRiniSnos epr-meTodis garkveuli SezRuduloba,

rac SemdegSi mdgomareobs: spinuri wonasworoba Seiswavleba sxvadasxva

temperaturaze sistemis magnituri Tvisebebis dadgeniT. oTaxis tempe-

raturaze am parametris gansasazRvravad aRniSnuli meTodi gamousade-

garia. hemuri kompleqsebis eleqtronuli relaqsaciis Zalian mcire

drois gamo oTaxis temperaturaze epr-is signali saerTod ar Cndeba.

am kompleqsebis spinuri mdgomareobebis kvleva SesaZlebelia upirate-

sad optikuri meTodebis gamoyenebiT, romlebic SesaniSnavad asaxaven

spinur mdgomareobas. methemoglobinis warmoebulTaTvis naCvenebia, rom

magnituri amTvisebloba xazovnadaa dakavSirebuli sores zolebis mde-

bareobasTan. magnituri amTviseblobis zrdisas sores zoli SedarebiT

mokle talRiani olqis mxares inacvlebs da piriqiT, am Tvisebis Sem-

cirebisas sores zoli grZeltalRovani ubniskenaa wanacvlebuli [154].

gamoricxuli araa, rom aseTi korelacia samarTliani iyos sxva hemo-

proteinebis, da kerZod, citoqrom P450-isTvisac [146]. sores zolebis

wanacvlebebi imis gamo xdeba, rom daJangul formaSi mocemuli hemo-

49

proteini erTdroulad ori spinuri mdgomareobiT arsebobs da Ti-

Toeuli maTgani sores zolis gansxvavebuli mdebareobiT xasiaTdeba:

maRalspinur mdgomareobas mokle talRebis mxares, xolo dabalspinurs

maqsimumi ufro grZeli talRebis mxares gaaCnia [155]. gansazRvrul

spinur formasTan dakavSirebuli sores zolis zusti mdebareoba TviT

hemoproteinzecaa damokidebuli, kerZod, spinuri mdgomareoba mniSvne-

lovanwilad hemuri rkinis aqsialuri ligandebiTaa ganpirobebuli. spi-

nuri wonasworobis arsebobisas sores zolis mdebareoba ori spinuri

formis SedarebiTi wiliT ganisazRvreba. sxvadasxva temperaturaze di-

ferenciuli speqtrebis registrirebisas dabal- da maRalspinuri for-

mebis sores zolebi SeiZleba erTmaneTisagan iqnes dayofili [156].

sxvadasxva temperaturaze STanTqmis zolebis intensivobiT adgenen spi-

nuri wonasworobis konstantebs da hemoproteinis gansazRvruli spinu-

ri formis Semcvelobas, Tu, ra Tqma unda, am formis eqstinqciis koe-

ficienti cnobilia.

citoqrom P450-is unikalur Tvisebas warmoadgens is, rom igi Tavis

substratebTan da inhibitorebTan speqtrofotometrulad advilad re-

gistrirebad kompleqsebs warmoqmnis. mikrosomul membranebSi aseT kom-

pleqsTa SesaZlo warmoqmnis gamosavlenad gansazRvraven STanTqmis di-

ferenciul speqtrebs da erTmaneTs udareben kompleqsisa da Tavisufa-

li hemoproteinis STanTqmis speqtrebs. citoqrom P450-is am kuTxiT kvlevis dawyeba dakavSirebulia narasimhulusa da misi TanamS. [157]

mier miRebul SedegebTan, romlebmac aCvenes, rom Tirkmelzeda

jirkvlis qerqis mikrosomul suspenziaze 17-oqsiprogesterolis dama-

tebisas adgili aqvs speqtrul cvlilebebs. SemdgomSi dadgenil iqna,

rom sruliad gansxvavebuli bunebis mqone naerTebi RviZlis mikroso-

mebze damatebisas STanTqmis diferencialur speqtrebSi damaxasiaTebel

cvlilebebs iwveven da es cvlilebebi dakavSirebulia citoqrom P450-Tan sxvadasxva nivTierebebis uSualo urTierTqmedebasTan [158-160]. amis

mixedviT yvela speqtruli cvlileba, romlebic citoqrom P450-Tan sxvadasxva nivTierebis dakavSirebisas registrirdeba, sami tipisaa: I, II da modificirebuli-II (nax. 1.6). Sesabamisad xdeba am cvlilebebis ga-

momwvevi substratebis klasifikaciac [158, 161].

50

0.04

0.03

0.02

0.01

0.00

0.01

0.02

0.03

0.04

0.05

_

_

_

_

_

360 390 420 450

ABS

III

III

nm

_

_

_

_

_

_

_

_

_

_

nax. 1.6. speqtrul cvlilebaTa tipebi mikrosomebis (1 mg/ml-ze cilis

Semcvelobis mixedviT) urTierTqmedebisas sxvadasxva substra-

tebTan. I tipis speqtruli cvlilebebi xasiaTdeba STanTqmis

maqsimumiT 390 nm-ze da minimumiT 420 nm-ze (mrudi I, sub-strati _ 8.4 nmoli heqsabarbitali; II tipis speqtruli

cvlilebebi xasiaTdebian maqsimumiT 420-430 nm-ebze da minimu-

miT 390 nm-ze (mrudi II, substrati _ 2.5 nmoli anilini);

modificirebuli II tipi warmoadgens “Sebrunebul I tips”, anu I tipis speqtruli cvlilebebis sarkisebur asaxvas (mrudi III, substrati _ 1.6 nmoli agroklavini).

I tipis substratebs, iseve rogorc aRdgenas, citoqrom P450-is

rkina dabalspinuridan maRalspinur mdgomareobaSi gadahyavT, xolo II tipis substratebi piriqiT, ukugadasvlas iwveven [162, 163]. es uCveu-

lo Tviseba sxva hemoproteinebisTvis damaxasiaTebeli araa [130, 163].

aseTi gadasvlis meqanizmis detalurma Seswavlam aCvena, rom igi unda

xorcieldebodes dabalspinuri citoqrom P450-is aRdgenis stadiis

gavliT, romelic Sualedi formaa Semdeg reaqciaSi:

51

Fe3+

Fe2+

Fe2+

dabalspinuri dabalspinuri maRalspinuri

I tipis speqtruli cvlilebebi mWidrodaa dakavSirebuli im nivTie-

rebaTa metabolizmTan, romlebic citoqrom P450-is induqcias iwveven da praqtikulad mikrosomebis mahidroqsilirebeli sistemis substra-

tebs warmoadgenen. maT miekuTvneba: najeri da aromatuli naxSirwyal-

badebi, mesameuli aminebi, eTerebi, alicikluri da heterocikluri

naerTebi da sxv. yvela es nivTiereba I tipis ligandTa klass Seadgens.

hilisa da misi TanamS. [164] mier postulirebulia, rom I tipis nivTierebaTa damatebisas daJanguli citoqrom P450-is maRalspinuri

mdgomareobis matebas Tan axlavs rkinis ionsa da ligands Soris kom-

formaciul-inducirebuli manZilis gazrda, romelic merkaptilur

jgufTan trans-mdgomareobaSi imyofeba. am dros rkina gamodis porfi-

rinis birTvis sibrtyidan gogirdis atomis mimarTulebiT. jefkoti da

misi TanamS. [165] citoqrom P450-is daJanguli formis speqtrebze I tipis nivTierebaTa gavlenas gansxvavebuli interpretaciiT warmoadge-

nen. maTi mosazrebiT, am nivTierebaTa damateba iwvevs konformaciul

cvlilebebs, rasac saboloo jamSi koordinaciuli kompleqsis cvlile-

basTan mivyavarT. mravalricxovani monacemebis analizis safuZvelze aR-

niSnulma mkvlevarebma gamoTqves varaudi, rom metalsa da gogirdis

atoms Soris manZili unda izrdebodes da ara mcirdebodes. rogorc

Cans, am ori ganxiluli hipoTezidan upiratesoba meores unda mieniWos:

aRdgenil citoqrom P450-Tan naxSirbadis monooqsidis mierTebisas CO-

s molekula koordinaciuli sferodan gamodevnis merkaptilur jgufs,

romelic mas daJanguli formis hemoproteinSi ekava. amis Sedegad mii-

Reba kompleqsi, romelSic metali gamosulia porfirinis birTvis

sibrtyidan CO-s mimarTulebiT, xolo trans-mdgomareoba an Tavisufa-

lia, an dakavebulia metalTan sustad bmuli ligandiT. fermentuli

sistemis funqcionirebisaTvis, rogorc Cans, aucilebelia, rom Jangba-

dis molekula hemTan amgvaradve iyos dakavSirebuli.

I tipis substratebis mierTebisas citoqrom P450-is redoqs-

potenciali -300 mV-dan -230 mV-mde izrdeba. amis Sedegad eleqtro-

nis satransporto JaWvSi misi aRdgena advildeba. Jangbadisadmi maRali

swrafva (KM=0.1_0.2 µM) da didi autooqsidabeloba (meore rigis

reaqciis siCqaris konstanta _ 2_4.106 moli.wm-1, naxevraddaJangvis pe-

52

riodi _ 300 moli.wm-1) citoqrom P450-is aerobulad aRdgenisas sta-

cionaruli mdgomareobis dabal dones ganapirobebs [166, 167]. mocemuli

parametrebiT igi citoqromoqsidazas waagavs [167], magram misgan

gansxvavebiT ufro maRali swrafva aqvs CO-s mimarT. imisaTvis, rom

citoqromoqsidazas aqtivoba 50%-iT daiTrgunos, saWiroa fardoba

CO2:O2=10, maSin rodesac citoqrom P450-isaTvis es koeficienti 1-is tolia. orive fermenti Cu2+

-s Seicavs [47, 168].

nivTierebebi, romlebic II tipis speqtrul cvlilebebs iwveven, ro-

gorc wesi, aminebs warmoadgenen da mcire gamonaklisis garda (anilini,

imidazoli da sxv.), ar miekuTvnebian mikrosomuli sistemis substra-

tebs. es citoqrom P450-Tan maTi urTierTobis xasiaTiTaa ganpirobebu-li. iTvleba, rom II tipis ligandebi (substratis aminojgufebi) uSua-

lod ukavSirdebian citoqromis hemur rkinas, rogorc tipiuri hemo-

qromogenebi da fermentis meeqvse aqsialuri ligandebi (ferihemoqrome-

bi) xdebian [158, 169]. diferenciul speqtrebSi es ∼420 nm-ze

STanTqmis maqsimumis da ∼390 nm-ze STanTqmis minimumis gaCenas iwvevs [170]. citoqrom P450-Tan II tipis ligandebis urTierTqmedebisas da-

balspinuri kompleqsebi warmoiqmnebian, ramdenadac hemuri rkina koor-

dinaciul sferoSi eqvs ligands Seicavs. II tipis aminebi konkurencias uweven CO-s, da SesaZloa, O2-sacc rkinis erTsa da imave centrTan da-

kavSirebisaTvis. mTel rig SemTxvevaSi (anilini, nikotinamidi) mikroso-

mul suspenziasTan urTierTqmedebisas SeiniSneba diferenciuli speq-

trebi ori maqsimumiT da ori minimumiT, rac SeiZleba am nivTierebaTa

gardaqmnaSi citoqrom P450-is sxvadasxva formis monawileobas asax-

avdes [159, 170]. amasTan dakavSirebiT SevniSnavT, rom sadReisod yve-

laze detaluradaa daxasiaTebuli RviZlis mikrosomebis citoqrom

P450-is ori forma: erTi maTgani (P450LM2), rogorc zeviT aRiniSna,

fenobarbitaliT, xolo meore _ 3-meTilqolantreniT an β-naftol-

flavoniT inducirdeba da aRiniSneba, rogorc P450LM4 [171, 172]. maTi

homogenuri formebis zogierTi maxasiaTebeli moyvanilia 1.1 cxrilSi

[173, 174]. cnobilia P450LM2-formis 30 rgolis N-kidura aminomJavuri

Tanmimdevroba, romlebic 20-dan 17 hidrofobul aminomJavur naSTs da

or klasterSi gaerTianebul xuT Tanmimdevrulad dakavSirebul lei-

cins Seicavs. varaudoben, rom P450LM2-is es fragmenti pasuxismgebelia

53

mikrosomul membranasTan dakavSirebaze, an sistemis funqcionirebisas

citoqrom P450-is kompleqsis Tavisi reduqtazisadmi da fosfolipide-

bisadmi orientaciaze [171].

cxrili 1.1

bocvris RviZlis mikrosomebis citoqrom P450-is homogenur formaTa zogierTi maxasiaTebeli [173, 174]

Tvisebebi LM2 LM4

fermentis molekuluri masa (kD) 48.7 55.3

polipeptidis molekuluri masa (kD) 47.6 54.2

Semcveloba:

P450 nmoli/mg cilaze 20 17

hemi nmoli/nmol cilaze 1 1

lipidebi nmoli/nmol cilaze 0.3 0.5-1.6

detergenti mg/mg cilaze 0.05 0.05

C-kidura aminomJava arginini lizini

N- kidura aminomJava meTionini _

naxSirwylebi nmoli/nmol cilaze:

glukozamini 0.7 0.8

manozebi 1.7 2.0

glukoza 0.2 0.9

Tavisi speqtruli da sxva maxasiaTeblebiT citoqrom P450LM4

Zlier gansxvavdeba P450LM2 formisagan [175, 176]. pirveli ar ikavSi-

rebs organul hidrozeJangebs da mikrosomuli sistemis iseT tipiur

substratebs, rogorebic benzoli, cikloheqsani da antipirinia. es imiT

aixsneba, rom citoqrom P450LM4 induqtorTan (3-meTilqolantrenTan)

an mis metabolitebTan kompleqsis saxiT arsebobs. aseTi kompleqsebi

maRalspinur mdgomareobaSi imyofebian da zRudaven hemoproteinTan Ca-

moTvlil nivTierebaTa dakavSirebas [175, 176]. zogierTi nivTiereba

(anilini, naftalini, dimeTilanilini) efeqturad ukavSirdeba citoqrom

P450LM4-s, magram praqtikulad am hemoproteiniT ar iJangeba. es faqti

54

imaze miuTiTebs, rom unda arsebobdes am citoqromis dabalspinuri

kompleqsis ori tipi, romlebSic ligandebis intensiuri dakavSireba

xdeba, magram isini Jangvis produqtiulobis TvalsazrisiT ar gamoirCe-

vian [175]. anilini, piridini, imidazoli da, SesaZloa, sxva azotSemc-

veli organuli fuZeebic dabalspinuri formis kompleqss warmoqmnian,

romelSic ukve SeuZlebelia molekuluri Jangbadis an organuli hi-

drozeJangebis mierTeba. P450LM2-sa da P450LM4-s Soris arsebiTi

gansxvaveba wriuli diqroizmis meTodiTac dasturdeba [177]. am meTo-

diT miRebuli Sedegebis safuZvelze gakeTebulia daskva, rom P450LM4-

is formis hemi ufro mWidro hidrofobuli “jibiT” aris garemoculi,

vidre P450LM2-is formis hemi.

iseve rogorc sxva citoqrom P450-ebi P450LM2 da P450LM4 advilad

ganicdian aRdgenas diTionitiT da warmoqmnian CO-sTan aRdgenili hemo-

proteinebis kompleqsebs STanTqmis maqsimumebiT Sesabamisad 450 da 448

nm-ebze. zogjer P450LM4-is identur citoqrom P450-is formas cito-qrom P448-s uwodeben [170].

arsebobs speqtruli cvlilebebis tipi STanTqmis maqsimumebiT 390

da 420 nm-ze. rogorc aRiniSna, faqtiurad igi I tipis speqtrul

cvlilebaTa sarkisebul gamosaxulebas warmoadgens da amitom mas

“modificirebuli II” tips akuTvneben (literaturaSi igi inversiuli an

reversiuli speqtris saxelwodebiTacaa cnobili).

aseTi cvlileba substratis hidroqsilis jgufisa da maRalspinuri

rkinis urTierTqmedebis Sedegia. am dros STanTqmis maqsimumis 394 nm-

idan 416 nm-mde gadanacvleba xdeba.

modificirebuli II tipis speqtruli cvlilebebis gamomwvevi niv-

Tierebebis jgufi Tavisi SemadgenlobiT sakmaod araerTgvarovania. masSi

Sedian spirtebi, ketonebi, alkaloidebi, sterolebi da sxv. [158, 176,

178, 179]. mcire gamonaklisis garda, isini mikrosomuli Jangvis siste-

mis substratebs warmoadgenen. citoqrom P450-is spirtebTan ur-

TierTqmedeba rTuli bunebisaa da, upirveles yovlisa, am nivTierebaTa

alifaturi jaWvis sigrZezea damokidebuli. mcire zomis molekulebis

mqone spirtebis (meTanolis, eTanolis, propanolis) hemur rkinasTan

koordinireba meeqvse aqsialur mdgomareobaSi xorcieldeba, xolo Tu

spirtis molekulas grZeli, an gantotvili jgufi gaaCnia, maSin mas I

55

tipis substratebis msgavsad citoqrom P450LM2-Tan hidrofobuli ur-

TierTqmedebiT dakavSireba SeuZlia. imis gamo, rom spirtebi ukve Seica-

ven polarul OH-jgufebs, principSi isini citoqrom P450-is sub-

stratebs ar unda warmoadgendnen. miuxedavad amisa, rogorc irkveva, am

klasis naerTebis JangviT metabolizmSi citoqrom P450 mainc monawi-leobs [179].

citoqrom P450-is jgufis fermentebze arsebul monacemebs Tu ga-

vaanalizebT, SeiZleba garkveuli daskvnebis gamotana: 1) am cilebSi he-

mi hidrofobul garemocvaSi imyofeba; 2) neitralur gamxsnelSi sub-

stratis aryofnisas citoqrom P450-is daJanguli formebi upiratesad

dabalspinur mdgomareobaSia; 3) aseT dabalspinur kompleqsSi cisteinis

merkaptiluri jgufi rkinasTanaa dakavSirebuli; 4) daJangul formaSi

darCenil koordinaciul mdgomareobas cilis sxva polaruli jgufi,

SesaZloa histidinis naSTis imidazoluri jgufi ikavebdes; 5) I tipis naerTTa Tanamyofobisas SeiniSneba konformaciulad inducirebuli ga-

dasvlebi, rasac daJanguli formis maRalspinur mdgomareobaSi gardaqm-

nasTan mivyavarT; 6) aRdgenili citoqrom P450 maRalspinur mdgomareo-

baSi arsebobs, maSin rodesac misi rogorc daJanguli, aseve aRdgenili

formebi dabalspinur kompleqsebs warmoqmnian [96].

1.3.4.1 mikrosomuli hidroqsilirebis calkeuli stadiebi

(monooqsigenazuri cikli)

ucxo naerTTa mikrosomuli Jangvis SeswavlaSi centralur pozi-cias dResac hidroqsilirebis rTuli procesis meqanizmis kvleva

warmoadgens. sadReisod araviTar eWvs ar iwvevs is faqti, rom

mikrosomebSi da rekonstruirebul sistemebSi mimdinare yvela hidro-qsilazur reaqcias monooqsigenireba udevs safuZvlad. es imas niSnavs,

rom am gardaqmnisas gaaqtiurebuli molekuluri Jangbadis erTi atomi

wylis warmoqmnaze ixarjeba, xolo meore dasaJangi substratis

molekulaSi inergeba [181, 182]. monooqsigenazuri gardaqmnis cikluri

buneba da misi calkeuli stadiebis Tanmimdevroba pirvelad 1968 wels

estabrukis, hildebrantisa da ulrixis [183] mieraa SemoTavazebuli.

56

zogierTi damatebebiT igi sqema 1.2-zea mocemuli. Semdgomma gamokvle-vebma naTlad aCvenes am sqemis samarTlianoba da cocxal samyaroSi misi

universaloba sayovelTaod iqna aRiarebuli [184-188]. sqemidan vxedavT,

rom hidroqsilireba kooperatiuli, mravalstadiuri procesia.

OH2

ROH

2H+

1_k=0.1_1.5c

RH FeO +3

Fe3+

1_

O2 Fe RH2+._

O2 Fe RH

e_

O2 Fe RH

O2

Fe RH RH Fe CO

CO

1_k>1000 c

1_k=2_10ce_

1_k=50c

H2O2

2H+

O2 Fe RH3+=

1_k=3_4c

Fe RH3+

1_k=0.2c

H2O2 + O2O2._

O2._

+ +2H+

k=1_2c 3+._

2+

2+ 2+

RH

hν

XOOH

XOH

sqema 1.2. monooqsigenazuri cikli [52].

I stadiaze citoqrom P450-Tan (ferocitoqromTan) substratis

mierTeba da ferment-substratis kompleqsis warmoqmna xdeba. amas Tan

sdevs hemoproteinis Jangva-aRdgeniTi potencialisa da spinuri mdgoma-reobis cvlilebebi. redoqs-potenciali -300 mV-dan +170 mV-mde izr-deba. amis gamo Termodinamikurad momgebiani xdeba NADPH-is aRmd-geneli eqvivalentebis ferocitoqromze gadatana.

substratTa umetesobas citoqromi dabalspinuridan (jamuri spini

+1/2) maRalspinur formaSi (jamuri spini +5/2) gadahyavs, xolo zogi-erTi amini ukugadasvlas iwvevs. varaudoben, rom maRalspinuri forma

meeqvse mdgomareobaSi kargavs aqsialur ligands, magram mexuTe ligan-dad cisteinis naSTs inarCunebs [156]. ferment-substratis kompleqsTa

57

warmoqmnis siCqareebi maRalia da xasiaTdebian 102±106 konstantTa mniS-vnelobebiT, rac TviT substratTa bunebazea damokidebuli [189]. fer-ment-substratis kompleqsis warmoqmna detaluradaa Seswavlili qafu-ris damJangvel citoqrom P450-isaTvis (P450cam) [153, 190-192].

naCvenebia, rom hemoproteinis daJanguli formis am substratTan

urTierTqmedebis Sedegad STanTqmis speqtrSi 417, 539 da 569 nm-ebze

maqsimumebis mqone zolebis nacvlad Cndeba axali _ 391, 500 da 640

nm-ebze maqsimumebis mqone, anu dabalspinuri 2.46, 2.26 da 1.91 g-faqto-riani forma gadadis maRalspinur formaSi da Cndeba 3.4 da 1.8 g-faqtoriani signalebi.

kompleqsis warmoqmna meore rigis siCqaris (4.1.106 moli.wm-1), xolo

misi daSla pirveli rigis siCqaris konstantiT (6.0 moli.wm-1) xasi-

aTdeba. garda amisa, kompleqss ufro maRali redoqs-potenciali 140mV gaaCnia, vidre Tavisufal hemoproteins. kompleqsi-

rebisas citoqromis ciklur nawilSi konformaciuli cvlilebebi

mimdinareobs [193]. dakavSirebisaTvis absoluturad aucilebelia misi

karboniluri naSTis arseboba. naxSirbadis monooqsids unari aqvs

daTrgunos, an sulac kompleqsidan gamoaZevos dakavSirebuli subst-rati [149, 194-196].

cnobilia naerTTa klasi, romelsac hidroqsilirebis gaTiSva SeuZ-lia. am klasis naerTebi citoqromTan kompleqsebs warmoqmnian, magram

Semdgom aRar hidroqsilirdebian [197-200]. am dros hemoproteinTan

substratis urTierTqmedebis yvela damaxasiaTebel Sedegs (diferen-ciuli speqtrebis warmoqmnas, NADPH-isa da O2-is moxmarebis gazrdas)

aqvs adgili, magram TviT gamTiSvelis JangviTi gardaqmnebi ar

registrirdeba. nivTierebaTa am jgufs mravali ftorSemcveli naxSir-

wyalbadi miekuTvneba [199, 201]. maTi moqmedebis asaxsnelad saWiroa

gaTvaliswinebul iqnas cnobili faqti ortinol- da salicil-

athidroqsilazur reaqciebSi substratuli analogebiT _ rezorciniTa

da benzoatiT hidroqsilirebis gaTiSva, romlebic eleqtronebis nakads

Jangbadis aqtivaciisaken mimarTaven, wyalbadis zeJangis Semdgomi warmo-qmniT [198, 200]. mikrosomul mahidroqsilirebel sistemaSi gaTiSvas

SeiZleba wylis daCqarebuli warmoqmna axldes [197]. daJangul

citoqromTan arahidroqsilirebadi gamTiSvelis kompleqsireba imis

58

TvalsaCino ilustraciaa, Tu ramdenad sworia warmodgena imis Sesaxeb,

rom ferment-substratis dakavSireba monooqsigenirebis pirveli etapia.

meore mxriv, jerac daudgenelia, SeiZleba Tu ar es stadia sruli

ciklis siCqaris malimitirebel ubnad CaiTvalos da ganisazRvros misi

siCqare.

literaturaSi arsebobs miTiTebebi kompleqsirebis saxeobriv speci-fikurobaze. mag., kortizoli zRvis goWebis RviZlis mikrosomebis

citoqrom P450-Tan I tipis, xolo virTxis mikrosomebis hemoprotein-

Tan II tipis kompleqsebs warmoqmnis [202]. substratebTan urTierT-

qmedeba gavlenas axdens citoqrom P450-is, rogorc eleqtronTa

aqceptoris Tvisebebze da es, Tavis mxriv, asaxvas poulobs NADPH-

reduqtazul reaqciaSi misi aRdgenis siCqareze; kerZod, citoqromTan

substratTa dakavSireba aadvilebs ferment-substratis kompleqsis

pirveli eleqtroniT aRdgenas [170, 184].

II stadiaze citoqrom P450-substratis kompleqsis aRdgena xdeba.

Tavis adreul samuSaoebSi jiletma da misma TanamS. [203-205] SeZles

gaezomaT eleqtronTa gadatanis jaWvSi NADPH-citoqrom P450-reduq-tazuli reaqciis siCqare. swrafi reaqciebis Camweri, rTuli orsxiviani

speqtrofotometruli teqnikis gamoyenebiT am mkvlevarebma aCvenes, rom

anaerobul pirobebSi mikrosomul suspenziaze NADPH-is damateba ci-

toqrom P450-is TiTqmis srulad aRdgenas iwvevs, da es procesi orfa-

ziani xasiaTisaa. pirveli, sawyisi faza, romelic uaRresad swrafad

xorcieldeba, araa damokidebuli anaerobiozis siRrmeze; meore, Senele-

buli faza ki sainkubacio xsnarSi Jangbadis kvalis saxiT Tanamyofobas

moiTxovs. I tipis substratebi aCqareben, xolo II tipis substratebi aneleben sawyisi fazis siCqares da igi hidroqsilirebis saerTo reaq-

ciis siCqaresTan srul SesabamisobaSia. meore, Senelebuli faza, ro-

gorc Cans, sxva araferia, Tu ara citoqrom P450-is Zalze dabali siC-

qariT aRdgena, romlis drosac substratis dakavSireba da misi hi-

droqsilireba uSualod ar xorcieldeba. igi aseve ar warmoadgens he-

moproteinis ori qvefraqciis Jangbadisadmi gansxvavebuli swrafvis Se-

degs [206]. reaqciis swraf fazaSi aRdgenili citoqrom P450-is raode-noba optimalurad korelirebs citoqrom P450-substratis (cikloheq-

sani) kompleqsis SemcvelobasTan [178]. reaqciis srafi faza pirveli

59

rigis, Senelebuli ki meore rigis reaqciis kinetikiT xasiaTdeba. amave

avtorebma [203] aCvenes, rom mikrosomul fraqciaSi citoqrom P450-is garkveuli nawili (10-20%-mde) SeiZleba romelime I tipis endogenur substratTan iyos kompleqsirebuli. miRebuli Sedegebis safuZvelze

gakeTebul iqna daskvna, rom citoqrom P450-is aqtiur centrSi pirve-li eleqtronis aqceptireba hidroqsilirebis malimitirebeli stadiaa.

mogvianebiT es daskvna araswori aRmoCnda. macubaram da Tanaavt. [207]

gamoiyenes ra ufro srulyofili Seyovnebuli nakadis meTodi, naxes,

rom mikrosomebSi hemoproteinis aRdgena marTlac orfaziani reaqciaa.

samagierod, orive faza pirveli rigis reaqciis tips miekuTvneba, siCqa-

ris konstantebiT Sesabamisad Cqari fazisaTvis 2 wm-1 da Senelebuli-

saTvis 0.3 wm-1. swraf fazaSi aRdgenas ganicdis mTeli hemoproteinis

Semcvelobis 50% da amasTan reduqtazuli reaqciis siCqare (150 nM

wT-1/mg cilaze) gacilebiT metia, vidre hidroqsilirebis siCqare (mag.,

eTilmorfinisaTvis 8.6 nM wT-1/mg cilaze) [207]. aqedan gamomdinare,

pirveli eleqtroniT hemoproteinis aRdgena ar SeiZleba oqsigenazuri

ciklis malimitirebel rgolad CaiTvalos. sayuradReboa aRiniSnos is-

ic, rom citoqrom P450-is aRdgenis orfazianoba mikrosomuli sistemis

membranasTan integraciis yvela donezea (intaqtur membranaSi, deter-

gentiT solubilizebul mikrosomuli membranis komponentTa narevSi da

gasufTavebuli flavoproteiniTa da citoqrom P450-iT rekonstruire-bul sistemaSi) SenarCunebuli [189, 208]. reaqciis orfazianoba vlin-

deba diTionitis aRmdgenelad gamoyenebisas; mxolod am SemTxvevaSi au-

cilebelia fosfolipidis Tanamyofoba. xsnad rekonstruirebul siste-

maSi fosfolipidi SeiZleba detergentebiT Seicvalos [209-211]. rekon-

struirebul membranul sistemaSi uaryofiTad damuxtuli fosfolipi-

dis damateba NADPH-citoqrom P450-reduqtazuli reaqciis stimuli-

rebas iwvevs. aseT sistemebSi citoqrom b5-s unari aqvs citoqrom

P450-s pirveli eleqtroni miawodos, magram ara 10-20%-iT imaze meti

siCqariT, rasac NADPH-specifikuri flavoproteini aRwevs. samagie-

rod, meore eleqtroni b5-dan gacilebiT swrafad gadaitaneba [211]. sa-

reaqcio areSi erT- da orvalentian metalTa ionebis damateba NADPH-

citoqrom P450-reduqtazas gaaqtiurebas iwvevs [212, 213]. aRmoCnda,

rom hidroqsilirebis reaqciaze Mg2+-is mastimulirebeli moqmedeba am

60

flavoproteinis gaaqtiurebiT realizdeba. es dasturdeba orive reaq-

ciis temperaturaze erTnairi damokidebulebiT da maTze D2O-is erT-

nairi mainhibirebeli moqmedebiT [214].

saerTod, umravles SemTxvevebSi reduqtazuli da hidroqsilazuri

reaqciebis siCqareebi erTmaneTTan dadebiT korelaciaSia. maT Soris

gansxvavebebi (mag., eTilmorfinis demeTilirebisas) sxvadasxva saxis

cxovelebSi advilad SeiZleba aixsnas NADPH-citoqrom P450-reduq-tazas gansxvavebuli aqtivobebiT. gacilebiT xSirad hidroqsilirebis

siCqare ferment-substratis aRdgenis siCqaresTan ufroa SesabamisobaSi,

vidre Tavisufali citoqrom P450-is aRdgenis siCqaresTan [215, 216]. NADPH-citoqrom P450-reduqtazuli reaqciis meqanizmis kvlevam

aCvena, rom hemoproteinis molekulas pirveli tipis substratebisaTvis

(eTilmorfinisaTvis) dakavSirebis araerTi ubani gaaCnia. swored es ub-

nebi arian pasuxismgebelni mocemuli reaqciis gaaqtivebaze [217]. erTi

maTgani substratisadmi maRali, xolo meore dabali swrafviT (Sesaba-

misad Ks – 0.016 mmoli da Ks – 0.60 mmoli) xasiaTdeba. miuxedavad ami-

sa, arc es konstantebi da arc maTgan gamoTvlili saSualo sidideebi

(0.13 mmoli) ar emTxveva am substratisaTvis damaxasiaTebel hidroqsi-

lirebis KM-s da disociaciis speqtrul konstantebs (0.23 da 0.062

mM Sesabamisad). es ki imaze miuTiTebs, rom NADPH-is Jangvis jaWvSi

eTilmorfinisaTvis dakavSirebis sul mcire, sami ubani mainc unda ar-

sebobdes [217]. pirvels, rogorc Cans, fiziologiuri funqcia akisria,

ramdenadac substratisadmi maRali swrafva aqvs da misi dabali koncen-

traciebisas reduqtazuli reaqciis aqtivacias akontrolebs. Zalian

dabali swrafvis mqone ubans mniSvneloba SeiZleba substratis toqsi-

kuri koncentraciebisas eniWebodes. maTgan gansxvavebiT, mesame ubani

speqtruli cvlilebebis aRZvras ganapirobebs. uaRresad mniSvnelovania

avtorTa mier gakeTebuli daskvna imis Taobaze, rom hidroqsilirebis

reaqciis siCqare unda ganisazRvros ara Tavisufali an substratTan

dakavSirebuli citoqrom P450-is aRdgenis siCqariT, aramed sididiT,

romelic substratis moqmedebiT reduqtazuli reaqciis daCqarebas

axasiaTebs.

citoqrom P450-is molekulaSi dakavSirebis gansxvavebuli centre-

bis arsebobaze miuTiTeben Sromebic, romlebSic naCvenebia rekonstrui-

61

rebul sistemebSi hemoproteinis fosfatidilqolinis absoluturi au-

cilebloba maSin, rodesac fermentis substratTan dakavSireba da da-

maxasiaTebeli speqtruli cvlilebebis aRZvra aseT sistemaSi fosfoli-

pids ar saWiroebs [218, 219].

arCakovisa da misi TanamS. mier [40, 41] Catarebul iqna gamokvleva,

romelic miznad isaxavda N,N-dimeTilanilinTan (DMA) kompleqsSi ci-

toqrom P450-is stimulirebuli aRdgenis gavlenis dadgenas am sub-

stratis N-demeTilirebis, p-hidroqsilirebisa da N-Jangvis reaqciaTa

siCqareebze. NADPH-citoqrom P450-reduqtazuli reaqciis mastimuli-

rebel faqtorad gamoiyeneboda Mg2+-is ionebi [212, 213]. aseTi midgoma

sakmaod originaluri iyo hidroqsilirebis meqanizmSi kompleqsis aRd-

genis reaqciis rolis srulad gamosavlenad: hidroqsilirebis jaWvSi

reduqtazuli reaqcia Tu marTlac malimitirebelia, maSin Mg2+-is dax-

marebiT misi siCqaris gazrdam erTi substratis Jangvis samive tipis

reaqciis stimulireba unda moaxdinos. am SemTxvevaSi substratad

DMA-s gamoyeneba amis srul saSualebas iZleva. misi gardaqmnis SesaZ-

lo gzebi mocemulia naxazze. 1.7.

Mg2+-is sxvadasxva koncentraciebis moqmedebis Seswavlam DMA-s

demeTilirebisa da p-hidroqsilirebaze gansxvavebuli mastimulirebeli

efeqtebi gamoavlina. aqtivaciis koeficientebi Sesabamisad 1.5 da 2.2-s

Seadgenda. N-demeTilirebis maqsimaluri stimulacia miiRweoda 15 mmo-

li MgCl-is Tanamyofobisas. p-hidroqsilirebisaTvis Mg2+-is ufro ma-

Rali koncentraciebi aRmoCnda aucilebeli. rac Seexeba DMA-s N-

Jangvas, masze am metalis gamaaqtiurebeli moqmedeba ar gamovlinda da

am Sedegs avtorebma orgvari axsna misces: demeTilireba da hidroqsi-

lireba an NADPH-specifikuri flavoproteinis meSveobiT ar xor-

cieldeba, an N-JangvaSi monawileobs sxva, Mg2+-sadmi aramgrZnobiare

flavoproteini [40].

eleqtronTa gadatanis reaqciebze magniumis gavlenis Seswavlisas

dadginda, rom am metalis ionebi 2–2.5-jer zrdian NADPH-is Jangvis

siCqares sainkubacio narevSi rogorc DMA-s Tanamyofobisas, aseve mis

gareSe [40]. garda amisa, DMA-c iwvevs NADPH-is Jangvis siCqaris

62

NCH3 CH3

NCH3 CH3

NCH3 CH3

OH

NH CH3

O

NADPH + O2

N,N-dimeTilanilini

(DMA)N-oqsid-N,N-dimeTilanilini

N-monomeTilanilini

N,N-dimeTil-p-aminofenoli

+ CH2ON-demeTilireba

N-Jangva

p-hidroqsilireba

+ H2O

+ H2O

nax. 1.7. DMA-s Jangvis gzebi RviZlis mikrosomebSi.

matebas, Tumca am SemTxvevaSi stimulaciis koeficienti gacilebiT da-

balia. miuxedavad amisa, rogorc gamoirkva, Mg2+-isa da DMA-is Tana-

myofobisas NADPH-is Jangvis siCqaris gazrdis efeqti aditiuria.

hidroqsilirebis meqanizmis pirveli ori stadiis rolis ganxilvis

Sedegad SeiZleba met-naklebad sicxadiT iTqvas, rom arc citoqrom

P450-substratis kompleqsis warmoqmnis da arc NADPH-is Jangvis

jaWvSi am kompleqsis aRdgenis reaqcia umravlesi substratebis hi-

droqsilirebis siCqaris limitirebas ar axdenen. mxolod zogierTi

maTganis (mag., aminopirinisa da DMA-saTvis) demeTilirebis siCqare

utoldeba citoqrom P450-substratis aRdgenis siCqares, da amdenad, am

63

substratebis mier kompleqsis warmoqmnas SeuZlia Jangvis siCqaris li-

mitireba. saerTo procesSi “viwro adgils” romeliRac sxva stadia

unda warmoadgendes. gamoTqmulia mosazreba, rom es rgoli _ sammagi

kompleqsis (aRdgenili citoqromi P450_substrati_O2) meoradi aRdge-

naa [185]. citoqrom P450-is substratTan urTierTqmedebis kinetika im-denad rTulia, rom SeuZlebeli xdeba KS-is im WeSmariti sididis dad-

gena, romelic lainuiver-berkis meTodiT hemoproteinis substratTan

swrafvas axasiaTebs.

III stadiaze erTi eleqtroniT aRdgenili kompleqsi maRali siCqa-

riT urTierTqmedebs molekulur JangbadTan da sammag kompleqss (aRd-

genili citoqromi P450_O2_substrati) warmoqmnis. sxvadasxva kom-

pleqsebisaTvis O2-is mierTebis siCqaris konstantebi 104-106 moli-1.wm

-1-

is diapazonSi icvleba [189]. hidroqsilirebis meqanizmis sqema am

kompleqsis producirebis aucileblobas iTvaliswinebs, radgan meore

eleqtroniT Semdgomma aRdgenam Jangbadis uSualo gaaqtiveba unda

gamoiwvios. sammagi kompleqsis warmoqmnis SesaZlebloba rogorc aRniS-

nuli iyo pirvelad Pseudomonas putida-s hidroqsilazur sistemaSi iqna

gamovlenili [153]. estabruki da Tanaavtorebi [220] NADPH-iT

aRdgenil RviZlis mikrosomul suspenziaSi heqsobarbitalis damate-

bisas akvirdebodnen speqtrul cvlilebebs. maT mier miRebul dife-

rencialur speqtrSi STanTqmis ori zoli Cndeba 440 da 590 nm-ze. es

cvlilebebi Seafases rogorc Sualedi kompleqsis (−•

2O _Fe3+ _ I tipis

substrati) warmoqmnis Sedegi. meore tipis substratebi am SemTxvevaSi

araefeqturni aRmoCndnen. mogvianebiT SesaZlebeli gaxda Pseudomonas putida-s kulturidan citoqrom P450-is oqsigenirebuli stabiluri

formis gamoyofa, romlis SemadgenlobaSic Sedioda TviT citoqrom

P450cam, qafuri da O2. absolutur speqtrSi kompleqsis STanTqmis

zolebi 355, 418 da 555 nm-ebis olqSi mJRavndeboda [187].

oqsicitoqromis sammagi kompleqsis Seswavlam cxadyo, rom oqsi-

hemoglobinis analogiurad eleqtronuli simkvrive Jangbadis atomzea

gadanacvlebuli da amis gamo rkina kvlav daJangul mdgomareobaSi

rCeba. kompleqsma SeiZleba disproporcionireba ganicados superoqsid-

uri anion-radikalis warmoqmniT. arc isaa gamoricxuli, rom ferment-

substratis kompleqsma, Jangbadis molekulis nacvlad, wyalbadis

64

zeJangidan an araorganuli hidrozeJangebidan ukve ori eleqtroniT

aRdgenili Jangbadatomebi miierTos [184]. sammag kompleqsSi Jangbadis

mdgomareoba da misi konfiguracia SedarebiT detalurad jerac araa

gamokvleuli. gamoTqmuli varaudiT, rom misi mdgomareoba ukeT

Seesatyviseba vaisis [221] mier SemoTavazebul struqturas: Fe3+ −2O da

masSi

OFe O

kuTxe 120°-s uaxlovdeba.

zogierTi mkvlevari cdilobda qvantur-meqanikuri meTodebis gamo-

yenebas oqsigenirebuli citoqrom P450-is yvelaze momgebiani kon-

figuraciis gansasazRvrad [222, 223]. Catarebulma gamokvlevebma aCvenes,

rom hemoproteinis Jangbaduri da karboniluri kompleqsebis uCveulo

speqtruli Tvisebebi mTlianadaa dakavSirebuli mexuTe aqsialur

mdgomareobaSi myof Tiolur ligandTan da Zlieraa damokidebuli hemur

rkinasa da am ligands Soris manZilze. 2 nm-iT am manZilis gazrda

savsebiT sakmarisia citoqrom P450-is araaqtiur citoqrom P420-ad gardasaqmnelad, romelic pirvelisagan aRdgenil mdgomareobaSi karbo-

niluri kompleqsis sores zolis maqsimumiT gansxvavdeba [223]. amis

safuZvelze garkveuli daskvnis gakeTeba zogierTi avtoris mier naad-

revad da saxifaTod iqna miCneuli, ramdenadac oqsihemoglobinisaTvis

Catarebuli Fe_O2-is kavSiris Teoriuli gaTvlebi aradamajerebeli

aRmoCnda. mxolod am hemoproteinis rentgenostruqturulma gamokv-

levebma aCvenes, rom O2-is molekuluri RerZi porfirinis birTvis

sibrtyesTan marTlac qmnis kuTxes, romelic 120°-s utoldeba [224].

mikrosomul mahidroqsilirebel sistemaSi sammagi kompleqsi

aramgradia da TviTJangvas ganicdis. substratis monooqsigenirebisaTvis

aucilebelia am kompleqsis kidev erTi eleqtroniT aRdgena, rac

procesis momdevno stadiaze xorcieldeba.

arsebuli monacemebis safuZvelze damaxasiaTebeli speqtruli Tvise-

bebis mqone sammagi kompleqsis warmoqmna araviTar eWvs ar iwvevs.

miuxedavad amisa, warmodgenili meqanizmis srulad misaRebad saWiroa

gairkves, Tu: 1) hemoproteinis oqsigenirebuli forma ratom warmoiqm-

neba maincdamainc mxolod I da ara II tipis substratebis SemTxvevaSi? 2) kompleqsis gasaaqtiureblad saidan miiReba meore eleqtroni? mra-

vali avtoris [212, 217, 225] Tanaxmad kompleqsSi myofi citoqrom

65

P450-is aRdgenis reaqciis siCqare hidroqsilirebis reaqciis saerTo

siCqares utoldeba, maSin rodesac igi araumcires orjer meti unda

iyos, radgan erTi molekula hemoproteinis aRdgenas ori eleqtroni

esaWiroeba. mxolod am kiTxvebze pasuxis gacemis Semdeg SeiZleba

sabolood sarwmunod iqnas miRebuli zeviT ganxiluli warmodgenebis

siswore hidroqsilirebisas Sualedi sammagi kompleqsis warmoqmnis

Sesaxeb, romlis Semdgom aRdgenasac Jangbadis aqtiuri formis gene-

rirebasTan mivyavarT.

IV stadia “Jangbadis aqtiuri formis” warmoqmnas da dasaJang

substratTan mis dakavSirebas moicavs. am formis Jangbadis Semcveli

sammagi kompleqsis warmoqmna da misi Semdgomi daSla imdenad rTulia,

rom xSirad am etapebs aradiskretulad ixilaven.

substratis hidroqsilirebisaTvis sammagi kompleqsis aRdgenaa

saWiro da amisaTvis meore eleqtronis wyarod NADPH-citoqrom b5-reduqtazas, xolo gadamtanad citoqrom b5-s miiCneven. NADPH-

citoqrom P450-reduqtaziT citoqrom b5-is aRdgenis SesaZlebloba

eqsperimentulad dadasturebulia, da amaze Cven adre gvqonda saubari

(ix. 1.3.2 da 1.3.3). eleqtronis transportSi citoqrom b5-is mniSv-

nelovani roli naCvenebia rogorc mikrosomul [225-228], aseve

rekonstruirebul sistemebSi mTeli rigi substratebis Jangvis Seswav-

liT [229-235]. iTvleba, rom eleqtronTa transportis NADH-speci-

fikur jaWvSi aRdgenili citoqromi b5 meore eleqtrons uSualod

gadascems sammag kompleqsSi oqsicitoqrom P450-s, ris Sedegadac

peroqsicitoqrom P450 warmoiqmneba. amas adastureben uamravi eqsperi-mentuli monacemebi, romelTa analizi detaluradaa ganxiluli or

SesaniSnav monografiaSi [126, 175]. pirveli faqtori, romelic uTuod

yuradRebas imsaxurebs, isaa, rom I tipis substratebis (naxSirwyal-

badebis, mesameuli aminebis, aliciklebis) Jangvisas ori piridinnuk-

leotidis (NADH-isa da NADPH-is) erTdroulad Tanamyofobisas

adgili aqvs sinergizmis efeqts, rac ar SeiniSneba II tipis substratTa mimarT. es Sedegi mikrosomul JangvaSi orive eleqtronul-satrans-

porto sistemis SesaZlo Tanamonawileobaze miuTiTebs [225, 226].

meore faqtori, romelic aranakleb sayuradReboa: sinergizmis fenomenis

aRZvraSi mxolod endogenuri da ara mikrosomul membranaSi CaSenebuli

66

egzogenuri citoqromi b5 erTveba, romelic araTu ar astimulirebs

hidroqsilirebas, aramed aminopirinisa da dimeTilanilinis JangviTi

gardaqmnebis damuxruWebasac ki iwvevs [226]. es Sedegebi SeiZleba imiT

aixsnas, rom membranaSi CaSenebuli hemoproteini im klasterSi ar

“jdeba”, romelSic citoqrom P450 da misi reduqtaza mdebareobs. inhi-bitorul moqmedebas rac Seexeba, es SesaZloa imiT iyos gamowveuli,

rom citoqrom b5 NADPH-citoqrom b5-reduqtazadan eleqtronebis

aqceptirebis miuxedavad, hidroqsilirebis TvalsazrisiT SemdgomSi maT

araproduqtiulad xarjavs.

arsebobs meore alternativac mikrosomul Jangvaze endogenuri da

egzogenuri citoqrom b5-is gansxvavebuli zemoqmedebis axsnis Sesaxeb,

rac natiuri da damatebuli hemoproteinis gansxvavebul redoqs-poten-

cials ukavSirdeba. SesaZloa, pirveli jgufis substratebi citoqrom

P450-Tan iseT kompleqsebs qmnian, romelTa Jangva-aRdgeniTi potenci-

alebi dadebiTad moqmedeben citoqrom b5-iT sammagi kompleqsis aRdge-

naze, maSin rodesac aminopirini da dimeTilanilini warmoqmnian komp-leqsebs, romelTa Jangva-aRdgeniTi potencialebi am process zRudaven

[226].

rekonstruirebul sistemaSi mimdinare Jangvis procesebze citoqrom

b5-is gavlena mniSvnelovnadaa ganpirobebuli TviT citoqrom P450-is bunebiT. mag., identificirebulia am hemoproteinis forma, romelsac

citoqrom b5-isadmi mometebuli swrafva gaaCnia [236], da p-nitro-anizolis JangviTi demeTilirebis reaqcia efeqturad Cqardeba am ci-

toqrom P450-iT [231, 234]. citoqrom b5 gansxvavebuli doniT aCqarebs

erTi da igive substratis (3,4-benzpirenis) Jangvas identur pirobebSi,

procesSi monawile ori gansxvavebuli formis citoqrom P450-iT (P450LM2-iT da P450LM4-iT), anu misi buneba arsebiTad cvlis cito-

qrom b5-is mastimulirebel SesaZleblobas.

amrigad, SeiZleba misaRebad CaiTvalos, rom mikrosomebSi da rekons-

truirebul sistemebSi sxvadasxva substratis JangvaSi citoqrom b5-is

gavlena ramodenime faqtoriTaa ganpirobebuli: citoqrom b5-is war-

moSobiT (ujredSi arsebulia Tu damatebuli) da misi Jangva-aRdgeniTi

potencialiT; dasaJangi substratis bunebiT da sammagi kompleqsis

Jangva-aRdgeniT maxasiaTeblebze misi gavleniT; TviT citoqrom P450-is

67

bunebiT da citoqrom b5-sadmi misi gansxvavebuli swrafviT. aRniSvnis

Rirsia agreTve fermentul sistemaTa rekonstruqciis meTodebis, maTSi

detergentebis narCenebis Semcvelobisa da reaqciis msvlelobis piro-bebis faqtorebi. citoqrom b5-isa da sammagi kompleqsis Jangva-

aRdgeniTi parametrebis optimaluri Tanafardobisas hemoproteini asti-

mulirebs substratis Jangvas; arakeTilsasurveli Tanafardobisas ki

citoqrom b5-s NADPH-citoqrom P450-reduqtazidan SeuZlia eleqt-

ronebis miReba, magram citoqrom P450-ze maT gadatanas veRar axor-cielebs, ris gamoc Jangvis procesi iTrguneba.

gansja imis Sesaxeb, Tu romeli sistemaa mikrosomebSi mimdinare

hidroqsilirebis procesis yvelaze ukeTesi modeli, ekuTvniT Staudin-

gersa da mis TanamS. [237, 238], agreTve iudenfrendsa da mis TanamS.

[239, 240]. amis safuZvelze maT mier SemoTavazebulia ramodenime

kriteriumi, romelTaganac yvelaze popularobiT sxvadasxva mdgoma-

reobaSi Canacvlebuli birTvis mqone cikluri naerTebis hidroqsi-

lirebis siCqaris SedarebiTi Seswavla sargeblobs. mag., modelur

sistemebSi acetanilidisa da anizolis o-, m- da p-mdgomareobebSi hidroqsilirebis siCqareTa Tanafardobis gazomva aRniSnul avtorebs

miaCniaT mikrosomuli hidroqsilirebis meqanizmTan Sedarebis saukeTeso

midgomad. aRmoCnda, rom gamoyenebuli substratis miuxedavad, mikro-

somebSi hidroqsilirebis produqtTa Tanafardoba iseTia, rom saerTo

raodenobis araumcires 90%-s p-warmoebulebi, ∼10%-s o-warmoebulebi

da araumetes 1%-s m-warmoebulebi Seadgenen. rac Seexeba •HO -

radikalebis generirebad modelur sistemebs (fentonis _ Fe2+, EDTA,

askorbati, H2O2, N2; da iudenfrendis _ Fe2+, EDTA, O2), maTSi p-

hidroqsilirebul produqtTa gamosavali ~55 %-s ar aRemateba xolo

o- da m-warmoebulebi Sesabamisad ~45 da ~10%-s Seadgenen. maSasadame,

hidroqsilirebul produqtTa gamosavliT es modeluri sistemebi met-

naklebad Seesatyvisebian mikrosomuls, Tumca Sesabamisoba mainc

arasrulia.

Jangbadis radikalebis generirebad sistemebSi reaqciebis Tanmim-

devroba, savaraudod, ase gamoiyureba:

68

._O2 + e_O2 + e_

H+

O2 + e_2_

2H+ 3H+

+ e _

2_

4H+

2_O + O

_

H2O2

2O

2H2OHO2. HO. + H2O

Jangbadis oTxeleqtroniani aRdgenis Tanmimdevroba [126].

aqedan Cans, rom •HO radikalebis generaciisaTvis aucilebelia

Jangbadis sam-eleqtroniani aRdgena. Sualed produqtebad −2

2O da H2O2

warmoiqmnebian. katalazas an Ti-is saSualebiT H2O2-is mudmivi mocile-

ba Tu moxdeba, aseT sistemaSi •HO -radikalebis koncentracia mkveTrad

moiklebs, xolo Zalze arastabiluri •

2HO radikalebis wili sagrZ-

noblad moimatebs [241]. sistemaSi, romelSic •HO - da

•

2HO -mahid-

roqsilirebel agentebs Soris Tanafardoba •

2HO warmoqmniskenaa gada-

weuli, sxvadasxva mdgomareobebis hidroqsilirebul produqtTa gamosa-

valSi Tanafardobrivi cvlileba moxdeba da am dros samive saxis pro-

duqtis daaxloebiT Tanabar warmoqmnas eqneba adgili, anu •

2HO radi-

kaliT hidroqsilirebis Sedegad miRebul produqtTa gamosavali mikro-

somebisagan gansxvavebuli iqneba.

mahidroqsilirebeli nawilakis WeSmariti bunebis dadgenaSi aucile-

beli aRniSvnis Rirsia hamiltonis [242] mier wamoyenebuli hipoTeza.

monooqsigenazuri reaqciebis gaanalizebisas man SeniSna maTi gasaocari

msgavseba karbenebis (CR2) da nitrenebis (NR) damaxasiaTebel reaqcieb-

Tan. es naerTebi mraval qimiur gardaqmnebSi warmoiqmnebian rogorc ma-

Ralreaqciisunariani Sualeduri formebi. hamiltonma paraleli gaavlo

da amasTan dakavSirebiT gamoiTqva mosazreba, rom ferment-mono-

oqsigenazebs Jangbadi substratze gadaaqvT karbenoidebis msgavsad, e.w.

oqsenoiduri meqanizmiT [243, 244]. zogierT dioqsigenazasac axasiaTebs

substratze Jangbadis gadatanis oqsenoiduri gza. miuxedavad imisa, rom

Znelia damtkicdes yvela monooqsigenazas oqsenoiduri meqanizmiT funq-

cionireba, mainc SeiZleba iTqvas, rom am klasis fermentTa umravlesoba

procesis sxvadasxva stadiaze msgavsebis arsebiT niSnebs amJRavnebs. ha-

miltonis azriT, oqsigenazebiT katalizebul reaqciebSi aqtiur dam-

69

Jangvel nawilaks warmoadgens Jangbadis atomi, romelsac gare energe-

tikul doneze 6 eleqtroni (Jangbadis singleturi mdgomareoba) gaaC-

nia, anu esaa ara Tavisufali Jangbadis atomi, aramed nawilaki (oqse-

noidi), romelsac substratze aseTi Jangbadi gadaaqvs. sailustraciod

moviyvanT ramodenime magaliTs [182, 245]:

1. karbenebs SeuZliaT mraval alkanSi Canergva da alkiluri warmoe-

bulebis warmoqmna maTi konfiguraciis Seucvlelad:

HC CR2HC+ CR2 (1)

arcTu iSviaTad aris SemTxvevebi, roca monooqsigenazebi alkanTa mole-

kulebSi Jangbadis atoms nergaven da maTi pirveladi konfiguraciis

SenarCunebiT spirtebs warmoqmnian:

HC OHCmonooqsigenaza

+ O2 (2)

2. karbenebi uerTdebian alkenebs ciklopropanebis warmoqmniT:

RR

+ CR2 (3)

am reaqciis analogiurad mimdinareobs monooqsigenazebiT katalizebuli

alkenebis epoqsidirebac:

Omonooqsigenaza

+ O2 (4)

3. karbenebi advilad reagireben aromatul naerTebTan alkilbenzolisa

da norkaradienis warmoqmniT, romlebic wonasworobaSia ciklohep-

tatrienebTan:

X

CHR2

X

RR

X X

RR

+ CR2 + (5)2

mravali oqsigenaza akatalizebs aromatuli naerTebis fenolebad da

70

arenoqsidad gardaqmnas, romlebic wonasworobaSia oqsipinTan:

X

OH

X

O

H

HX

O+ O2 + 22 (6)

aRniSnul reaqciaTa analogiam Camoayaliba Sexeduleba imis Sesaxeb,

rom karbenebi oqsigenazebis Soreul models warmoadgenen. aman, Tavis

mxriv, gamoiwvia gacxovelebuli kvlevebis dawyeba oqsigenazebis moqme-

debis imitaciis unaris mqone modeluri sistemebis moZiebisa da Ses-

wavlisaTvis.

oqsenoiduri meqanizmis realobaze arsebuli mTeli rigi sarwmuno

mtkicebulebebis miuxedavad, Znelia garkveulad msjeloba mis bunebasa

da struqturaze. erTi ram mainc naTelia: sxvadasxva SemTxvevaSi oqse-

noids gansxvavebuli struqtura unda gaaCndes. marTlac, bunebrivia,

rom iseTi rTuli fermentuli sistemisaTvis, romelic citoqrom P450-sa da flavoproteins Seicavs, arsebobdes substratze Jangbadis gadam-

tani erTi aqtiuri universaluri nawilaki. erT SemTxvevaSi oqsenoidi

erTi eleqtroniT aRdgenil JangbadTan hemuri rkinis kompleqss, an mi-

si gardaqmnis produqts warmoadgens, romelic O_O bmis daSlis Sede-

gad miiReba. meore SemTxvevaSi oqsenoidi SeiZleba iyos flavinis hi-

drozeJangi an misi gardaqmnis produqti, romelic substratSi Casanerg

nawilakSi eleqtronis deficitiT xasiaTdeba. citoqrom P450-is hemiT an sxva hemSemcveli cilebiT Jangbadis aqtivaciis Teoriuli ganxilva

[Fe3+_

−22O ] struqturis sasargeblod metyvelebs. aq Jangbadis atomebi

120°-ian kuTxes qmnian. gardamaval mdgomareobaSi es nawilaki sub-

strats rkinisagan kidura Jangbadis atomze gadascems [222].

[Fe3+_

−2

2O ] → [Fe3+_O2-] + O[(1s2)2(2s)2 2z

22x

2 )p()p( ] (7)

arsebobs monacemebi, romelTa mixedviTac oqsenoidi SeiZleba iyos

nawilaki 3+ ..Fe _ O

..

.. , romelic O_O bmis daSlisa da wylis molekulis

gamoTavisuflebis Sedegad warmoiqmneba. aseTi mtkiceba efuZneba daJan-

gul mdgomareobaSi myof citoqrom P450-is katalizur aqtivobas

iodozobenzolTan SeTanawyobaSi. am SemTxvevaSi oqsenoidi ar SeiZleba

71

Jangbadis or atoms Seicavdes, radgan iodozobenzoli mxolod Jangba-

dis erTi atomis donoria [246].

iseT modelur sistemebze Catarebuli cdebiT, romlebSic mahidroq-

silirebel nawilakad warmoiqmneboda an Jangbadis eqvseleqtroniani

atomi _ [N2O → O + N2] (oqsenoiduri meqanizmi) an OH+-ionebi _

(CF3COOH + H2O2). naCvenebia, rom maTgan arc erTi produqti ar miiRe-

ba iseTi TanafardobiT, rogorc mikrosomebis mahidroqsilirebel sis-

temaSi. CamoTvlili modelebi principSi hidroqsilirebis yvela SesaZ-

lo meqanizms (radikalurs, oqsenoidurs da eleqtrofilurs) moicaven.

amdenad miRebuli Sedegebi ori daskvnis gakeTebis saSualebas iZlevian:

1). yvela Seswavlili modeli ar asaxavs mikrosomuli hidroqsilirebis

meqanizms; 2). mikrosomebSi substratTa p-mdgomareobaSi hidroqsilireba

uzrunvelyofilia apofermentiT, an membranul lipidur fazaSi reaq-

ciis msvlelobis TaviseburebebiT. meore varaudis sisworis SemTxvevaSi

zeviT moyvanili meqanizmebidan erT-erTma mainc srulad unda asaxos

mikrosomuli hidroqsilirebis meqanizmi [126]. am arCevanis gasakeTeblad

sxvadasxva mkvlevarebi ramdenime meTodur xerxs iyeneben da maTgan yve-

laze efeqturi iudenfrendis [247-249] laboratoriaSi dadgenili

movlena aRmoCnda, romelic “NIH-Shift”-is saxelwodebiTaa cnobili.

misi azri imaSi mdgomareobs, rom substrati dasaJang atomTan Tu raime

Camnacvlebels (deiteriums, triTiums an halogens) Seicavs, maSin Cam-

nacvleblis nawili mikrosomebSi hidroqsilirebisas substrats ki ar

moscildeba, aramed mezobeli mdgomareobisaken gadainacvlebs. aseTi

retencia ki (molekulaSi Camnacvleblis Sekaveba) mxolod oqsenoiduri

da OH+-hidroqsilirebisTvisaa damaxasiaTebeli [250], da absoluturad

ar xdeba radikalebis Semcvel sistemaSi. am ukanasknels kidev imitom

ar SeiZleba hqondes adgili, rom fentonis sistemaSi ar registrirdeba

cikloheqsanis hidroqsilireba, maSin rodesac es substrati mikroso-

mebSi sakmaod maRali siCqariT iJangeba [238, 251]. radikaluri meqanizmi

gamoyenebulia O-dealkilirebisTvisac [238]. moyvanili monacemebi imis

dadasturebaa, rom Tavisufali radikalebisa da OH+-ionebis generire-

badi sistemebi srulad ver xsnian mikrosomuli hidroqsilirebis meqa-

nizms. safiqrebelia, rom Jangbadis aqtivacia midis misi oreleqtronia-

72

ni aRdgeniT. warmoqmnili kompleqsi O O

Fe2+

Seicavs labilur (O_O)

kavSirs, romlis gaxleCisas an uSualod pirdapir SeiZleba ganxor-

cieldes erT-erTi Jangbadis Canergva dasaJang substratSi (polarul-

mahidroqsilirebeli meqanizmi), an winaswar warmoiqmnas rkiniT stabi-

lizebuli Jangbadovani radikali da mxolod amis Semdeg ganxor-

cieldes es reaqcia (radikalur mahidroqsilirebeli meqanizmi). mod-

elur sistemebSi am tipis reaqciaTa Soris sxvaobebi jerac araa ga-

movlenili.

superoqsiduli radikalis (−•

2O ) warmoqmnis SesaZlebloba eqsperi-

mentulad Semowmda xsnad rekonstruirebul mikrosomuli hidroqsili-

rebis sistemaze [252, 253]. aRmoCnda, rom aseT sistemaSi hidroqsila-

zuri aqtivoba mkveTrad qveiTdeba eriTrokupreinis Tanamyofobisas, ro-

melic superoqsiduli radikalebis dismutacias iwvevs. amave dros su-

peroqsidebis generirebad ferments _ qsantinoqsidazas NADPH-

reduqtazis Secvlis unari aRmoaCnda. avtorebis mier Catarebuli am

gamokvleviT faqtiurad daixvewa Sexeduleba Jangbadis aqtiuri formis

bunebis Sesaxeb da igi superoqsid-anions warmoadgens. samwuxarod re-

konstruirebul sistemebSi mimdinare hidroqsilirebis reaqciebi, am

radikalis rolze miRebuli monacemebi membranuli sistemebisaTvis ar

iqna dadasturebuli.

amgvarad, arsebuli faqtebis safuZvelze SeiZleba davaskvnaT, rom

biologiur JangvaSi •HO -s da O·

-s aqtiuri formebis monawileoba

erTob saeWvoa maTi araseleqtiurobis gamo. •

2HO radikalic ar unda

iRebdes monooqsigenirebaSi monawileobas dabali reaqciisunarianobis

gamo. Jangbadis atomi O(3p) maRalaqtiuria, magram molekuluri

Jangbadidan misi pirdapiri generireba SeuZlebelia. savaraudod rCeba

is, rom substratebs Jangbadis atomebi gadaecema gardamavali mdgo-

mareobis nawilakebiT da aseTebs warmoadgenen oqsenoidebi, romlebic

Jangbadis aqtiuri formis stabilizebas axdenen. oqsenoiduri agentebis

bunebisa da Tvisebebis kvleva, agreTve oqsenoiduri meqanizmebiT moqmedi

modeluri sistemis Seqmna aris is erT-erTi mTavari mizani, romelic

Tavis aucilebel gadawyvetas elodeba, raTa sabolood gaixsnas

73

fermentuli sistemebiT molekuluri Jangbadis aqtivaciis meqanizmi.

mikrosomuli Jangvis IV stadiaSi savaraudod monawileoba SeuZlia

miiRos am sistemis ramdenime makromolekulurma biokomponentma. mxo-

lod qafuri_P450cam_O2 sammagi kompleqsis meore eleqtroniT aRdgenis

stadiaa siCqaris konstantebiT daxasiaTebuli [254, 255]. es procesi

efeqturad putidaredoqsins iyenebs da baqteriul sistemaSi qafuris

Jangvis malimitirebel safexurad iTvleba.

IV stadiis Semdeg sammag kompleqsSi O_O kavSiris daSla da

Jangbadis erTi atomis Semcveli mahidroqsilirebeli agentis (oqsenis)

warmoqmna xdeba. reaqcias Tan axlavs wylis gamoyofa. amis kvaldakval

damJangavi nawilaki substratSi xleCs C_H bmas, ris Sedegadac reaq-

ciis produqti (ROH) da regenerirebuli sawyisi katalizatori (Fe3+)

miiReba [256]. O_O kavSiris daSla citoqrom P450-is aqtiuri centris erT-erTi aminomJavis karboqsiluri jgufis monawileobiT mimdinareobs.

kuenis [256] azriT, sammagi kompleqsis karboqsilis jgufTan urTierT-

qmedebiT warmoqmnili zeJanguri mJavas struqtura keTilsasurvelad

moqmedebs O_O kavSiris daSlaze da mahidroqsilirebeli nawilakis

Camoyalibebaze. es mosazreba dasturdeba aromatul da alifatur

naxSirwyalbadebTan SedarebiT zeJangur mJavaTa maRali mahidroqsili-

rebeli unariT, romelic metalTa kompleqsebTan maTi koordinirebisas

izrdeba. SemTxveviTi araa, rom zeJanguri mJavebi monooqsigenazebis

moxerxebul models warmoadgenen da mikrosomul sistemaSi subst-

ratSi Jangbadis Canergvis aqtis imitacias axdenen [175, 243]. oqsenoidi

stabilizdeba citoqrom P450-is Tioluri ligandiT, romelic Tavise-

bur eleqtronebis rezervuars warmoadgens mahidroqsilirebeli nawi-

lakis CamoyalibebaSi da mis reaqciaSi:

S_FeV FeIV FeIIIO

_2 S_

O_

S_

FeIIIO_.

O S (8) mahidroqsilirebeli agentis warmoqmnaSi hemoproteinis aqtiuri

centris aminomJavis karboqsiluri jgufis SesaZlo monawileobis faqti

eqsperimentulad naCvenebia sligarisa da misi TanamS. [257] mier cito-

qrom P450cam-is magaliTze. mZime Jangbadis (18O2) gamoyenebiT maT daad-gines, rom P450cam_

18O2-is oqsigenirebul kompleqsSi dihidrolipois

mJava acilebs distalur (kidura) Jangbads da warmoqmnis Sualed ze-

74

Jangur naerTs, romelSic O_O bmis gaxleCa Zalze gaadvilebulia. amis

gamo 18O-is erTi atomi qafurSi, xolo meore dihidrolipois mJavas

karboqsilur jgufSi inergeba:

Fe O O Fe O O C RO

O C RO

FeO 3+ _

O2+ 2+_

RCOO_ =

+ (9)

maSasadame, acilzeJangis heterolituri daSlisas oqsenoidi Fe18O

da lipois mJava warmoiqmneba moniSnuli karboqsilis jgufiT

O18 C RO

.

aRniSnuli gardaqmnis alternativad kuunma da uaitma [256] ganix-

iles Sualedi zeJanguri naerTis homolituri daSlis SesaZlebloba,

romelic amiduri jgufis C NH2

O

monawileobiT xorcieldeba. am

SemTxvevaSi saWiroa daSvebul iqnas, rom substratSi C_H bma amiduri

radikalis NH C O

mxridan Setevas ganicdis.

zogierTi avtori [258] reaqciis aseT gzas nakleb albaTurad miiC-

nevs, radgan amiduri radikali SedarebiT mdgradia da fiziologiurTan

axlo myof temperaturaze citoqrom P450-is substratTa umetesobaSi mtkice C_H kavSiris darRveva ar SeuZlia. es avtorebi gamoricxaven

Sualedurad warmoqmnil acilze JangbadSic arsebuli O_O kavSiris

daSlis homolitur gzasac, radgan metalis kompleqsebisa da ionebis

monawileobiT am bmis homolizs Tan axlavs warmoqmnili acilperoqsi-

radikalis dekarboqsilireba [259]:

R C OOHO

R COOO

R C OOHO

R COO

. + +(n-1)+ M+n + H + M (10)

+ M+ . +(n-1)+ HO + M (11)

me-11 reaqciis Sedegad apofermentis mJava, romelic oqsigenirebul

75

citoqrom P450-Si Jangbadis kovalentur fiqsaciaSi monawileobs, Seuq-

cevadad daiwyebs xarjvas da katalizatori swrafad daiSleba.

amgvarad, srul mikrosomul sistemaSi oqsenoidis warmoqmnis gzas

yvelaze meti albaTobiT warmoadgens acetilzeJangebis homolituri

daSla. es naerTebi sammagi kompleqsis gaaqtivebul JangbadTan fermen-

tis aqtiuri centris erT-erTi aminomJavis karboqsilis jgufis ur-

TierTqmedebiT formirdeba. mniSvnelovania aRiniSnos, rom citoqrom

P450-iT koordinirebuli Jangbadi aramarto fiqsirdeba fermentis mat-

riciT, aramed kovalenturadac ukavSirdeba apofermentis erT-erT mJa-

vas, zeJanguri bunebis Sualedi naerTis warmoqmniT [260, 261].

procesis damamTavrebel stadiaze substratis molekulaSi C_H-bmis

gaxleCa xdeba. kuuni da misi TanamS. [256] Tvlian, rom es stadia radi-

kaluri meqanizmiT mimdinareobs. gamomdinare iqidan, rom oqsenoidis

struqtura da reaqciisunarianoba Fe O Fe O kvlavac ucnobia,

metelicam [170] dauSva, rom substratze Setevis oridan erTi meqanizmi

unda realizdebodes: pirveli _ oqsenoiduri, anu oreleqtroniani:

OHCC H FeO .... H

3++ 3+O.. (12)+ FeC

OFe3+

xolo meore _ farul-radikaluri, anu erTeleqtroniani:

OHCFeO ....H C. FeO

.... 3++ 3+O.. (13)+ FeC 3+O..

araswori iqneboda imis mtkiceba, rom reaqciis romelime erTi gza

universalurs warmoadgens. rodesac oqsenoidSi Jangbadis mdgomareoba

singleturia, maSin pirveli meqanizmi realizdeba, romlis mixedviTac,

kavSiris gawyvetis gareSe, Jangbadi C_H bmaSi inergeba. reaqciis aseTi gza yvelaze metad albaTuria aromatuli da ujeri naerTebis Jangvis

procesSi, radgan Zneli warmosadgenia radikaluri meqanizmiT ciklo-

heqsanisa da olefinebis oqsidebis warmoqmna. Tu oqsenoidi tripletur

mdgomareobaSi imyofeba, maSin substratSi Jangbadis pirdapiri Canergva

SeuZlebeli iqneboda, radgan aseTi reaqcia spin-akrZalulia. am SemTxve-

vaSi eleqtroniani farul-radikaluri meqanizmi amoqmeddeba.

mikrosomuli Jangvis malimitirebel stadias rac Seexeba, amTaviTve

76

unda aRiniSnos, rom erTi da igive substratisaTvis igi mikrosomebSi

da rekonstruirebul sistemebSi sxvadasxvaa. garda amisa, winaswarve

SeiZleba iTqvas, Tu romeli stadia ar SeiZleba iyos malimitirebeli.

daJanguli citoqrom P450-is urTierTqmedeba mraval substratTan ma-

Rali siCqariT mimdinareobs. ferment-substratis kompleqsis aRdgena

NADPH-citoqrom P450-reduqtaziT aseve swrafad xorcieldeba, da

amdenad, am stadias mTliani Jangvis procesis limitireba ar SeuZlia

[183, 259]. mesame stadia _ aRdgenili ferment-substratis kompleqsis

mier molekuluri Jangbadis mierTeba, siCqaris maRali konstantebiT

(103_106 M-1.wm-1) gamoirCeva. varaudoben, rom swored es stadia unda

alimitirebdes mTliani procesis siCqares. amasTan dakavSirebiT para-

lels avleben mikrosomuli da qafuris mJangav baqteriul mahid-

roqsilirebel sistemebs Soris, romelSic sammagi kompleqsis aRdgena

marTlac mTeli procesis malimitirebeli stadiaa. miuxedavad amisa,

aseTi analogia SeiZleba mcdari iyos, radgan baqteriul sistemaSi

kidev erTi damatebiTi cila _ putidaredoqsini monawileobs.

cxrili 1.2

RviZlis mikrosomebis rekonstruirebul sistemaSi benzfetaminis

N-demeTilirebis kinetikuri maxasiaTeblebi

procesis stadia kinetikuri maxasiaTeblebi

benzfetaminis mierTeba citoqrom P450-Tan K=50 wm-1 ferment_substratis kompleqsis aRdgena

pirveli eleqtroniT K2=4.12

ferment_substratis kompleqsTan Jangba-

dis mierTeba K3=(1.0_1.3).103

wm-1

sammagi kompleqsis daSla reaqciis pro-

duqtis warmoqmniT V≥2.3*

NADPH-is Jangva V=0.8_1.2* formaldehidis dagroveba V=0.5_0.7* * _ procesebis siCqaris ganzomilebaa nmoli.wm

-1.nmol-1 citoqrom P450-ze.

mikrosomul JangvaSi malimitirebeli stadiis dazustebaSi yvelaze

marTebulia am rTuli procesis calkeuli safexurebis siCqaris

77

gazomva da maTi Sepirispireba. aseTi samuSaoa Catarebuli satosa da

misi TanamS. [259] mier rekonstruirebul sistemebSi benzfetaminis

Jangvisas. cxrilSi 1.2 mocemulia procesis calkeuli stadiebis kine-

tikuri maxasiaTeblebi.

benzfetaminis Jangvis calkeuli stadiebis siCqareTa Sedarebam av-

torebi im daskvnamde miiyvana, rom malimitirebeli stadia SeiZleba

iyos an sammagi kompleqsis aRdgena meore eleqtroniT, an am aqtis Sem-

deg nebismieri, ramdenadac reaqciis produqtis dagroveba ufro naklebi

siCqariT xdeba, vidre sammagi kompleqsis daSla.

1.4 citoqrom P450-is mravlobiTi formebi da maTi Tanamedrove nomenklatura

bioqimiis zogadi principebidan gamomdinare, fermentebis subst-ratuli specifikuroba SeiZleba iyos absoluturi (erTi fermenti –

erTi substrati) an farTo (erTi fermenti – mravali substrati, rom-

lebsac gansazRvruli saerTo Tviseba gaaCniaT). fermentTa umrav-lesobisaTvis damaxasiaTebelia maRali specifikuroba. isini Cveulebriv

urTierTqmedeben mxolod erT substratTan da erT reaqcias akata-lizeben. arsebobs fermentebi, romlebic mkacrad gansazRvruli qimiuri

jgufebis Semcvel nivTierebebTan urTierTqmedeben. am TvalsazrisiT

citoqrom P450-is mravlobiTi formebi, rogorc fermentebi, bioqimiaSi

uprecendento movlenas warmoadgenen. citoqrom P450-is substratebis speqtri imdenad mravalferovania (maT Cven II da III TavebSi mimovixi-

lavT), rom Zneli warmosadgenia, amdeni sxvadasxvagvari qimiuri struq-

turebis naerTebis Jangva mxolod erTi izoformis mier kataliz-

debodes. amitom citoqrom P450-is kvlevis sawyisi etapebidanve warmo-

iSva mosazreba, rom es hemoproteini ramdenime izoformis saxiT

arsebobs. hipoTeza pirvel rigSi citoqrom P450-is induqtorebis mra-valgvarobas eyrdnoboda – im nivTierebebis saerTo raodenoba, rom-

lebic citoqrom P450-Semcveli monooqsigenazas induqcias iwveven,

ramdenime aseuls aRwevda [262]. es nivTierebebi gansxvavdeba rogorc

qimiuri bunebis, ise biologiuri moqmedebis mixedviT. induqtorebis

erTaderTi saerTo Tvisebaa lipofiluri buneba, rac Tavis mxriv,

78

citomembranebSi maT SerCeviTad SeRwevas da ujredis endoplazmur

badeSi dagrovebis unars ganapirobebs. es, Tavis mxriv, xels uwyobs

induqcias: rac ufro didxans imyofeba substrati organizmSi, miT

ufro xangrZlivia misi fermentTan kontaqti da, Sesabamisad, ufro

maRalia induqciis xarisxi. savaraudoa, rom induqciis movlena garemo

pirobebTan organizmis Seguebis erT-erT meqanizms warmoadgens, ramde-

nadac am dros izrdeba qsenobiotikebis metabolizmis siCqare da ufro

efeqturi xdeba maTi gauvnebelyofis (detoqsikaciis) procesi [263]. monooqsigenazuri sistemis molekuluri organizaciis zogadi prin-

cipebis ganxilvisas usaTuod wina planze gamodis sakiTxi imis Sesaxeb,

Tu ra simravliT arseboben da riT gansxvavdebian an emsgavsebian erT-maneTs citoqrom P450-is izoformebi. kvlevis sawyis stadiaze domini-rebda Sexeduleba, rom qsenobiotikTa Jangvis yvela reaqciaSi monawi-leobs Zlier farTo substratuli specifikurobis mqone erTi hemo-

proteini. SemdgomSi es Sexeduleba imis sasargeblod Seicvala, rom

arsebobs gamokveTili specifikurobis mqone citoqrom P450-is mrav-lobiTi formebi. mag., Tirkmelzeda jirkvlis mitoqondriebisa da mik-rosomebis fraqciebi Seicaven hemoproteinebs, romlebic sterolebs

sxvadasxva mdgomareobaSi Jangaven. mikrosomuli citoqrom P450 sterolis gverdiTi jaWvis C-21 mdgomareobas Jangavs; mitoqondriebi 2 hemoproteins Seicaven, romelTagan erTi qolesterols gverdiT jaWvs

acilebs C-20 da C-22 mdgomareobebSi, xolo meore – dezoqsi-kortikosterols Jangavs 11β- da 18-e mdgomareobebSi [264].

mravalwliani kvlevebis safuZvelze Camoyalibda zogadi xasiaTis

mqone debulebebi:

1. citoqrom P450 warmoadgens b-tipis CO-damkavSirebel hemoproteins,

romelic bunebaSi mravlobiTi formiT arsebobs;

2. citoqrom P450-is mravlobiTi formebi polipeptidur jaWvSi amino-

mJavaTa gansazRvruli TanmimdevrobiT xasiaTdebian, rac imaze miuTi-

Tebs, rom maTi biosinTezi sxvadasxva genebiT kontroldeba;

3. arsebobs iseTi substratebi, romelTa specifikuri metabolizmi ci-

toqrom P450-is gansazRvruli formebiT katalizdeba. amis parale-

lurad, substratTa umravlesobisaTvis fermentTa mkacrad dadgeni-

li specifikuroba ar arsebobs, Tumca hemoproteinis sxvadasxva

79

formebiT maTi gardaqmnis siCqareebi xSirad gansxvavebulia;

4. citoqrom P450-is izoformebs SeiZleba erTnairi molekuluri masebi da aRdgenil mdgomareobaSi CO-kompleqsis STanTqmis erTnairi maqsimumebi gaaCndeT, magram isini genetikurad sxvadasxva cilebs warmoadgendnen. yovelive aman citoqrom P450-is kvlevaSi axali nomenklaturis Se-

motanis aucileblobis sakiTxi daayena. genetikisa da molekuluri bi-

ologiis ganviTarebam pirobebi Seqmna naTeli gamxdariyo citoqrom

P450-is klasifikaciaSi arsebuli bundovaneba. problemis gadawyvetaSi

sawyis kriteriumebs sayovelTaod cnobili biologiuri kanonzomierebe-

bi warmoadgenen. rogorc cnobilia, genebi gansazRvraven ujredis re-

gulatoruli meqanizmebis arsebobas, xolo es meqanizmebi genSi kodire-

buli fermentiT realizdeba. ujredSi ganuwyvetlad mimdinareobs ram-

denime genis transkripcia, Tumca genomis nawili SeiZleba arc gamov-

lindes. faqtorebs, romlebic ganapirobeben ribosomebze fermentis sin-

Tezis siCqares transkripciis doneze rnm-is molekulebis degradaciis

siCqareebi alimitireben. amgvarad, aregulireben ra fermentebis biosin-

Tezs, genebi akontroleben gansazRvrul metabolur reaqciebs. es ga-

moixata mtkicebulebaSi: “erTi geni _ erTi fermenti”. aqedan gamomdi-

nare, citoqrom P450-is sxvadasxva izoformebis biosinTezi unda imyo-febodes genetikuri kontrolis qveS da es izoformebi erTmaneTisagan

mxolod aminomJavuri TanmimdevrobiT unda gansxvavdebodnen. maTi nawi-

li unda iyos konstituciuri, e.i. warmoiqmnebodes imis miuxedavad, Tu

ra pirobebSi imyofeba ujredi; nawili ki induqciuri, e.i. warmodgeni-

li unda iyos kvalis saxiT da gansazRvrul dones aRwevdes endogenu-

ri an egzogenuri substratebis Tanamyofobisas. citoqrom P450-is zo-gierTi izoformisaTvis SeimCneva e.w. imprintingis movlena, romlis ar-

si imaSi mdgomareobs, rom neonatalur periodSi induqtoris (fenobar-

bitalis) Seyvanisas cxovelis RviZlSi hemoproteinis Sesabamisi mniSv-

nelobebi SenarCunebulia sqesobrivi simwifis miRwevamde. am SemTxvevaSi

adgili aqvs fermentis Seuqcevad induqcias [265]. unda aRiniSnos, rom

konstituciuri fermentebis izoformebis nawils axasiaTebs induqciuri

bunebac, nawils ki ara. amitom citoqrom P450-is izoformebisaTvis

damaxasiaTebeli Taviseburebaa genTa eqspresia. calkeuli genebis trans-

kripciuli aqtivoba iwyeba organizmis ganviTarebis sxvadasxva sta-

80

diebze da damokidebulia cxovelis sqesze, saxeobasa da qsovilis

tipze [266]. zogierTi genis transkripciisaTvis aRmoCenilia fenobar-

bital-mgrZnobiare promotori, romelic Seicavs dadebiT da uaryofiT

maregulirebel elementebs [267]. maT SeiZleba warmoadgendes specifi-

kuri cila, romelic fosforileburi formiT moqmedebs, rogorc pro-

motoris dadebiTi, xolo defosforileburi formiT _ rogorc uaryo-

fiTi elementi. dadgenilia, rom glukokortikoidebs, zrdis, sqesobriv

da Tireoidul hormonebs genebis eqspresiis mniSvnelovani modulirebis

unari gaaCniaT [268].

sadReisod genebis raodenoba, romlebSic citoqrom P450-is izo-

formebia kodirebuli, 500-mdea. isini eukariotebis 85 saxeobaSi (xerxem-lianebi, uxerxemloebi, mcenareebi, sokoebi) da 20 saxeobis prokariotebSia aRmoCenili (ix. cxrili 1.3) [263]. citoqrom P450-is izofermentebis kla-

sifikaciisaTvis gaiTvaliswines fermentis aminomJavuri Sedgenilobis msgavsebis principi da genebis saerTo warmomavlobis kanonzomierebebi.

cxrili 1.3

sxvadasxva taqsonomiuri jgufebis organizmebis citoqrom P450-is ojaxebis saerTo daxasiaTeba [263]

CYP 1

xerxemlianebSi: inducirdeba dioqsiniT; mona-

wileobs policikluri naxSirwyalbadebis, he-

terocikluri naerTebis, aromatuli aminebis

metabolizmSi

CYP 2 xerxemlianebSi da uxerxemloebSi: monawi-

leobs wamlebis da garemos dambinZureblebis

metabolizmSi

CYP 3 xerxemlianebSi: monawileobs wamlebis da gare-

mos dambinZureblebis metabolizmSi

CYP 4 xerxemlianebSi: monawileobs cximovani mJavebis

hidroqsilirebaSi; uxerxemloebSi: funqcia

jerjerobiT ucnobia

CYP 5 xerxemlianebSi: tromboqsansinTaza;

mwerebSi: monawileobs mcenareuli warmoSobis

naerTebis da pesticidebis metabolizmSi

CYP 6 mwerebSi: monawileobs mcenareuli warmoSobis

81

naerTebis da pesticidebis metabolizmSi

CYP 7A xerxemlianebSi: qolesterin 7a-hidroqsilaza

CYP 7B xerxemlianebSi: funqcia jerjerobiT ucnobia

CYP 8 xerxemlianebSi: prostaciklinsinTaza

CYP 9 mwerebSi: funqcia jerjerobiT ucnobia

CYP 10 moluskebi: mitoqondriuli fermentebi

CYP 11 xerxemlianebSi: qolesterinis gverdiTi jaWvis

Jangva, steroid 11-b-hidroqsilaza da aldos-

teron sinTaza (mitoqondriebSi)

CYP 12 mwerebSi: mitoqondriuli fermentebi

CYP 13 nematodebSi

CYP 14 nematodebSi

CYP 15 nematodebSi

CYP 16 nematodebSi

CYP 17 xerxemlianebSi: steroid 7a-hidroqsilaza

CYP 18 mwerebSi

CYP 19 xerxemlianebSi: androgenebis aromatizacia

CYP 21 xerxemlianebSi: steroid 21-hidroqsilaza

CYP 24 xerxemlianebSi: steroid 24-hidroqsilaza (mi-

toqondriebSi)

CYP 27 xerxemlianebSi: steroid 27-hidroqsilaza (mi-

toqondriebSi)

CYP 51 cxovelebi, mcenareebi, safuvrebi, sokoebi:

sterolis biosinTezi

CYP 52 safuvrebi: alkanebis hidroqsilireba

CYP 53 – CYP 62 sokoebi

CYP 71 – CYP 92 mcenareebi

CYP 73 mcenareebi: dariCinmJava 4-hidroqsilaza

CYP 101 – CYP 118 baqteriebi

82

amJamad arsebobs citoqrom P450-ebis genebis 120 ojaxi, romel-

Tagan 11-s yvela ZuZumwovris dnm-Si vxvdebiT (ix. cxrili 1.3).

(am monacemTa bazis ganaxleba mudmivad xdeba da maTi gacnoba inter-

netis saSualebiT SeiZleba [272-274]) mag., „ojaxi 1“ _ policikluri

aromatuli naerTebiT, maT Soris 3-meTilqolantreniT inducirebad

citoqrom P450-ebs moicavs, xolo „ojaxi 3“-is warmomadgenlebi ste-

rolebiT inducirdeba. „ojaxi 2“ aerTianebs ramdenime qveojaxs, mag.,

„qveojax 2B“-Si fenobarbitaliT inducirebuli hemoproteinebia, xolo

„qveojax 2E“-s izoformebisaTvis induqtori eTanolia. zogjer, moxer-

xebulobisaTvis, ojaxisTvis iseTi nomeria SerCeuli, rom igi fermen-

tis specifikur aqtivobas ukavSirdeba. mag., „ojaxi 11“ Seicavs

citoqrom P450-ebs, romlebsac steroid-11-β-hidroqsilazuri aqtivoba

gaaCniaT. asevea danomrili „ojaxi 17“ (steroid-17-α-hidroqsilaza),

„ojaxi 21“ (steroid 21-hidroqsilaza) da sxv. Tumca, rogorc aRvniS-

neT, ojaxebsa da qveojaxebSi izoformebi erTiandeba ara substratuli

specifikurobis, aramed genetikuri homologiurobis xarisxis mixedviT.

unda aRiniSnos, rom citoqrom P450-is yvela mkvlevari sixaruliT

ar Sexvedria axal klasifikacias da aRniSnaven, rom igi mosaxmarad

arc ise martivia, rogorc erTi SexedviT Cans. pirvel rigSi, mouxer-xebelia, rom zogierT gens ukve miniWebuli hqonda trivialuri saxel-wodeba da mxolod Semdeg dadginda maTi roli citoqrom P450-is kodirebaSi. mag., geni Eig17-1, romlebic gvxvdeba mwerebSi (Drosophila melanogaster) da mcenareebSi (Arabidopsis thaliana) warmoadgens gens

Cyp18, e.i. gamodis, rom Eig17-1 da Cyp18 oficialuri sinonimebia, rac klasifikaciisaTvis miuRebelia. aseve xSiria gaugebroba publikaciebSi,

radgan bevri mkvlevari kvlav hemoproteinebis Zvel nomenklaturas

iyenebs, romelic citoqrom P450-is izoformebis saxeobriv Tavisebu-rebebzea dafuZnebuli: magaliTad ixmareba bocvris citoqrom P450olf 1-is nacvlad gamoyenebul unda iqnas CYP2C16, virTagvis citoqrom

P450II C16-is nacvlad _ CYP2C16. steroidhidroqsilazebSi citoq-romebi Р450c11b1, Р450cb1, Р450arom unda dasaxeldnen, rogorc

CYP11B1, CYP11B2 da CYP17. molekuluri biologiis Tanamedrove meTodebi genetikuri elemen-

tebiT manipulaciis saSualebas iZleva. erT-erTi aseTi meTodia in vitro

83

pirobebSi konstruirebuli dnm-is rekombinirebuli molekulebis klo-

nirebiT genebis Canergva. am dros miRebuli dnm-is hibriduli moleku-

la SemdgomSi SeiZleba klonirebul iqnas in vivo pirobebSi. axali

meTodologia (PCR _ Polymerase chain reaction _polimerazuli jaWvuri

reaqcia) saSualebas iZleva didi raodenobiT gamoiyos dnm-is indivi-

dualuri fragmentebi (genebi) nebismieri wyarodan. swored aseTi meTo-

debiT iqna miRebuli citoqrom P450-is iseTi izoformebi, romlebic

bunebrivi izoformebisagan aminomJavuri TanmimdevrobiT gansxvavdebian.

aseT SemTxvevaSi rekomendirebulia Semdegi nomenklaturis gamoyeneba:

jer unda mieTiTos citoqrom P450-is bunebrivi izoforma, Semdeg

abreviatura X###Y, romelic gviCvenebs, rom cilis ### ubanSi X

aminomJava Secvlilia Y-iT. magaliTad, aRniSvna CYP1A1I426V gviCve-nebs, rom CYP1A1 izoformaSi izoleicini (aRiniSneba I-T) 426-e mdgo-mareobaSi Secvlilia valiniT (aRiniSneba V-Ti).

citoqrom P450-is izoformebze mravalmxrivi informaciis dagro-

vebam xeli Seuwyo Seqmniliyo filogenetikuri xe, romelic aRwerda am

cilebis evolucias. cnobilia, rom filogenetikurad dacilebuli saxe-

obebis erTi da igive tipis fermentebs saerTo struqturuli fragmen-

tebi gaaCniaT, Tumca mniSvnelovnad gansxvavdebian aminomJavuri Tanmim-

devrobiT. aseT cilebs, rogorc wesi, aqtiuri centris ubanSi erT-

nairi aminomJavuri Tanmimdevroba aqvT. rogorc Cans, am ubanSi

nebismieri cvlileba arRvevs steriul Sesabamisobas substratTan, an

iwvevs im prosTetuli jgufis dakargvas, romelic katalizur

procesSi monawileobs. aqedan gamomdinare, aminomJavuri Tanmimdevrobis

divergenciis mixedviT SesaZlebelia kodonebSi momxdari mutaciebis

raodenobis gamoTvla da amiT advilad fasdeba modificirebuli niSan-

Tvisebebis memkvidrulad gadacema. rogorc Cans, mutaciis tempi,

romelic aminomJavur TanmimdevrobaSi mJRavndeba, droSi cvalebadia da

gansxvavebulia sxvadasxva ojaxebisaTvis [263]. arsebobs ramdenime sqema, romlebic aCveneben naTesaur kavSirebs ci-

toqrom P450-is calkeul ojaxebs Soris. [275-278]. maTgan yvelaze

farTod aRiarebul sqemaSi [277] ZiriTadi ojaxebi da calkeuli izo-

formebi gayofilia rva (I_VIII) jgufad, romlebic TanmimdevrobiTaa

erTmaneTTan dakavSirebuli. am kavSiris safuZvelia polipeptiduri

84

jaWvebis homologiuroba. I jgufs (5 geni) miekuTvneba 4 hemoproteini,

romlebic gvxvdeba xerxemliani cxovelebis organizmebSi da 1 _ mwe-

rebSi (CYP18). II jgufi warmodgenilia 13 genisgan Semdgari klasteris

saxiT da mxolod mcenareebSia gavrcelebuli. III jgufis (6 geni) hemo-

proteinebi damaxasiaTebelia uxerxemloebisaTvis. IV jgufs (5 geni)

miekuTvneba citoqrom P450-ebi, romlebic prokariotebsa da eukari-

otebSi cximovani mJavebis Jangvas akatalizeben. V jgufis 7 genSi ko-

direbulia mitoqondriuli citoqrom P450-ebi, romelTa ZiriTad

funqciasac steroidebis Jangva warmoadgens. VI jgufi aerTianebs mce-

nareuli hemoproteinis 4 gens. VII jgufis 6 geni miekuTvneba sokoebs,

xolo VIII jgufSi (7 geni) Tavmoyrilia sxvadasxva taqsonomiuri

jgufebis warmomadgeneli organizmebis citoqrom P450-ebi. sainteresoa, rom am ojaxis warmomadgenlis CYP19 geni, romelic ZuZumwovrebSi

gvxvdeba da akodirebs aromatazas (aromatuli birTvis mJangav citorom

P450-s), mcenare Arabidopsis thaliana-s (wiwmaturas) CYP86A1-is genis

homologiuria. taqsonomiurad gansxvavebuli organizmebis citoqrom

P450-ebis homologiurobis kargi magaliTia soko Neurospora-s CYP54, romelic akodirebs steroid 14-demeTilazas da gavrcelebulia cxove-

lebSic, mcenareebSic, sokoebsa da safuvrebSic. cxadia, rom baqterii-

dan adamianamde citoqrom P450-is genebs erTi saerTo winapari hyavT. unda aRiniSnos, rom citoqrom P450-is genebis aseTi filoge

unda aRiniSnos, rom citoqrom P450-is genebis aseTi filogenetiku-

ri ganlagebisas adgili ar moiZebna baqteria Pseudomonas putida-s CYP101 hemoproteinisaTvis, arada swored es citoqrom P450 iqna pir-velad gamoyofili kristaluri saxiT da citoqrom P450-is katalizu-

ri Tvisebebi swored am fermentzea pirvelad Seswavlili. garda amisa

am filogenetikur sqemaSi verafriT moiZebna adgili NO-sinTazasTvis,

romelic Tavisi katalizuri aqtivobiT citoqrom P450-is analogiuria.

NO-sinTazas aminomJavuri Tanmimdevrobis kompiuterulma analizma aCve-

na, rom es fermenti ar aris citoqrom P450-is superojaxis wevri. aRmoCnda, rom igi C-bolos domenis Sedgenilobis mixedviT _ cito-

qrom c reduqtazas ojaxs “enaTesaveba”. SesaZloa, rom funqciisa da

Sedgenilobis aseTi Seusabamoba evoluciis procesSi momxdari diver-

genciis Sedegia.

85

moyvanili faqtebi imaze migvaniSneben, rom baqteriebis, mcenareebisa

da cxovelebis divergenciisas 2 miliardi wlis win momxdarma evolu-

ciurma cvlilebebma citoqrom P450-is ZiriTad struqturul-funqciur

Taviseburebebs mniSvnelovani kvali ar daamCnia. es is etapia, rodesac

evoluciis erT-erTi yvalze ufro kritikuli _ efeqturi aerobuli

metabolizmis Camoyalibebis stadia mimdinareobda, rasac mitoqondrie-

bis warmoqmna mohyva [263]. qromosomebSi citoqrom P450-is genebis ganlagebis Seswavlam aCvena,

rom zogjer izoformebis makodirebeli genebi erTsa da imave qromoso-

maSia lokalizebuli. magaliTad, CYP1A1 da CYP1A2 adamianis me-15

qromosomaSi erTmaneTis axlos, erT klasterSia “Cawerili”. aseT SemT-

xvevaSi saxelwodebis miniWebisas rekomendebulia miTiTebul iqnas, ro-

mel ojaxs ekuTnis es klasteri, e.i. am SemTvevaSi klasteris saxel-

wodeba iqneba “CYP1A klasteri”, xolo genis adgilmdebareobis miniS-

nebisaTvis gamoyenebuli unda iyos molekulur biologiaSi miRebuli

aRniSvnebi, magaliTad, CYP1A 15q22-qter (MPI) [279]. klasterebis warmoqmnas mkvlevarebi dnm-s duplikaciis Sedegad dip-

loiduri ujredebis warmoqmnas ukavSireben, racr400 milioni wlis gan-

mavlobaSi xdeboda[277]. aseTi duplikaciis Sedegad diploidur uj-

redebSi TiToeuli fermentisaTvis oris nacvlad oTxi genia. Semdeg ge-

neraciebSi genTa TiToeuli wyvili erTmaneTisgan damoukideblad SeiZ-

leba icvlebodes: erTi wyvili darCes ucvleli, meorem ki mutacia ganica-dos. amis Sedegad meore wyvili igive funqciis ferments daasinTezebs, rasac pirveli, magram gansxvaveba specifikur aqtivobaSi gamovlindeba.

klasikuri genetikuri kvlevebiT dadginda, rom SesaZlebelia adgili

hqondes genTa translokacias _ qromosomis erTi nawilis sxva qromo-

somaSi gadatanas. cxadia, evoluciur procesSi citoqrom P450-is genTa duplikaciis dros aseT translokaciasac eqneboda adgili. aseTi tra-

nslokaciis magaliTad asaxeleben adamianis genebs, romlebic citoqrom

P450-is erT ojaxs qmnian: СYP2B (19g13.1-13.2), CYP2C (19g12-g13.2) da CYP2D (10g24.1-24.3). aseTivea citoqrom P450-is yvelaze “Zveli” geni

СYP51, romelic warmodgenilia Sesabamisi klasteriT adamianis me-7

qromosomaSi da aseve ori fsevdogeniT me-3 da me-13 qromosomebSi.

citoqrom P450-is izoformebs saxeobrivi specifikurobac axasia-

86

TebT. magaliTad, Р4501В1 adamianSi pasuxismgebelia izoformaze, ro-

melic eToqsirezorcinis O-demeTilirebas akatalizebs, magram Tagvebsa

da virTagvebSi es izoforma aRniSnuli reaqciis mimarT absoluturad

araaqtiuria [280].

citoqrom P450-is izoformebis mravlobiToba inhibitorebis mraval-

ferovnebasac ganapirobebs. amJamad citoqrom P450-is inhibitorebi, moq-medebis meqanizmis mixedviT, 4 jgufad iyofa [281]:

a jgufis inhibitorebi moqmedeben hemTan kompleqswarmoqmnis doneze,

kerZod, aseTia CO, romelic specifikurad ukavSirdeba hems, amitom igi

yvela izoformisaTvis universaluria;

b jgufis inhibitorebi SKF-525A (β-dieTilaminoeTildifenilpro-

pilacetati), piperonil buToqsidi, meTiraponi da sxvebi moqmedeben

reaqciis meqanizmis doneze da substratis msgavsad ukavSirdebian aq-

tiur centrs, magram ar gardaiqmnebian. am jgufis inhibitorebis moqme-

debis efeqti xanmoklea;

c jgufis inhibitorebi moqmedeben hemis biosinTezis doneze. mag.,

porfobilinogeni Trgunavs feroxelatazas (hemis sinTetazas); glicini

da suqcinil CoA erTad ablokireben ferment δ-aminolevulinmJava-

sinTetazas da a.S. metad mravalmxrivi efeqti aqvs am jgufis warmomad-

genel kidev erT inhibitors – kobaltis (II) ions. Co2+ ainhibirebs δ-

aminolevulinmJava-sinTetazas da feroxelatazas. igi xels uSlis hemis

normalur asocirebas apocitoqrom P450-Tan da amasTanave astimuli-

rebs hemis degradaciaSi monawile ferments – hemoqsigenazas;

d jgufis inhibitorebi warmoadgenen agentebs, romlebic hemis de-

struqciis doneze moqmedeben. mag., alilizipropilacetamidi reagirebs

citoqrom P450-Tan da warmoqmnis epoqsids, romelic hemis destruq-

cias iwvevs, aseTive efeqti gaaCnia oTxqlorian naxSirbadsac, romelic

citoqrom P450-isaTvis „gamanadgurebeli“ substratia, radgan hemisaT-

vis „saSiS“ triqlormeTilis Tavisufal radikals generirebs. hemis

destruqtoria agreTve zogierTi mZime metalis ionic, kerZod, Cd2+.

qsenobioqimiisaTvis sainteresoa is izoformebi, romlebic ucxo

naerTTa JangvaSi monawileoben. pirvel rigSi, aseTi izoformaa

P4501А1, romelic policikluri aromatuli naxSirwyalbadebis kon-

formaciulad daxuruli ubnebis hidroqsilirebas axorcielebs. am

87

reaqciis Sedegad arenebis iseTi epoqsidebi warmoiqmneba, romlebic

epoqsihidrolazebis saSualebiT aRar gardaiqmnebian Sesabamis diolebad.

amis gamo reaqciisunariani metabolitebi Semdgom gardaqmnebs aRar ga-

nicdian da iqmneba imis albaToba, rom isini kovalenturad daukavSird-

nen cilebsa da nukleinis mJavebs. yovelive es ganapirobebs aseTi meta-

bolitebis citotoqsikur, mutagenur da kancerogenul moqmedebas

[282]. analogiuri moqmedebis metabolitebi warmoiqmneba Р4501А2-is mier policikluri aromatuli aminebis Jangvis Sedegadac. rac Seexeba izo-

formas Р4501В1, substratuli specifikurobiT is axloa Р4501А1-Tan [280]. citotoqsikuri naerTebis formireba xdeba citoqrom Р4502Е1-is mier benzolis, stirolis, oTxqloriani naxSirbadis, qloroformis,

diqlormeTanis, qlorvinilis, eTilkarbamatis da akrilonitrilis

Jangvis Sedegadac [283]. sainteresoa, rom inhalaciuri anesTetiki (ma-

galiTad, narkozisaTvis gamoyenebuli qloroformi) swored am izo-

formis saSualebiT CasunTqvidan 3 wamSi hepatotoqsikuri metabolite-

bi warmoiqmneba [284]. citoqrom Р4502Е1-s ukavSireben eTanolis Jangvis

katalizebasac, ramdenadac es izoforma alkoholdehidrogenazas msgav-

sad eTanolidan acetaldehids warmoqmnis.

mcenareebSi yvelaze ukeT Seswavlilia CYP76B1, romelic klasiku-

ri fermentTa klasifikaciis mixedviT dariCinmJava-4-hidroqsilazas (EC 1.14.14.1) warmoadgens. es citoqrom P450 aqtiurad akatalizebs mravali

toqsikuri naerTis, maT Soris sxvadasxva qimiuri bunebis herbicidebis

metabolur gardaqmnebs. saerTod, mcenareebSi aRmoCenilia 120-mde dnm-

is Tanmimdevroba, romlebSic kodirebulia citoqrom P450 [271, 285-287]. es mcenareebia: xorbali (Triticum vulgare), avokado (Percea anerica-na), badrijani (Solanium melongela), katapitna (Nepeta racemosa), gvelis

suro (Catharanthus roseus), pitna (Mentha piperita), quTquTa (Thlaspi ar-vense), wiwmati (Arabidopsis thaliana), simindi (Zea mays), miwavaSla (Hel-lianthus tuberosus), maSa-lobio (Vigna radiata), ionja (Medicago sativa), mzesumzira (Helianthus annuus), barda (Pisum sativum), seli (Linum usita-tissimum), gvaiula (Parthenium argentatum), petunia (Petunia hybrida), jad-vari (Phalaenopsis sp. hibridi SM9108), sorgo (Sorghum bicolor), kowax-uri (Berberis stolonifera), mindvris mdogvi (Brassica campestris), cercvi (Vi-cia faba), Tambaqo (Nicotiana tabacum), soia (Glycine Max) da sxv.

88

1.4.1 citoqrom P450-is mravlobiTi formebis induqcia

sadReisod miRebulia, rom citoqrom P450-is mravlobiTi formebis

induqcia sami modelis mixedviT xdeba [288]:

I – benzpirenis modeli _ aseT induqcias iwveven 3-meTilqolantrenis

tipis induqtorebi: romlebSic Sedis TviT 3-meTilqolantreni, benzpi-

reni, benzantraceni, 2,3,7,8-tetraqlordibenzo-p-dioqsini, tetraqlor-

benzoli, β-naftoflavoni da sxv.;

II – fenobarbitalis modeli _ am jgufs miekuTvneba: TviTon feno-

barbitali, barbitali, Tiopentali, aldrini, dieldrini, poliqlori-

rebuli bifenilebi, reptaqlori da sxv.;

III _ qlofibratis modeli _ am tipis induqtorebia: safroli,

izosafroli, pregnenoloni, 16α-karbonitrili, aroqlori, heqsaqlor-

benzoli da sxv.

mikrosomul fermentTa induqtorebad yvelaze xSirad fenobarbitali

da sxva barbituratebi gamoiyeneba. maTi efeqti xasiaTdeba endo-

plazmuri membranebis raodenobisa da cilebSi aminomJavebis CarTvis

siCqaris zrdiT. es efeqti gansakuTrebiT mkveTrad gluv membranebSia

gamosaxuli [289-291]. fenobarbitaliT cxovelis winaswari damuSavebis

Semdeg miRebul mikrosomul fraqciaSi cilis Semcveloba TiTqmis

gasammagebulia. aseTi mikrosomebisaTvis damaxasiaTebelia agreTve mrava-

li qsenobiotikis, kerZod, benzfetaminisa da aminopirinis N-demeTi-lirebis reaqciaTa gaZliereba. garda amisa, adgili aqvs citoqrom

P450-is raodenobis mniSvnelovan matebas da igi cxovelSi feno-

barbitalis Seyvanis sixSireze, cxovelis saxeobaze, sqesze da sxva

damatebiT faqtorebzea damokidebuli. dadgenilia isic, rom subst-

ratTa metabolizmis specifikurobas mxolod citoqrom P450, da ara monooqsigenazuri sistemis sxva romelime komponenti gansazRvravs [289].

mikrosomuli monooqsigenazuri sistemis mZlavri stimulatoria 3-

meTilqolantreni [290]. in vivo pirobebSi gamoyenebisas citologiuri

maxasiaTeblebis cvlilebebiT da zogierTi qsenobiotikis metabolizmis

specifikiT es induqtori fenobarbitalisagan arsebiTad gansxvavdeba. 3-

89

meTilqolantreniT inducirdeba hemoproteini, romelsac CO-aRdgenil

mdgomareobaSi STanTqmis maqsimumi speqtris mokletalRiani olqisaken

(448 nm-ze) odnav win aqvs gadaweuli. Tavdapirvelad varaudobdnen,

rom speqtris aseTi cvlileba citoqrom P450-isa da 3-meTilqolan-

trenis urTierTqmedebas unda gamoewvia. SemdgomSi es varaudi ar da-

dasturda da am individualurma hemoproteinma citoqrom P448-is sax-elwodeba miiRo. 3-meTilqolantreniT inducirebuli citoqrom P448-is metaboluri aqtivobis yvelaze damaxasiaTebel Tvisebas benzpirenis hi-

droqsilirebis siCqaris mkveTri zrda warmoadgens. rac Seexeba β-naf-toflavons [291], SeiZleba dabejiTebiT iTqvas, rom mocemuli qsenobi-

otiki induqciis iseTive efeqts iZleva, rogorsac 3-meTilqolantreni,

xolo poliqlorirebuli bifenilebis warmomadgeneli aroklor 1254-is

induqciuri efeqti fenobarbitalisa da 3-meTilqolantrenis erTd-

rouli moqmedebiT aRZruli efeqtis identuria [292]. meTilqolantre-

nuli tipis induqtorebia sxva policikluri aromatuli naxSirwyalba-

debic. analogiuri unari aRmoaCndaT poliqlorirebul bifenilebs da

zogierT qlorirebul dibenzodioqsins [293]. gamokvlevebma aCvenes, rom

am nivTierebaTa induqciis efeqts molekulaSi qloris atomis mdgoma-

reoba gansazRvravs. meTilqolantrenuli tipis Zlieri induqtoria

2,3,7,8-tetraqlordibenzo-p-dioqsini. meTilqolantrenuli efeqtis mi-

saRebad sakmarisia am qsenobiotikis 4 rigiT (10 000-jer) ufro nak-

lebi koncentracia.

izosafroliT inducirdeba individualuri hemoproteini, romelsac

eleqtroforezuli ZvradobiT Sualeduri mdgomareoba ukavia fenobar-

bitaliTa da meTilqolantreniT inducirebul hemoproteinebs Soris.

mas citoqrom P450d ewoda [291]. am hemoproteinis ganmasxvavebel Tvi-

sebad is iTvleba, rom gamoiyofa izosafrolis metabolizmis produq-

tebTan kompleqsSi. igi akatalizebs cikloheqsanis, eTilbenzolisa da

p-nitroanizolis JangviT gardaqmnebs.

zogierTi sinTezuri steroli in vivo zemoqmedebisas astimulirebs

mikrosomul reaqciebs da zrdis citoqrom P450-is raodenobas. pregne-nolon-16α-karbonitriliT induqcia ar miekuTvneba arc fenobarbita-

lur da arc meTilqolantrenul tips. speqtrulad gansazRvruli hemo-

proteini fenobarbitaliT inducirebulis identuri aRmoCnda, magram

90

misgan gansxvavebulad aaqtiurebda benzpirenis hidroqsilirebisa da

eTilmorfinis N-demeTilirebis reaqciebs [291]. maSasadame, sarwmunod SeiZleba iTqvas, rom mikrosomebSi fermentul

aqtivobaTa induqciis saerTo suraTi, romelic mravali qsenobiotikis

zemoqmedebis Sedegia, upirveles yovlisa, citoqrom P450-is gansxvave-buli formebis biosinTezze daiyvaneba.

rekonstruirebul sistemebSi ama Tu im qsenobiotikis metabolizmis

Seswavlisas sakiTxi ar dgas imis Sesaxeb, Tu konkretulad citoqrom

P450-is romeli forma axorcielebs mas, radgan es piroba winaswar

TviT eqsperimentatoris mieraa dazustebuli. organos an organizmis

doneze mTliani mikrosomebis kvlevisas ki sakiTxi gacilebiT rTula-

daa, radgan metabolur reaqciebSi citoqrom P450-is ramdenime izo-

formas SeuZlia erTdroulad CarTva da mocemuli qsenobiotikis Jang-

viT degradaciaSi TiToeuli am formis Tanamonawileobis donis dadge-

nas uaRresad didi mniSvneloba eniWeba.

molekuluri masebis zrdisa da Sesabamisad, SDS-eleqtroforezuli

Zvradobis Semcirebis mixedviT hemoproteinis yvela cnobil izoformas

(maT aRniSnaven, rogorc LM) aseTi saxiT alageben [291]: 1. konstituciuri qvefraqcia – citoqrom P450LM1; 2. fenobarbitaliT inducirebuli qvefraqcia – citoqrom P450LM2; 3. konstituciuri qvefraqcia – citoqrom P450LM3; 4. 3-meTilqolantreniT an β-naftoflavoniT inducirebuli qvefraqcia

– citoqrom P450LM4; 5. 2,3,7,8-tetraqlordibenzo-p-dioqsiniT inducirebuli qvefraqcia –

citoqrom P450LM6. hemoproteinis CamoTvlili izoformebi speqtruli da sxva maxasia-

TeblebiT Zlier gansxvavdebian erTmaneTisagan. mag., citoqrom P450LM2-

isagan P450LM4 imiT gansxvavdeba, rom igi ar ikavSirebs mikrosomuli

Jangvis iseT tipiur substratebs, rogorebic benzoli, cikloheqsani an

antipirinia [294]. zogierTi qsenobiotiki (anilini, naftalini, dimeTi-

lanilini) citoqrom P450LM4-s advilad ukavSirdeba, magram am izo-

formiT praqtikulad ar iJangeba. am faqts xsnian citoqrom P450LM4-is

kompleqsTa ori dabalspinuri formis arsebobiT, romlebiTac ligande-

bis (qsenobiotikebis) intensiuri dakavSireba mimdinareobs, magram Jang-

91

vis procesis TvalsazrisiT es ligandebi substratebs ar warmoadgenen.

anilini, piridini, imidazoli da, SesaZloa, sxva organuli azotSemc-

veli fuZeebi warmoqmnian dabalspinur kompleqsebs, romlebTanac ukve

SesaZlebelia molekuluri Jangbadis, an organuli hidrozeJangis dakav-

Sireba, raTa CamoTvlili substratebis (ligandebis) Jangva ganxor-

cieldes. naftalins da dimeTilanilins SeuZliaT citoqrom P450LM4-

Tan urTierTqmedeba da maRalspinuri kompleqsis warmoqmna, romelSic

ligandebi porfirinuli birTvis sibrtyis gaswvrivaa ganlagebuli.

miuxedavad amisa, maTi Jangva ar xdeba, radgan I-tipis substratTa dama-

kavSirebeli ubnebi induqtoriT an misi metabolitebiTaa dakavebuli.

citoqromebis – P450LM2-isa da P450LM4-is konformaciuli gansxvavebe-

bi wriuli diqroizmis meTodiTaa dadgenili. aRmoCnda, rom P450LM4-is

formis hemi ufro mWidro hidrofobuli „jibiTaa“ garemoculi. hemo-

proteiniT aRniSnul formaTa struqturuli gansxvavebebi Sesabamisad

did sxvaobebs iwveven maT substratul specifikurobaSi, katalizur

aqtivobasa da imunoqimiur TvisebebSi.

induqciis dros citoqrom P450-is formireba warimarTeba cilis

sinTezisa da misi “momwifebis”, hemis warmoqmnisa da apofermentTan mi-

si dakavSirebis gziT [295]. dadgenilia, rom qsenobiotik-induqtoris

moqmedebiT represoruli cila genis represorul ubanSi gadainacvlebs,

rac cilis sinTezs inicirebs. citoqrom P450-is apofermentis sinTezis es momenti yvela organizmisaTvis saerToa, Tumca meqanizmis calkeul

detalebs Soris mniSvnelovani gansxvavebebia [296]. rac Seexeba hemis

sinTezs, cxovelebis, safuvrebisa da baqteriebisagan gansxvavebiT,

umaRles mcenareebSi es procesi mxolod glutamat-damokidebuli gziT

mimdinareobs da ara 5-alanin-sinTazas saSualebiT [297].

92

1.5 mikrosomuli fosfolipidi _ mahidroqsilirebeli

kompleqsis aucilebeli komponenti

rTul membranadakavSirebul fermentul sistemaTa specifikuri moq-

medebis regulaciaSi uaRresad didi roli eniWeba biologiuri

membranebis fosfolipidur komponentebs. karga xania, rac Cveni war-

modgenebi gascda im arsebul sqemebs, romelTa mixedviTac biomembranaTa

saerTo konstruqciaSi am klasis naerTebs mxolod struqturul

(“Stativis” an “misamagrebeli blokis”) adgils uTmobdnen [298].

amJamad CvenTvis cnobilia, rom maT sxva, gacilebiT mravalmxrivi

datvirTva gaaCniaT; kerZod, dadgenilia, rom fosfolipidis saerTo

fondis garkveul fraqcias kofaqtoruli funqciac SeiZleba gaaCndes.

am nivTierebaTa amfifaturi buneba amis mtkicebis garkveul safuZvels

gvaZlevs. mogvyavs is arasruli CamonaTvali (Tu ar CavTvliT kidev

iseT funqciebs, romlebic mkvlevarTa yuradRebis miRma darCa),

romelTa erTianoba fosfolipidTa monawileobiT aRZruli mravali

efeqtis axsnis saSualebas iZleva:

• cilebis aqtiuri konformaciis Seqmna da stabilizacia;

• fermentul kompleqsebSi calkeul komponentTa agregacia;

• membranaTa yvela damaxasiaTebeli Tvisebebis (seleqtiuri gan-

vladobis, kooperatiuli efeqtebisa da a.S.) mqone Caketili

uwyveti struqturis warmoqmnis unari;

• membranaSi mimdinare reaqciebisaTvis optimaluri hidrofobuli

garemos Seqmna;

• cximSi xsnadi nivTierebebis transporti [299] da sxv.

endoplazmuri retikulumis “araSemauRlebel” (energiis arageneri-

rebad) membranebs rac SeexebaT, cnobilia, rom isini fosfolipiduri

masalis simdidriT, maTi maRali denadobiT, membranis sigrZivi dagegma-

rebis mxriv heterogenuli ganawilebiT, xolo ganivi dagegmarebiT asi-

metriulobiT xasiaTdebian. amasTan erTad, TvalsaCinodaa gamokveTili

mikrosomul fosfolipidebsa da monooqsigenazebs Soris cila-lipi-

duri urTierTqmedebis ganmsazRvreli roli. gansakuTrebiT xazgasas-

melia is garemoeba, rom arsebobs sarwmuno mtkicebulebebi monooqsi-

genazuri reaqciebis kinetikis TaviseburebaTa Sesabamisobaze, romelic

93

damaxasiaTebelia mxolod bifazuri sistemisaTvis _ lipidi-wyali

[300].

hidroqsilirebis rTuli procesis praqtikulad yvela stadiaze

fosfolipidebis moqmedebis punqtebi safuZvlianadaa gaanalizebuli

liaxoviCisa da cirlovis monografiasa [301] da mimoxilviT statiaSi

[302]. zogadi suraTi ase gamoiyureba (nax. 1.8) [301]:

P_4503+_S

P_4503+ P_450 2+_S

S

CO

P_4502+

S

P_4502+

O2_

S

CO

b5

C

FP2 NADH

"X"

SOH

glukuronidi

H2OO2

FP1 NADPH

nax. 1.8. membranuli fosfolipidebis monawileoba monooqsigenazuri

sistemis funqcionirebaSi. isrebiT naCvenebia fosfolipid-

damokidebuli monooqsigenazis komponentebi da reaqciebi [301].

• fosfolipidebi membranasTan dakavSirebuli monooqsigenazebisa da

maTi fragmentebisaTvis hidrofobul garemocvas qmnian;

• fosfolipidebi xels uwyoben hidrofobuli substratebis cito-

qrom P450-Tan dakavSirebas; • fosfolipidebi optimalur pirobebs qmnian citoqrom P450-isa da

NADPH-citoqrom P450-reduqtazas urTierTqmedebisaTvis; • fosfolipidebi gavlenas axdenen mikrosomul redoqs-jaWvSi

eleqtronTa transportze.

94

sadReisod ar gagvaCnia sarwmuno monacemebi fosfolipidebis mo-

nawileobis Sesaxeb hidroqsilirebis procesis iseT yvelaze idumale-

biT mocul stadiaSi, rogoric substratSi gaaqtivebuli Jangbadis Ca-

nergvaa.

arsebuli monacemebis safuZvelze dabejiTebiT SeiZleba iTqvas, rom

fosfolipidebis erT-erTi uZiriTadesi funqcia citoqrom P450-is sta-bilizaciaa. evoluciuri ganviTarebis gansazRvrul safexurze membrana-

Si CaSenebis procesma ferments substratis gareSe aqtiuri konforma-

ciis SenarCunebis unari SesZina. amasTan dakavSirebiT maSinve ibadeba

bunebrivi kiTxva molekuluri organizaciis im TaviseburebaTa Sesaxeb,

romlebic membranul da wyalSi xsnad citoqrom P450-Semcvel monooq-

sigenazur sistemebs axasiaTebT: molekulur-organizaciuli Tvalsazri-

siT hemoproteinis membranaSi CaSenebam gamoiwvia Tu ara monooqsigena-

zas raime axali formis Camoyalibeba? principSi, SesaZlebelia fermen-

tul sistemaTa ori tipis organizacia. pirvelis Tanaxmad, membranaSi

arsebobs gansazRvruli steqiometriis mqone mtkice mravalkomponentia-

ni kompleqsebi da eleqtronTa gadatana mxolod aseTi “klasterebis”

SigniT SeiZleba xdebodes. meore tipi iTvaliswinebs membranaSi yvela

gadamtanis uwesrigo ganawilebas, romlebic Tavisi Zvradobis gamo mis

sibrtyeSi advilad difundireben. SemTxveviTi dajaxebebisas SeiniSneba

eleqtronTa gadatana. am dros bunebrivia muxtis gadamtani Sesabamisi

binaruli kompleqsebis warmoqmna. maTi sicocxlis xangrZlivoba Tu

ufro mcirea moreagire molekulebis birTvebs Soris eleqtruli mux-

tis gadatanis drosTan SedarebiT, maSin aseTi reaqciebi difuziur-

malimitirebadi iqnebian. winaaRmdeg SemTxvevaSi reaqcia difuziaze da-

mokidebuli aRar iqneba [52].

urTierTmoqmed molekulaTa saerTo raodenobasTan SedarebiT, kom-

pleqsebis ricxvi maTi warmoqmnis konstantis sididezea damokidebuli.

imis gamo, rom endoplazmuri retikulumis membrana Txevadkristalur

struqturas warmoadgens da masSi cilis molekulebs lateraluri

Zvradobis unari aqvT [303], konstanta, romelic gadamtanTa erTmaneTis

mimarT swrafvas axasiaTebs, membranaSi fermentuli sistemis moleku-

lur-organizacias unda axasiaTebdes. samwuxarod, mikrosomul membrana-

Si eleqtronTa gadamtanebisaTvis am sididis raodenobrivi Sefaseba

95

jerac ar ganxorcielebula da amitom oqsigenazuri sistemis moleku-

luri organizaciis problema kvlav gadauwyveteli rCeba.

mikrosomebis membranebSi monooqsigenazuri sistemis wyobis Sesaxeb

sxvadasxvagvari varaudebi arseboben; ase mag., ori gadamtanisaTvis, ker-

Zod NADPH-specifikuri flavoproteinisa da citoqrom P450-isaTvis miRebulia, rom isini membranaSi Tavisuflad difundireben da mtkice

xangrZliv sicocxlisunarian kompleqsebs ar warmoqmnian [88, 90, 304,

305]. oqsigenazuri sistemis ori sxva komponentisaTvis monacemebi imde-

nad winaaRmdegobrivia, rom saboloo daskvnebis gakeTebis saSualebas ar

iZlevian. gamokvlevaTa umetesoba aris imis Sesaxeb, rom unda arse-

bobdes NADPH-specifikuri flavoproteinisa da citoqrom P450-is ramodenime molekulisgan warmoqmnili mtkice fermentuli klasteri

[301, 306-308]. rekonstruirebul proteoliposomebze Catarebuli cde-

bi ki gadamtanTa Tavisuflad difundirebis sasargeblod metyveleben

[309-312]. naCvenebia, rom aseT sistemaSi eleqtronTa gadatanis siCqare

NADPH-specifikuri flavoproteinisa da citoqrom P450-is binaruli

kompleqsis koncentraciiTaa ganpirobebuli [313].

ganxilul warmodgenaTa sisworis Sesamowmeblad Seswavlil iqna

mikrosomul oqsigenazur sistemaSi eleqtronTa gadatanis siCqaris da-

mokidebuleba lipiduri biSris mikrosiblanteze [314]. gamomdinare im

mosazrebidan, rom membranaSi qolesterolis CaSeneba-mocilebiT fos-

folipiduri membranis mikrosiblante Seicvleboda, SesaZlebeli gaxde-

boda gamovleniliyo eleqtronTa gadatanis difuziaze damokidebuli

reaqciebi da agreTve is gadamtanebi, romlebic membranaSi funqcionire-

ben, rogorc Tavisuflad difundirebadi cilebi.

SeCerebuli nakadis meTodiT gamokvleul iqna citoqromebis _ b5-

isa da P450-is NAD(P)H-damokidebuli aRdgena. naxazze 1.9 moyvanili

monacemebi gviCveneben membranis mikrosiblantis cvlilebebis gavlenas

citoqrom b5-is NADH-damokidebuli aRdgenis siCqareze.

gamovlenilia ukudamokidebuleba lipiduri biSris mikrosiblantesa

da NADH-citoqrom b5-reduqtazuli reaqciis siCqaris konstantas So-

ris. qolesterolis CaSenebis difuziis koeficientis Semcirebas 2.4.10-

8-dan 1.5.10-8

sm2.wm

-1-mde (anu 1.6-jer) Tan sdevs am reaqciis siCqaris

konstantas Semcireba 3.103-dan 5.103

moli-1.wm

-1-mde (mrudi 2). qoleste-

96

rolis mocilebisas membranis mikrosiblantis daqveiTebas Tan axlavs

difuziis 3.3.10-3sm

2.wm-1-mde (anu 1.4-jer) da, Sesabamisad, reaqciis siC-

qaris konstantas gazrda 15.103moli

-1.wm-1-mde (mrudi 3). am reaqciisagan

gansxvavebiT, NADH-citoqrom P450-reduqtazas aqtivoba membranis mi-krosiblanteze damokidebuli ar aRmoCnda. amgvarad, NADPH-citoqrom

P450-reduqtazuli reaqcia damokidebulia difuziaze, xolo NADH-

citoqrom P450-reduqtazuli ki _ ara. es Sedegi SeiZleba ganxilul

iqnas, rogorc pirdapiri eqsperimentuli dadastureba Jangvis jaWvis

ori sawyisi komponentis _ NADH : flavoproteinisa da citoqrom b5-is

lateraluri Zvradobis Sesaxeb. es monacemebi sarwmunod gviCveneben

membranaSi am gadamtanTa uwesrigo ganawilebs. NADH-citoqrom P450-reduqtazuli reaqciis siCqareze biSris mikrosiblantis araefeqturoba

imaze metyvelebs, rom es reaqcia araa difuziaze damokidebuli. am

reaqciis SedarebiT dabali siCqare (siCqareTa konstantebs Soris sxva-

oba 3 rigiT ufro maRalia), gvafiqrebinebs, rom am SemTxvevaSi limi-

tirebad stadias citoqrom b5-is hemuri rkinidan citoqrom P450-is hemur rkinaze eleqtronebis gadatana unda warmoadgendes.

Na2S2O4

Na2S2O4

0 10 20 dro, mwm

∆A450_490b

_

_

_

_

_

1,2,3

0.01

0.03

0.05

0.200.20

0.10

0.05

0 100 200 dro, mwm

∆A424_408

a

_

_

_

12

3

_~ _~

nax. 1.9. citoqromebis _ b5-isa (a) da P450-is (b) NADH-damokidebuli

aRdgena virTxis RviZlis mikrosomebSi lipiduri biSris

gansxvavebuli mikrosiblantisas.

1. sakontrolo mikrosomebi;

2. mikrosomebi CaSenebuli qolesteroliT;

3. mikrosomebi mocilebuli qolesteroliT [315].

97

substratad NADPH-is gamoyenebisas lipidis mikrosiblanteze aR-

niSnul hemoproteinTa aRdgenis siCqaris damokidebulebis Seswavlam

zustad iseTive kanonzomiereba gamoavlina, rogorsac NADH-is Sem-

TxvevaSi hqonda adgili (nax. 1.10) [315]. NADPH-citoqrom b5-reduq-

tazuli reaqciis siCqare damokidebuli aRmoCnda mikrosiblanteze, xo-

lo NADPH-citoqrom P450-reduqtazuli _ ara. am reaqciebis siCqare-

Ta konstantebi araumetes erTi rigiT gansxvavdebian erTmaneTisagan.

Na2S2O4Na2S2O4

0 1.0 2.0dro, mwm

∆A450_490

b

_

_

_

_

_

1,2,3

0 100 200 dro, mwm

∆A424_408

a

_

_

_

12

3_

_

0.050

0.100

0.150

0.02

0.04

0.06

0.18

_

__

~ _~

nax. 1.10. citoqromebis _ b5-isa (a) da P450-is (b) NADPH-damoki-

debuli aRdgena virTxis RviZlis mikrosomebSi lipiduri bi-

Sris gansxvavebuli mikrosiblantisas.

1. sakontrolo mikrosomebi;

2. mikrosomebi CaSenebuli qolesteroliT;

3. mikrosomebi mocilebuli qolesteroliT [315].

amgvarad, NADPH-specifikuri flavoproteinisagan, citoqromebis

b5-isa da P450-isagan ufro mosalodnelia mtkice xangrZlivsicocxlia-

ni kompleqsebis warmoqmna, vidre am flavoproteinisa da citoqrom b5-

isagan.

fosfolipidebis fizikurma mdgomareobam SeiZleba Zlier imoqmedos

aramxolod Jangvis siCqareze, aramed mTliani procesis malimitirebeli

stadiis Secvlac ki gamoiwvios. am SemTxvevaSi mniSvnelovania tempera-

turuli faqtori.

srul mikrosomul sistemaSi, romelic yvela komponents, agreTve

98

NADPH-s da O2-s Seicavs, mravali substratis Jangvis siCqaris kons-

tantas temperaturaze damokidebulebam aCvena, rom Jangvis aqtivaciis

energia mkveTrad icvleba 18-25°C temperaturisas [316-319]. am tempera-turis zeviT mTeli rigi substratebis Jangva xasiaTdeba aqtivaciis

energiiT, romelic 33-50 kj.moli-1-is sazRvrebSi imyofeba, maSin rode-

sac mravali substratis aqtivaciis energia 18-25°C-is qveviT 80-104 kj.moli

-1-mde izrdeba. am temperaturaze areniusis grafikebze Cndeba

moxriloba, da igi dakavSirebuli araa citoqrom P450-is an reduqta-zis mdgomareobis cvlilebebze: xsnarSi an liposomebSi individualuri

citoqrom P450-is monawileobiT hidrozeJanguri Jangvis reaqciis tem-

peraturuli sxvaoba aqtivaciis energiis mxolod erTi sididiT xa-

siaTdeba [317, 319]. reduqtazas Jangvis aqtivaciis energia 50-59 kj.mo-

li-1-is tolia da farTo temperaturul intervalSi raime mniSvnelovan

cvlilebebs ar ganicdis. areniusis damokidebulebebze arsebuli mo-

xriloba mTlianadaa dakavSirebuli mikrosomuli fosfolipidebis fa-

zur gadasvlebTan, rac zegavlenas axdens reduqtazisa da citoqrom

P450-is urTierTqmedebaze. fosfolipidebis fazuri gadasvlis wer-

tilze dabali temperaturisas (18-25°C-ze), reduqtazisa da citoqrom P450-is xistad organizebul kompleqsSi hemoproteinis aRdgena SeiZle-

ba Jangvis mTliani procesis siCqaris malimitirebeli stadiac ki aR-

moCndes; fazuri gadasvlis wertilis zeviT citoqrom P450-is aRdge-nis procesi advildeba misi da reduqtazis gazrdili Zvradobis gamo.

aseTi suraTi SeiniSneba ara mxolod RviZlis mikrosomuli Jangvisas,

aramed kurdRlis filtvebis mikrosomebSi naftalinis Jangvis SemTxve-

vaSic [320]. maSasadame, mikrosomul procesebze membranuli fosfo-

lipidebis zemoqmedeba “fizikur” xasiaTs atarebs. igi dakavSirebulia

maT Txevadkristaluridan Txevad mdgomareobaSi gadasvlasTan, rodesac

izrdeba mahidroqsilirebeli kompleqsis komponentTa lateraluri

difuzia.

mikrosomul Jangvaze fosfolipidebis gavlenis molekuluri meqa-

nizmebis Seswavla gacilebiT mosaxerxebelia rekonstruirebul siste-

maze, sadac SeiZleba icvlebodnen vezikulebis, micelebis da liposome-

bis warmomqmneli fosfolipidebis koncentraciebi da qimiuri buneba

[321-326]. rogorc wesi loposomebSi an rekonstruqciisas fosfolipi-

99

debis Semcvel sistemaSi citoqrom P450-is CarTva, am ukanasknelis ka-

talizuri aqtivobis gazrdas iwvevs, rac arsebiTad fosfolipiduri

vezikulis bunebazea damokidebuli. naCvenebia mag., rom citoqromi

P450LM2 gansakuTrebiT aqtiuria jamuri mikrosomuli fosfolipidebis,

an aramembranuli dilauroilfosfatidilqolinis Tanamyofobisas [327].

rekonstruirebul sistemebSi did rols asrulebs fosfolipidis mux-

ti. uaryofiTad damuxtuli fosfolipidebis aqtivoba Tanabar pirobeb-

Si neitralur fosfolipidebTan SedarebiT gacilebiT metia. p-nitroanizolis O-demeTilirebis reaqciaSi citoqrom P450LM2-is aqti-

voba uaryofiTad damuxtuli fosfolipiduri vezikulebis eleqtro-

forezuli Zvradobis pirdapirproporciulia [327].

gel-filtraciis meTodisa da 14C-dilauroilglicero-3-fosforil-

qolinis gamoyenebiT naCvenebia, rom citoqrom P450LM2-is suberTeuli

fosfolipidis 22 molekulas ikavSirebs, da aseTi kompleqsis diso-

ciaciis konstanta 7.10-6 molis tolia. am pirobebSi micelebis warmoq-

mnis kritikuli konstanta 4.5.10-5 mols udris [328]. rekonstruirebul

sistemebSi fosfolipidebi didi siWarbiTaa da amdenad yovelTvis

daculia micelebis warmoqmnis pirobebi. am SemTxvevaSi fosfolipidebis

zegavlena or _ molekulur da zemolekulur doneze xorcieldeba:

molekulur dones gansazRvravs hemoproteinis erTi molekulis mier

22 molekula fosfolipidis mierTeba; zemolekuluri done micela-

ruli katalizis efeqtebiT mJRavndeba _ fosfolipiduri vezikulebi

aadvileben citoqrom P450-is reduqtazasTan da agreTve hemoproteinis substratTan dakavSirebas. es viTareba nawilobriv migviTiTebs

rekonstruirebul sistemebSi fosfolipidebis arsebobis aucileblobas

masSi CaSenebuli citoqrom P450-is maRali katalizuri aqtivobis

gamosamJRavneblad. xsnarebTan SedarebiT micelarul sistemebs sxva

upiratesobebic gaaCniaT: fosfolipiduri micelebi aCqareben reduqta-

ziT citoqrom P450-is aRdgenas; micelaSi katalazis faqtori xSirad

gadamwyvetia, magram arc am faqtorebiT amoiwureba fosfolipidebis

roli, radgan isini Zlier da mravalmxriv zegavlenas axdenen TviT

hemoproteinze, rodesac sistemaSi Sesabamisi reduqtaza ar imyofeba.

ingelman-sandbergis [329] mier Seswavlilia fosfolipidebis

gavlena individualur citoqrom P450-is reaqciebze. natriumis

100

periodatis Tanamyofobisas androstedionis 6β-hidroqsilirebis siCqare

2.5-jer izrdeboda fosfolipid-citoqromis Tanafardobisas 10 : 1. am

mkvlevarma gamoTqva varaudi imis Sesaxeb, rom gamoyenebuli fosfati-

dilqolini, fosfatidileTanolamini da fosfatidilserini mniSvne-

lovnad unda gansazRvravdnen citoqrom P450-is aqtiur konformacias. aRniSnuli monacemebis safuZvelze SemdgomSi metelicasa da misi

Tanaavt. [329-330] mier gamokvleul iqna sxvadasxva fosfolipidis

gavlena citoqrom P450LM2-is monawileobiT kumolis hidrozeJangiT

substratTa Jangvaze. fosfolipidis citoqrom P450LM2-Tan 30 da meti

moluri Tanafardobisas fosfatidilserini, fosfatidilinoziti da

lizofosfatidilqolini naftalinisa da anilinis Jangvis siCqares 3-4-

jer zrdidnen maSin, rodesac fosfatidilqolini da sfingomielini

imave koncentraciebiT aRniSnuli substratebis Jangvis siCqareze ar

moqmedebdnen. rac Seexeba mikrosomebidan miRebul cximovan mJavebs,

isini am reaqciebis inhibirebasac ki axdendnen. maSasadame, anilinis,

naftalinisa da dimeTilanilinis hidrozeJangur JangvaSi citoqrom

P450LM2-is individualur reaqciaze gamaaqtiurebel moqmedebas avlenen

mJava, da ara neitraluri fosfolipidebi. aRniSnulidan gamomdinareobs

sagulisxmo daskvna: citoqrom P450-is aqtivaciaSi mniSvnelovan rols

asrulebs fosfolipidis muxti. amasTan dakavSirebiT Catarebulia

saintereso gamokvlevebi, romlebic citoqrom P450-is spinur formaTa wonasworobaze fosfolipidebis pirdapir gavlenas exeba [331-334].

naCvenebia, rom neitraluri fosfolipidebi zrdian citoqrom P450-is maRalspinuri formis Semcvelobas, rac speqtrebSi STanTqmis maqsimumiT

382-390 nm-ebze da minimumiT 417-418 nm-ebze dasturdeba. rac Seexeba

mJava fosfolipidebs, isini iwveven speqtrul cvlilebebs maqsimumebiT

435-440 da minimumebiT 416-419 nm-ebze [35]; e.i. lipidi gavlenas axdens

mxolod hemoproteinis spinur mdgomareobaze.

Cvens laboratoriaSi [335] mcenareul (soiasa da simindis lebnebis)

Txel anaTlebze naCvenebia triton X-100-is damTrgunveli (65-85%-iT)

moqmedeba aminopirinis, dimeTilanilinis N-demeTilirebaze da anilinis

p-hidroqsilirebaze; N-demeTilazuri aqtivobis sruli da p-hidro-qsilazuri aqtivobis 60%-iT aRdgena fosfatidilqolinis (“nedli”

lecitinis saxiT) damatebiT moxerxda. sayuradReboa erTi faqti:

101

fosfolipidis mastimulirebeli efeqti absoluturad erTnairia

sainkubacio xsnarSi detergentTan erTad misi Setanisas (inhibireba

aRar SeiniSneba) da detergentuli inhibirebis miRebis Semdegac. aqedan

gamomdinare, fosfatidilqolinis moqmedebas ornairi axsna miveciT: 1).

detergentTan erTad Tanamyofobisas es fosfolipidi mJangveli

fermentuli sistemis irgvliv lipid-damokidebul hidrofobul gare-

mocvas qmnis da hemoproteinis aqtiur centrs detergentisadmi

Seuvals xdis; 2). inhibirebis Semdeg fosfolipidi, rogorc Cans, fer-

mentis aqtiuri hidrofobuli ubnebidan aZevebs detergents da mis

irgvliv kvlav hidrofobul garemocvas aRadgens.

amgvarad, mikrosomebis lipiduri komponentis funqcias, upirveles

yovlisa, citoqrom P450-is konformaciul relaqsaciaze zegavlena

warmoadgens. garda amisa, lipidebi zrdian ferment-substratis komp-

leqsis mdgradobas; xels uwyoben am hemoproteinisa da misi reduq-

tazis kompleqsirebas da astabilizeben mas; keTilsasurvel garemos

qmnian Jangvis procesebisaTvis da hidrofobul zonaSi substratTa

koncentrirebas axorcieleben; uzrunvelyofen monooqsigenazuri sis-

temis komponentTa optimalur lateralur Zvradobas da a.S. zogadad

rom iTqvas, citoqrom P450-ze da mTlian monooqsigenazur sistemaze

fosfolipidebis gavlena uaRresad mravalwaxnagaa da rogorc mole-

kulur, aseve zemolekulur doneze xorcieldeba [336].

102

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333. Ахрем, А.А., Киселев, П.А., Кисель, М.А., Усанов, С.А., Метелица, Д.И. Влия-ние фосфолипидов на спектральные характеристики высокоочищенного цито-хрома Р-450 микросом печени кролика. ДАН СССР, 1980, 245, 234-237

334. Gibson, G.G., Cinti, D.L., Sligar, S.G., Schenkman, J.B. The effect of microsomal fatty acids and other lipids on the spin state of partially purified cytochrome P-450. J. Biol. Chem., 1980, 255, 1867-1873

335. Гордезиани, М.Ш., Дурмишидзе, С.В., Курашвили, Л.К. Восстановление фос-фатидилхолином ингибированных детергентом реакции гидроксилирования в целой растительной ткани. Сообщ. АН ГССР, 1981, 104, 465-468

336. Уваров, В.Ю., Бачманова. И.А., Арчаков, А.И., Сухомудренко, А.Г., Мясоедо-ва, К.Н. Конформация и тепловая стабильность растворимого и встроенного в мембрану липосом цитохрома Р-450. Биохимия. 1981, 46, 280-286

124

Tavi 2

oqsigenazebi Sidaujredul metabolur cvlaSi

2.1 citoqrom P450 mcenaris azotfiqsaciaSi

parkosan mcenareTa fesvebSi koJris baqteriebi baqteroidebad

transformirdebian. isini baqteriebis simbiozur formebs warmoadge-

nen. am dros nivTierebaTa cvlis iseTi Rrma cvlilebebi xorciel-

deba, romlebic simbiozuri pirobebisaTvis absoluturad axalia da,

rogorc Cans, srulad emsaxurebian azotis fiqsaciis process. gar-

da amisa, mimdinare cvlilebebi eleqtronebiT uzrunvelyofasa da

ATP-is energias saWiroebs [1]. N2-is biologiuri aRdgena anaerobu-

li procesia, magram intaqtur baqteroidebs Jangbadis gareSe funq-

cionireba mainc ar SeuZliaT. amasTan dakavSirebiT yvelaze mniSvne-

lovani isaa, rom baqteroidebSi citoqromuli komponentebis Semc-

veloba Tvisobrivadaa Secvlili: a-jgufis citoqromebis gaqrobis fonze maTSi TiTqmis gaormagebulia c-jgufis citoqromebis raode-noba da, rac mTavaria, Cndebian citoqromuli sistemis iseTi axali

komponentebi, rogorebicaa autooqsidabeluri CO-reaqtiuli cito-

qromebi P450 da c552. maTi Tanamyofoba mxolod koJris baqteriebis

baqteroiduli formebisTvis („efeqturi” baqteroidebisTvis) aris

damaxasiaTebeli da amitom safiqrebelia, rom maTi induqcia axlad

SeZenil funqciebTan iyos dakavSirebuli. mindvris mcenaris baqte-

roidebis centrifugatebis da qviSaze gazrdili xanWolas koJrebis

baqteroidebis suspenziaSi yovelTvis SeiZleba gamovavlinoT inten-

siuri STanTqmis zoli 425 nm-ze maqsimumiT, rac citoqrom

P450_CO kompleqsisTvisaa damaxasiaTebeli. aRmoCnda agreTve, rom

citoqrom P450 aq xsnadi saxiTaa da Tavisi speqtruli TvisebebiT

RviZlis endoplazmuri membranebis hemoproteinTan ufro axlos

dgas, vidre qafuris mahidroqsilirebel xsnad baqteriul citoqrom

P450-Tan [2, 3]. am citoqromis maqsimaluri Semcveloba mcenaris

yvavilobis, anu aqtiuri sunTqvisa da aqtiuri azotfiqsaciis fazas

emTxveva.

citoqrom P450-is metabolur funqciebsa da baqteroidebis

azotmafiqsirebel unars Soris uSualo kavSirze pirveli mosazreba

eplbis [4] ekuTvnis, romelmac azotis aRdgenis procesSi am hemo-

125

proteinis SesaZlo rolze gaamaxvila yuradReba.

soiis fesvebidan gamoyofili msunTqavi baqteroidebis (Rhizobium japanicum) suspenziaze oqsileghemoglobinis damatebisas eplbim da

Tanaavtorebma [5] gamoavlines ATP-is donisa da ATP/ADP-is sidi-dis zrda. paralelurad adgili hqonda nitrogenazas aqtivobis ma-

tebas. maT mier gamoyenebuli citoqrom P450-is specifikuri inhibi-toris N-fenilimidazolis koncentraciebis Semcirebisas fardoba

ATP/ADP da nitrogenazas aqtivoba erTmaneTs Soris xazovan damo-kidebulebas avlenda. am monacemebze dayrdnobiT avtorebma gamoTqves

mosazreba imis Sesaxeb, rom citoqrom P450 warmoadgens JangviTi

fosforilebis komponents, sadac igi funqcionirebs rogorc mole-

kuluri Jangbadis gadamtani da ara rogorc terminaluri oqsidaza.

2.2 oqsigenazebi cximovan mJavaTa zeJangur JangvaSi

cnobilia, rom ujreduli membranebi ujeri cximovani mJavebiT

mdidar fosfolipidebs Seicavs. isini membranaTa maRal Zvradobas

uzrunvelyofen. stresuli zemoqmedebisas (mag., qsovilTa hipoqsii-

sas an iSemiisas) adgili aqvs cximovan mJavaTa zeJanguri Jangvis

reaqciebis gaaqtiurebas. garda amisa, membranebSi Tavisufali radi-

kalebis iniciacias Tan axlavs cximovan mJavebTan maTi urTierTqme-

deba, romelic warmoqmnili ujeri bmebis ricxvis zrdis Tanazomie-

rad Zlierdeba. ujer cximovan mJavaTa zeJanguri Jangvis Sedegad

saxeze gvaqvs erTi mxriv, membranis gazrdili siblante, meore mxriv

ki misi uaRresad daqveiTebuli barieruli funqcia.

sadReisod ar SegviZlia saboloo pasuxi gavceT kiTxvas: raSi

sWirdeba lipidTa zeJanguri Jangvis procesi ujreds? misi WeSma-

riti funqciuri rolis Seucnoblobis miuxedavad, fiziologiuri

da gansakuTrebiT paTologiuri cvlilebebisas am tipis reaqciebis

aucilebloba araviTar eWvs ar iwvevs. mogvepoveba savsebiT sarwmu-

no faqtobrivi masala imis Sesaxeb, rom lipidTa zeJanguri Jangva

damaxasiaTebelia yvela normaluri metabolizmis mqone qsovilisaT-

vis. rac ufro aqtiurad xorcieldeba nivTierebaTa cvla, miT maRa-

lia lipoperoqsidaciis done. arsebobs mosazreba (Tumca jer kidev

sadiskusio) imis Sesaxeb, rom es procesi membranaTa mudmiv TviTgan-

126

axlebas emsaxureba da gansakuTrebiT intensiurad im membranebSi mim-

dinareobs, romlebic eleqtronis satransporto sistemebs Seicavs.

jer kidev gasuli saukunis samocian wlebSi hoxSteinis,

ernsterisa da Tanaavtorebis mier [6-8] naCvenebi iqna cxovelis mi-

krosomebSi unikaluri fermentuli sistemis arseboba, romelic mem-

branaTa lipidebis zeJangur Jangvas akatalizebs. maT miervea Camoya-

libebuli debuleba, romlebic dRemde ZalaSi rCeba: arsebobs pe-

roqsidaciis ori _ fermentuli da arafermentuli sistema. pirveli

sistema reducirebadi eqvivalentebis wyarod aRdgenil NADPH-s

iyenebs da inaqtivdeba gacxelebiT an p-qlormerkuribenzoatiT. ara-

fermentuli lipoperoqsidaciis sistema aRmdgenelad askorbats iye-

nebs da aramgrZnobiarea temperaturisa da sulfhidriluri Sxamebis

mimarT.

fosfolipidTa SemadgenlobaSi myofi ujeri rigis cximovan mJa-

vaTa naSTebis fermentuli zeJanguri Jangva im erT gansakuTrebul

reaqciaTa klass miekuTvneba, romelic endoplazmur membranebSi lo-

kalizebuli fermentuli kompleqsiT xorcieldeba. am SemTxvevaSi

gansakuTrebul interess iZens am procesSi mikrosomuli NADPH-

specifikuri flavoproteinisa da citoqrom P450-is monawileobis

sakiTxi.

pirveli monacemebi, rom lipidTa zeJanguri da qsenobiotikTa

Jangva erTi da igive fermentebiT katalizdeba, ernstersa da mis

TanamSromlebs [9-10] ekuTvniT. am daskvnamde isini Semdeg faqtebze

dayrdnobiT mividnen:

1. qsenobiotikTa Jangvis produqtebi ainhibireben NADPH-

damokidebul lipidTa peroqsidacias;

2. mikrosomuli preparatebis “daberebisas”, rodesac qsenobiotikTa

Jangvis intensivobis mkveTri daqveiTeba xdeba, maTi mxridan

NADPH-damokidebuli zeJanguri Jangvis inhibireba ixsneba.

aRniSnuli faqtebis asaxsnelad am mkvlevarTa mier gamoiTqva

mosazreba, romlis Tanaxmadac orive procesSi NADPH-is mJangveli

eleqtron-satransporto fermentuli kompleqsi monawileobs. axlad

gamoyofil mikrosomebSi qsenobiotikebi am kompleqsze dominireben,

ris gamoc NADPH-is ZiriTadi fondi maT degradaciaSia CarTuli,

xolo lipiduri peroqsidacia daTrgunulia. “asakovan” mikrosomebSi

“monopolirebis” efeqti qreba da NADPH-is mJangveli sistema aq-

127

tiurad erTveba lipidur peroqsidaciaSi. am SemTxvevaSi NADPH-

specifikuri flavoproteini gansakuTrebiT efeqturi xdeba orva-

lentiani rkinis kompleqsTan (ADP_Fe2+-is, an EDTA_Fe2+

-is Tana-

myofobisas).

lipidTa zeJangur Jangvasa da qsenobiotikTa hidroqsilirebaSi

NADPH-specifikuri flavoproteinis monawileobis mtkicebulebebi

SeiZleba Semdegi debulebebis saxiT Camoyalibdes [11]:

1. orive procesi specifikuria NADPH-is mimarT da misi NADH-iT

Secvlisas ar mimdinareobs;

2. arsebobs korelacia NADPH-citoqrom P450-reduqtazul aqtivo-

basa da cximovan mJavaTa NADPH-specifikuri zeJanguri Jangvis

reaqciis siCqareTa Soris;

3. NADPH-citoqrom P450-reduqtazas konkurentuli inhibitori –

daJanguli NADP+ aseve warmatebiT Trgunavs NADPH-damokide-

bul zeJangur Jangvas;

4. fermentuli reaqciebis samive tipi _ NADPH-citoqrom P450-is oqsidoreduqcia, NADPH-specifikuri hidroqsilireba da NADPH-

damokidebuli zeJanguri Jangva erTnairad stereospecifikuria;

5. menadioni (vitamini K3) – xelovnuri aqceptori, romlis mimar-

Tac NADPH-specifikuri flavoproteinis swrafva uaRresad ma-

Ralia, erTnairad Trgunavs dimeTilanilinis demeTilirebas da

lipidTa zeJangur Jangvas;

6. NADPH-specifikuri flavoproteini, qsenobiotikTa hidroqsili-

reba da lipidTa zeJanguri Jangva erTnairad eqstremalurad

mgrZnobiarea proteazebis moqmedebis mimarT. tripsini da pronaza

dabal temperaturazec ki SerCeviTad axdenen flavoproteinis

solubilizacias da mkveTrad amuxruWeben hidroqsilirebisa da

lipidTa peroqsidaciis procesebs;

7. cximovan mJavaTa zeJanguri Jangvis sistema SeiZleba rekon-

struirdes liposomebisa da NADPH-specifikuri flavoprotei-

nisagan;

8. NADPH-citoqrom P450-reduqtazis antisxeuli ainhibirebs ze-

Jangur Jangvas.

CamoTvlili mtkicebulebebi calsaxad miuTiTeben imaze, rom

NADP-citoqrom P450-reduqtaza sawyis rgols warmoadgens hi-

droqsilirebisa da zeJanguri Jangvis reaqciebSi.

128

saWiroa aRiniSnos, rom arsebobs monacemebi, romelTa Tanaxmadac

am fermentis nacvlad orive procesSi eleqtronTa gadatanis jaWvis

sxva komponenti monawileobs. amis sasargeblo mniSvnelovani faqti

imaSi mdgomareobs, rom mikrosomebSi lipidTa peroqsidacia da qse-

nobiotikTa hidroqsilireba p-qlormerkurobenzoatiT iTrguneba,

maSin rodesac am Sxamis mimarT NADPH-flavoproteini aramgrZno-

biarea [6-8]. meore faqtis Tanaxmad proteazebiT solubilizebuli

fermenti supernatantSi aqtivobis daukargavad gadadis, magram am

dros hidroqsilirebisa da peroqsidaciis procesebi damuxruWebu-

lia. rogorc Cans, proteazebis mier eleqtronis satransporto

jaWvidan am sawyisi komponentis “amogleja” jaWvis momdevno ubnebs

aRdgenis unars akargvinebs. cnobilia, rom es fermenti membranis

zedapirzea lokalizebuli da masTan dasakavSireblad gamoiyeneba

ara lipidebi, aramed membranuli cilebis aminomJavuri naSTebi.

swored amis mizezi unda iyos proteazebis maRali masolubilize-

beli efeqti. membranul cilebTan susti kontaqti safiqrebels

xdis, rom zeJanguri Jangvis iniciaciaSi flavoproteini uSualod

ar monawileobs. amisaTvis saWiroa sivrcobrivi siaxlove radikale-

bis warmomqmnel centrebsa da fermentul zeJangur Jangvis ubnebs

Soris. am mosazrebidan gamomdinareobs, rom lipidTa fermentuli

peroqsidaciis warmatebiT ganxorcielebisaTvis NADPH-specifikuri

flavoproteini aucilebelia, magram procesis iniciacia ara am pir-

vel komponentTan, aramed ufro moSorebiT, sadRac Sualed, an jaW-

vis terminalur ubanze xdeba.

mikrosomuli membranebis NADPH-specifikuri redoqs-sistemis

calkeuli komponentis TvisebaTa ganxilva ar iZleva romelime maT-

ganze lipidTa peroqsidaciis zusti lokalizaciis dadgenis saSua-

lebas, radgan Tavisufali radikalebi, romlebic zeJangur Jangvas

inicireben, SeiZleba warmoiqmnas rogorc sawyis (TviTJangvadi fla-

vinis), aseve terminalur (citoqrom P450-is) ubnebze. Znelia am

procesSi eleqtronis satransporto jaWvis Sualeduri ubnis

rolze msjeloba, miT umetes Tu CavTvliT, rom aq citoqromi b5

imyofeba.

cximovan mJavaTa peroqsidaciaSi mniSvnelovania sakiTxi cito-

qrom P450-is monawileobis Sesaxeb. pirveli monacemebi amis sasar-

geblod arCakovsa da mis TanamSromlebs [11] ekuTvniT. aRniSnul

129

mkvlevarTa Tanaxmad hemoproteinis aqtiur centrSi myofi apofer-

mentis SH-jgufTan rkinis pirofosfatis (FePP) dakavSirebas Tavi-sufali radikalebis generirebadi axali centrebis Camoyalibeba

mosdevs (nax. 2.1). am SemTxvevaSi ar xdeba naxSirbadis monooqsidiT

aqtivobis daTrgunva, xolo citoqrom P450 funqcionirebs ara ro-gorc hemoproteini, aramed rogorc arahemuri gogirdSemcveli rki-

naproteini. mikrosomul eleqtronTa satransporto sistemaSi ci-

toqrom P450 erTaderTi gadamtania, romelsac aqtiur centrSi SH-

jgufebi gaaCnia da eleqtronebis transportSi monawileobis miReba

SeuZlia. zeJanguri Jangvis fermentuli sistema ki mgrZnobiarea SH-

jgufebis iseTi Sxamebis mimarT, rogorebic p-qlormerkuribenzoati,

HgCl2 da mersalilia.

NADPH

O2

O2

Fp citoqrom b5

SH

hemi

citoqrom P450-isapofermenti

hidroqsilireba

CO

EDTA

Fe3+PP

Fe2+PP

zeJanguri Jangva

nax. 2.1. citoqrom P450-is funqcionireba hidroqsilirebisa da

zeJanguri Jangvis reaqciebSi [11].

rogorc Cans, SH-jgufi lipidur peroqsidaciaSi mxolod endo-

genur rkinasTan kompleqsirebisas erTveba. askorbatdamokidebul

arafermentul zeJangur JangvaSi SH-jgufebis roli gamoricxulia,

radgan es procesi sulfhidriluri jgufebis Sxamebis mimarT

aramgrZnobiarea.

soios nazardebidan miRebul mikrosomul fraqciaze Cvens labo-

130

ratoriaSi Catarebuli gamokvlevebiT [12] dadgenil iqna, rom lipo-

peroqsidaciis (malonis dialdehidis warmoqmnis mixedviT) rogorc

fermentuli, aseve arafermentuli procesi dimeTilanilinis N-

demeTilirebis gaZlierebas iwvevs. Fe2+-isa da NADPH-is Tanamyofo-

bisas lipiduri peroqsidaciis orive tipis reaqciebi TiTqmis siner-

giulad jamdeba. paralelurad adgili aqvs fraqciis N-

demeTilazuri aqtivobis zrdas. fosfatidilqolini, romelsac an-

tioqsidanturi Tvisebebi gaaCnia, lipidTa zeJangur Jangvas ainhibi-

rebs, xolo N-demeTilirebis reaqcias astimulirebs. fosfatidile-

Tanolamins aseTi gamokveTili moqmedeba ar gaaCnia. safiqrebelia,

rom misi naxSirwyalbadovani naSTis ujeri bmebi TviTon xdebian

Tavisufali radikalebisaTvis moxerxebul samizned. miRebuli mona-

cemebis safuZvelze dasaSvebad miviCnieT mosazreba, rom mcenareSi

mocemuli qsenobiotikis Jangvis procesi radikalur stadias Sei-

cavs, Tumca igi lipiduri peroqsidaciisagan garkveuli damoukideb-

lobiT xasiaTdeba. TavisTavad es bunebrivicaa: mcenaris intaqtur

ujredSi am or process Soris NADPH-isaTvis aSkara konkurencia

ar unda vlindebodes, radgan am kofermentis iseTi mZlavri generi-

rebadi sistema funqcionirebs, rogoric fotosinTezia.

gamoTqmulia mosazreba imis Sesaxeb, rom fermentuli da arafer-

mentuli lipoperoqsidaciis iniciacia analogiuri meqanizmebiT unda

xdebodes [11]. reaqciaSi moqmedi ADP_Fe3+ an pirdapir aRdgenas ga-

nicdis reduqtaziT, an qsantinoqsidazas SemTxvevaSi arapirdapir

aRdgeba superoqsid-radikaliT. warmoqmnili perfenilioni SemdgomSi

poliujer cximovan mJavebTan reagirebs da zeJangur Jangvas inici-

rebs. peroqsidaciis yvela sistema emorCileba superoqsiddismutazas

mainhibirebel gavlenas, Tumca TavisTavad superoqsid-radikals misi

inicireba ar SeuZlia. hidroqsilis radikalebi fiziologiur piro-

bebSi lipidTa zeJangur JangvaSi mniSvnelovan rols ar unda asru-

lebdnen. superoqsiddismutazas mainhibirebeli moqmedeba qolatiT

solubilizebul fosfatidilqolinisa da fosfatidileTanolaminis

Tavisufal Jangvaze, agreTve liposomebSi fosfatidilqolinis Jang-

vaze gamovlenilia eriTrocitebSi [13]. es faqti arapirdapir miuTi-

Tebs, rom lipidTa peroqsidaciaSi superoqsiduri radikalebi mona-

wileoben.

lipidTa zeJanguri Jangvis NADPH-damokidebul fermentul

131

sistemaSi Seiswavleboda Jangbadis aqtiuri formebis warmoqmnis me-

qanizmi [14]. spinuri damWerebis meTodiT, agreTve superoqsiddis-

mutazisa da katalazas gamoyenebiT dadginda, rom NADPH-damoki-

debuli lipoperoqsidaciis inicireba RviZlis mikrosomebSi xor-

cieldeba Jangbadis aqtiuri formebis saSualebiT, romlebic mikro-

somul redoqs-jaWvSi Jangbadis Tanmimdevruli erTeleqtroniani

aRdgenis gziT warmoiqmnebian. Jangbadis radikalebis generirebaze

pasuxismgebel ubans NADPH-citoqrom P450-reduqtaza warmoadgens. mis gareSe NADPH-damokidebuli zeJanguri Jangva praqtikulad ar

mimdinareobs. egzogenuri rkinis Tanamyofobisas zeJanguri Jangva

eleqtronis gadatanis swored am ubanze dafiqsirda.

2.2.1 jaWvis gagrZelebis stadia

lipidTa peroqsidacia im Tavisufal-radikaluri jaWvuri meqa-

nizmiT xorcieldeba, romelic molekuluri JangbadiT nebismieri

organuli naerTis uSualo Jangvis reaqciebisTvisaa damaxasiaTebeli.

procesi mxolod im SemTxvevaSi daiwyeba, Tu sistemaSi Tavisufali

radikalebi gaCndeba [15, 16]. magaliTad, molekuluri JangbadiT or-

valentiani rkinis daJangvisas warmoiqmneba •2HO -tipis radikali:

Fe2+ + O2 + H+ → Fe3+ + •2HO (1)

maRali reaqciisunarianobis gamo warmoqmnili radikali reagirebs garemomcvel molekulebTan, kerZod, cximovan mJavebTan da naxSirwyal-

badovani )R( • radikali miiReba:

RH + •2HO → H2O2 + •R (2)

am ori reaqciiT iwyeba Jangvis jaWvuri procesi. amitom maT

jaWvis inicirebis stadias uwodeben.

lipidTa JangvaSi Fe2+ ormag rols TamaSobs: erTi mxriv, isini

Slian hidrozeJangebs da amiT aCqareben Jangvis process; meore

mxriv ki aCereben jaWvuri procesis dawyebas hidrozeJangebis pir-

veladi warmoqmnis damuxruWebis gziT da amdenad antioqsidantis

Tvisebas avlenen [17, 18]. rodesac [ROOH]≫[Fe2+], rkinis ionebi,

rogorc katalizatorebi, lipiduri zeJangebis warmoqmnaSi monawi-

132

leoben, xolo Tu [Fe2+]≫[ROOH], rkinis ionebi moqmedeben, rogorc

antioqsidantebi. ujredSi rkinis SesaZlo aRmdgen naerTebs miekuTv-

neba cisteini da askorbinis mJava. gamoricxuli araa isic, rom aRd-

genaSi cilis sulfhidriluri jgufebic monawileobdnen [19].

antioqsidanturi aqtivoba gaaCnia tokoferolisa da difenilur

warmoebulebs, romlebic tipiur radikalur “damWerebs” warmoad-

genen. maTi gavleniT zeJanguri Jangvis damuxruWeba umniSvnelovanesi

argumentia am procesSi Tavisufali radikalebis warmoqmnis mtki-

cebis sasargeblod [6].

Tavisufal radikalTa mniSvnelovani Tviseba isaa, rom ramden-

jerac ar unda moxdes molekulebTan urTierTqmedeba, maTi ricxvi

mainc ucvleli rCeba (Tavisufali valentobis mouspoblobis prin-

cipi). Jangbadis Tanamyofobis pirobebSi •R radikalebi masTan ur-

TierTqmedeben, ris Sedegadac axali •2RO radikali miiReba, romel-

sac zeJangur radikals uwodeben:

•R + O2 → •2RO (3)

zeJangur radikals ujer cximovan mJavas axal molekulebTan

reagireba SeuZlia. am dros warmoiqmneba hidrozeJangi (ROOH) da

isev •R radikali miiReba:

•2RO + RH → ROOH + •R (4)

am ori reaqciis monacvleobas iqamde mivyavarT, rom procesSi

lipidisa (RH) da Jangbadis axali molekulebi erTveba. amis Sede-

gad warmoiqmneba hidrozeJangebi, xolo •R da

•2RO radikalebis

jamuri raodenoba praqtikulad ucvleli rCeba (nax. 2.2). procesis

am stadias “prolongaciis” (gaxangrZlivebis) stadias uwodeben.

mTliani procesis siCqares zeJanguri radikalebisa da lipidis

molekulebis SedarebiT neli urTierTqmedebis reaqcia gansazRvravs.

Tavisufali radikalebi ujeri cximovani mJavis mxolod axal-

axal molekulebTan rom reagirebdnen, maSin JangbadiT sakmarisad

uzrunvelyofisas erTaderTi radikalic ki mocemul nimuSSi arse-

bul yvela cximovan mJavas zeJangad gardaqmnida. sinamdvileSi es ase

ar xdeba, radgan adgili aqvs radikalebis „Caqrobas“ [8].

133

NADPH + 4RH + 4O2 NADP + 4ROOH + Heleqtronis gadatanis jaWvi + +

NADPH + Fe-kompleqsieleqtronis gadatanis jaWvi

NADP + Fe-kompleqsi + H3+ 2++ +

Fe-kompleqsi + R + H3+ +.Fe-kompleqsi + RH

2+

R .

RO2.

O2

ROOHRH

R .

RO2.

R .

RO2.

RO2.

H+

O2

ROOH

O2

ROOHRH

R .

O2

ROOHRH

nax. 2.2. mikrosomebSi zeJanguri Jangvis reaqciis meqanizmi.

Fe2+-kompleqsi – radikalinicirebadi centri;

RH – erTi ormagi kavSiris mqone ujeri cximovani mJava [20].

es im SemTxvevaSia SesaZlebeli, Tu radikalebi moqmedeben:

_ erTmaneTTan, mag.:

R + R. .

RO2 + RO2. .

molekuluri produqtebiRO2 + R ..

_ cvalebadi valentobis mqone metalebTan (mJave areSi), mag.:

134

•2RO + Fe2+ + H+ → ROOH + Fe3+

(4)

•R + Fe2+ + H+ → RH + Fe3+ (5)

_ antioqsidantebis molekulebTan, mag.: α-tokoferolTan (AnH-Tan)

•2RO + AnH → ROOH + •An (6)

pirvel or SemTxvevaSi molekuluri produqtebi warmoiqmneba,

xolo mesameSi mcired aqtiuri radikalebi miiReba, romelTac axal

cximovan mJavebTan reaqciaSi Sesvlis unari dakarguli aqvT. nebis-

mier SemTxvevaSi xdeba jaWvuri reaqciebis SeCereba, anu gawyveta.

Tavisufali radikalis warmoqmnis momentidan mis gaqrobamde •R -isa

da •2RO -is monawileobiT ramdenime Tanmimdevruli cikluri reaqcia

mimdinareobs. amis Sedegad sistemaSi yovel warmoqmnil radikalze

hidrozeJangis ramdenime molekula Cndeba. es ricxvi jaWvuri

reaqciebis raodenobas, anu jaWvis sigrZes axasiaTebs. rogorc Cans,

jaWvis sigrZe misi gawyvetiT isazRvreba da igi antioqsidantebis

Tanamyofobisas mokldeba. amiT xsnian Tavisufal-radikalur proce-

sebze antioqsidantebis inhibitorul moqmedebas.

ganxiluli suraTi Jangvis ganutotveli jaWvebisTvisaa damaxasi-

aTebeli. cvalebadi valentobis mqone metalTa ionebis, kerZod,

orvalentiani rkinis Tanamyofobisas jaWvur reaqciaSi warmoqmnili

hidrozeJangis daSla xdeba:

ROOH + Fe2+ → •RO + −OH + Fe3+ (7)

am dros axali Tavisufali radikalebi Cndeba da Jangvis axali

jaWvis iniciacia xdeba. am SemTxvevaSi zeJanguri Jangvis procesis

gantotva xdeba jaWvuri reaqciebis meqanizmiT. zeJanguri Jangvis

siCqares amjerad gantotvis zemoT moyvanili reaqcia (7) gansaz-

Rvravs. modelur cdebSi Fe2+-is siWarbisas reaqciis siCqare upira-

tesad reagentis (ROOH) koncentraciazea damokidebuli. misi swra-

fi dagroveba imas iwvevs, rom mTlian process TviTaCqarebuli

(feTqebadi) xasiaTi eZleva. afeTqebasTan zeJanguri Jangvis am

stadiis ganviTarebis analogias is ganapirobebs, rom process Tan

axlavs naTeba (qemiluminescencia), romlis registrirebiTac reaq-

ciis kinetika da meqanizmi Seiswavleba [19].

135

RO2 + RO2. .

qemiluminescencia (8)

.P + O2

.P + O2 + hν1

R2 + 1/2(O2)2 R2 + O2 + hν2

aq P – reaqciis produqtia (SesaZloa _ ketoni), xolo hν1 da hν2-sinaTlis energiis (fotonis) TiTo qvantia. oleinmJavas Jangvisas

qemiluminenscenciis speqtrSi 390, 460, 550 da 600 nm-ebze STanT-

qmis maqsimumebi Cndeba, romelTagan nawili, rogorc Cans, karbo-

nilis jgufebs, nawili ki _ Jangbadis dimerebs ekuTvnis.

ujredSi jaWvebis gantotvis reaqciis siCqare upiratesad

ionebis koncentraciiT da lokalizaciiT ganisazRvreba; daukavSire-

beli an daJanguli rkina araaqtiuria, amitom zeJanguri Jangvis

siCqare saboloo jamSi damokidebuli iqneba sistemaSi rkinis

mdgomareobaze da im agentebze, romlebic mis SekavSirebasa da

aRdgenas uzrunvelyofen (mag., membranuli cilebis sulfhidriluri

jgufebi):

R_SH + Fe3+ → 1/2R_S_S_R + Fe2+ + H+ (9)

benzpirenis metabolizmis reaqciaSi rkinis ionebi astimulireben

intermediatis warmoqmnas, romelic mWidrod unda iyos dakavSi-

rebuli lipidTa zeJangur JangvasTan [21]. prooqsidantebi Fe2+ da

CCl4 erTnair mastimulirebel moqmedebas avlenen aramarto

lipidur peroqsidaciaze, aramed monooqsigenazur sistemaze da

aaqtiureben NADPH-damokidebul benzpirenis hidroqsilirebas; e.i.

lipiduri radikalebi saerTo intermediats warmoadgenen orive

tipis reaqciaSi. mTlianobaSi zeJanguri Jangva SeiZleba ganxilul

iqnas, rogorc im lipiduri radikalebis utilizaciis alternatuli

gza, romlebic policikluri naxSirwyalbadebis hidroqsilirebaSi

ar monawileoben. sayuradReboa, rom Tirkmlis mikrosomebSi identi-

ficirebulia citoqrom P450-is msgavsi hemoproteini, romelic

P450k-daa wodebuli [22]. igi monawileobs laurinmJavas JangvaSi da

CO-sTan kompleqsirebisas STanTqmis piks iZleva 453-454 nm-ze.

NADPH-isa da molekuluri Jangbadis Tanamyofobisas am hemoprote-

inis Semcveli Tirkmlis qerqis mikrosomebi akatalizeben laurin-

mJavas hidroqsilirebas da warmoiqmneba ω- da (ω_1)-hidroqsiwarmo-ebulebi. Sedegad miiReba ori produqti 2 : 1 SefardebiT. laurin-

136

mJavas hidroqsilireba citoqrom P450-is funqciaa. am reaqciis

msvleloba naxSirbadis monooqsidiT Seqcevadad inhibirdeba sinaT-

leze. garda amisa, mikrosomul suspenziaze laurinmJavas damateba I-tipis speqtrul cvlilebebs iZleva, rac hemoprotein_laurinmJavas

kompleqsis warmoqmnis maCvenebelia.

orvalentian rkinis ionTan kompleqsirebuli ADP-s Tanamyo-

fobisas lipiduri peroqsidaciis stimulireba naCvenebia mcenareSic,

kerZod, bardis eTiolirebuli nazardebidan da mwvane foTlebidan

gamoyofil mikrosomul suspenziaSi [23]. aRdgenili piridinnuk-

leotidebi iwveven zeJanguri Jangvis process da, rogorc Cans, igi

fermentulad mimdinareobs, radganac mgrZnobiarea gacxelebisa da

sulfhidriluri reagentebis mimarT.

mikrosomebSi NADPH-is Jangvaze ADP-s mastimulirebeli moqme-

deba pirvelad italieli mecnierebis mier [24] iqna gamovlenili.

odnav mogvianebiT maTive [25] da agreTve ernsterisa da

Tanaavtorebis [6-7] gamokvlevebiT dabejiTebiT iqna naCvenebi am or

process (NADPH-is Jangvasa da ujeri rigis cximovan mJavaTa

Jangvas) Soris uSualo pirdapiri kavSiri. eWvgareSe gaxda, rom

mastimulirebeli efeqti damaxasiaTebelia nebismieri pirofos-

fatuli da ara fosfaturi dajgufebebisaTvis. zeJangur Jangvaze

maTi gansxvavebuli moqmedebis axsna ar SeiZleba arc am ionebis

gavleniT jaWvur reaqciebze (modelur sistemebSi dabali koncent-

raciebiT isini aCqareben, xolo maRali koncentraciebiT ainhibireben

lipiduri zeJangebis warmoqmnas) da ara NADPH-specifikur jaWvSi

eleqtronTa gadatanis siCqareze maTi gavleniT, sadac orive ioni

sakvlevi koncentraciebis SemTxvevaSi mikrosomuli hidroqsilirebis

siCqareze araviTar zemoqmedebas ar avlenen [7, 19, 26].

pirofosfatis mastimulirebel moqmedebaze da fosfatis

araefeqturobaze yvelaze savaraudo axsnad SeiZleba gamoyenebul

iqnas daSveba imis Sesaxeb, rom es ionebi gansxvavebul zegavlenas

axdenen membranuli cilebis funqciur jgufebze, rkinis ionebis

damatebisas radikalwarmomqmneli centrebis formirebis procesSi.

imdenad, ramdenadac piro- da orTofosfatis efeqturi koncent-

raciebi fiziologiuris sazRvrebSi imyofebian. savsebiT bunebrivia,

rom am anionebs NADPH-damokidebul zeJangur JangvaSi reaqciis

siCqaris regulirebaSi monawileobis miReba SeeZlo.

137

fermentul sistemaSi fosfatisa da pirofosfatis moqmedeba

arafermentuli sistemisagan gansxvavebiT SeiZleba kargad aixsnas,

Tu modelur sistemebze miRebuli monacemebidan gamovalT, sadac

Fe2+-is haeris JangbadiT Jangvis aqtivaciis xarjze am nivTierebaTa

dabali koncentraciebi qemiluminescenciis induqciis ganviTarebis

periods kvecaven. SedarebiT maRali koncentraciebi qemilumi-

nescencias da zeJangur Jangvas Trgunavdnen, radgan am dros Fe2+-is

katalizatori araaqtiur Fe3+-formaSi gadadioda [19, 27].

bidlaksa da tapels [28] aqvT gamoTqmuli gansxvavebuli

mosazreba, romlis Tanaxmadac dakavSirebuli rkinis ionebi NADPH-

specifikur jaWvSi funqcionirebs, rogorc radikalwarmomqmneli

centrebi, romlebic zeJanguri Jangvis inicirebas iwveven. aqedan

gamomdinare, am mkvlevarebma varaudi gamoTqves, rom rkinis roli

radikaluri gziT hidrozeJangebis daSlaSi mdgomareobs. warmoqm-

nili zeJanguri radikalebi Tavis mxriv hidrozeJangebis warmoqmnis

axal reaqciebs inicireben. maTi warmodgeniT rkinis ionebi radikal-

warmoqmnisas jaWvis dagrZelebis stadiaze muSaoben. amgvarad Camoya-

libda kompromisuli Sexeduleba, romlis Tanaxmadac dakavSirebul

rkinas SeuZlia ifunqcioniros rogorc zeJanguri Jangvis

„iniciatorma“, aseve jaWvis „damagrZelebelma“.

lipidebis Tavisufalradikaluri Jangvis inducirebisas mikro-

somul membranaSi struqturul-funqciuri Tanafardobebi Seswavli-

lia in vitro pirobebSi [29]. naCvenebia, rom membranis lipidur fazaSi

mimdinare zeJanguri reaqciebi upirveles yovlisa arRveven

fosfolipidebis poliujer cximovan acilebs (C22:6; C22:6). amis

Semdeg procesSi erTveba ormagi kavSirebis naklebi ricxvis (C18:2; C18:0) mqone cximovani mJavuri naSTebi. lipidTa Jangvis produqtebis

dagrovebis paralelurad SeiniSneba membranadakavSirebuli fermen-

tebis aqtivobaTa cvlilebebi. monotonurad klebulobs citoqrom

P450-is Semcveloba (nawilobriv misi citoqrom P420-ad konversiis xarjze). Sesabamisad qveiTdeba aminopirinis demeTilirebisa da 3,4-

benzpirebis hidroqsilirebis siCqare. glukozo-6-fosfatazis cvli-

leba 3-fazian bunebas amJRavnebs: zeJanguri Jangvis induqciis sawyis

stadiaze SedarebiTi inhibirebis Semdeg aqtivobis Tavdapirvel

donemde aRdgena xdeba. misi aqtivacia dakavSirebulia lipidTa

zeJangur JangvasTan da rogorc Cans mikrosomuli membranis ganv-

138

ladobis cvlilebiTaa ganpirobebuli. arsebiTi isaa, rom membranis

mTlianobaze damokidebul fermentTa aqtivobebis cvlilebebi gaci-

lebiT ufro adre SeiniSneba, vidre membranis eleqtronul-mikro-

skopuli ultrastruqturuli cvlilebebi.

mikrosomebSi ujeri rigis cximovan mJavaTa zeJanguri Jangvis

reaqciis steqiometriis sakiTxi gansakuTrebiT detalurad fermen-

tuli sistemebisTvisaa Seswavlili, ramdenadac swored misTvisaa

damaxasiaTebeli im gansakuTrebuli meqanizmebis arseboba, riTac igi

arafermentuli sistemebisagan gansxvavdeba. mocemuli sakiTxis

gamokvlevebis dawyeba hoxSteinisa da ernsteris [6] kvlevebs

ukavSirdeba. maT SeZles daedginaT, rom sareaqcio areSi zeJanguri

Jangvis iniciatoris (ADP_Fe2+) Tanamyofobisas NADPH-is erTi

molis Jangva 10-20 moli Jangbadis aRdgenas iwvevs. ADP_Fe2+-kompleqsis gareSe mikrosomul NADPH-damokidebul eleqtronTa

gadatanis procesSi erTi moli NADPH 1 mol O2-s aaqtivebs.

mogvianebiT amave laboratoriaSi miRebul iqna ufro dabali

koeficienti: ∆NADPH : ∆O2=1 : 4 [9]. meisa da makkeis [30] gamokv-levebma erTi metad saintereso faqti gamoavlina. maT cdebSi

∆NADPH : ∆zeJanguri Jangva : ∆O2 fardoba 1 : 1 : 4-s Seadgenda. amas-Tan koeficienti ∆O2 : ∆NADPH mudmivi ar iyo da reaqciis msvle-

lobaSi 1-dan (reaqciis dasawyisSi) 4-mde (reaqciis dasasruls)

icvleboda. koeficientis aseTi cvlileba ar SeiZleba aixsnas

reaqciis warmoqmnili karboniluri naerTebis Semdgomi gardaqmniT,

anu es araa warmoqmnili arasruli Jangvis produqtebis Semdgom

daJangvaze Jangbadis xarjvis Sedegi. is, rom aseTi produqtebi ar

warmoiqmneba, dasturdeba zeJangebis gaqrobis qromatografiulad

Semowmebuli SedegebiTa da ioduri ricxvis cvlilebebiT.

gamoTvlebma aCvenes, rom arsebobs garkveuli Sesabamisoba gamqral

ormagi kavSirebis ricxvsa da reaqciis msvlelobaSi STanTqmul O2-s Soris. maSasadame, mikrosomebSi zeJanguri Jangvis ganviTarebis

Tanazomierad yovel molekula daJangul NADPH-ze sul ufro

meti warmoqmnili lipidis zeJangis molekulebis ricxvi modis. es

situacia Zlier waagavs procesebs, romlebic jaWvuri reaqciebisas

SeiniSneba; e.i. sadac iniciatoris yovel molekulaze produqtis

ramdenime molekula modis, romelTa raodenoba jaWvis sigrZezea

damokidebuli [19]. jaWvuri procesis Sesabamisad jaWvis sigrZis

139

zrda, romelic saboloo jamSi sistemaSi antioqsidantebis daSlas-

Tanaa dakavSirebuli, aseve tipiuria jaWvuri reaqciebisaTvis, ker-

Zod, orvalentiani rkiniT katalizirebuli fosfolipidebis zeJan-

guri JangvisaTvis [19, 31].

amgvarad, antioqsidantebis mainhibirebeli moqmedeba da lipiduri

zeJangebis warmoqmnis steqiometria savsebiT sarwmunod miuTiTebs

NADPH-Tan SeuRlebul lipidTa zeJanguri Jangvis zogierTi sta-

diebis jaWvur, Tavisufal-radikalur bunebaze. am gagebiT fermen-

tuli da arafermentuli zeJanguri Jangvis sistemebi erTmaneTisagan

faqtobrivad ar gansxvavdeba. aRniSnul procesebs Soris sxvaoba,

rogorc Cans, inicirebis adreul stadiebze mTavrdeba, xolo Semd-

gom isini erTi da igive meqanizmiT viTardebian da erTnair prod-

uqtebs warmoqmnian. orive sistemas qemiluminescenciis erTnairi

kinetika gaaCnia, rac imaze metyvelebs, rom lipiduri zeJangebis

fermentul da arafermentul warmoqmnebs Soris gansxvaveba mTavr-

deba procesis sawyis stadiebze, anu manamde, vidre zeJanguri

Jangvis jaWvuri reaqciebi „CairTveba“.

detaluradaa Seswavlili NADPH-specifikuri zeJanguri Jangvis

produqtebi [30, 32-34]. aRmoCnda, rom am procesis Sedegad esTe-

ruli kavSirebis ricxvi ar icvleba. samagierod mkveTrad mcirdeba

ormagi kavSirebis raodenoba da aminuri azotis Semcveloba. ormagi

bmebis Semcveli cximovani mJavebis zeJanguri Jangvis analizma aCvena,

rom Jangvas upirveles yovlisa 6, 4 da 2 ormagi bmebi ganicdian.

xSir SemTxvevaSi aseTi mJavebia Sesabamisi dokozoheqsenmJava, araqi-

donmJava da linolenmJava. swored es ujeri cximovani mJavebi war-

moadgenen fermentuli zeJanguri Jangvis ZiriTad substratebs. imis

gamo, rom cximovan mJavaTa naSTebi glicerinis ConCxis mimarT β-mdgomareobaSia, bunebrivia, Jangva maTze ganxorcieldeba. amas adas-

turebs fosfolipaza A-s SerCeviTi „Seteva“ β-esTerul bmebze. mem-

branuli fraqciis fosfolipaziT winaswari damuSaveba zeJangur

Jangvas ainhibirebs, xolo ukve daJanguli fraqciis am fermentiT

damuSaveba warmoqmnili polaruli produqtebis srul gamoTavi-

suflebas iwvevs [35].

NADPH-Tan inkubirebul mikrosomebidan lipiduri eqstraqtis

STanTqmis speqtrebs Tu gavaanalizebT, davinaxavT, rom masSi war-

moiqmneba SeuRlebuli ormagi da sammagi kavSirebis sistema STanT-

140

qmis maqsimumebiT 230-232 da 280 nm-ebze. amasTan, pirveli zoli

lipidebis hidrozeJangebs ekuTvnis [35].

zeJanguri Jangvis saboloo produqti lipofuscinmJavas msgavsi

pigmenti warmoiqmneba lizinis ε-aminojgufTan malonis dialdehidis

urTierTqmedebiT. igi koniugirebul Sikis fuZes (_N=CH_CH2_

HC=N_) Seicavs. misTvis damaxasiaTebelia fluorescenciis aRgz-

nebis maqsimumi 345-360 nm-ze da gamosxivebis maqsimumi 425-450 nm-

ze. procesis steqiometria aseTia: ∆O2 : ∆malonis dialdehidi : ∆li-

pofuscini = 436 : 16 : 1 [36]. warmoqmnil lipidTa hidrozeJangebs,

Jangvis pirvelad produqtsa da malonis dialdehids Soris urTi-

erTkavSiris Seswavlam aCvena, rom lipidTa zeJangebi malonis

dialdehidTan SedarebiT erTi rigiT meti warmoiqmneba da swrafadve

iSleba: lipiduri zeJangebis maqsimaluri raodenoba reaqciis sawyis

periodSi vlindeba. maTi raodenoba mcirdeba malonis dialdehidis

mudmivad mzardi koncentraciebis fonze. lipiduri zeJangebis didi

nawilidan (Tu mTlianad ara) zustad iseve, rogorc poliujeri

cximovani mJavebidan, malonis dialdehidi iq warmoiqmneba, sadac

isini fosfolipidebTan β-mdgomareobaSi imyofebian. fermentuli

zeJanguri Jangvis procesSi inhibitorebis moqmedeba ar gansxvavdeba

mTliani zeJanguri Jangvis procesze maTi moqmedebisagan [37, 38].

amrigad, zeJanguri Jangvis substratebad gamoiyenebian dokozo-

heqsen-, araqidon- da linolenmJavebi, romlebic fosfatidil, eTa-

nolaminisa da fosfatidilqolinis molekulebSi β-mdgomareobaSi imyofebian. reaqciis dasawyisSi unda arsebobdes gansazRvruli sxva-

obebi membranebSi zeJanguri Jangvis inicirebis centrebis gansxva-

vebuli ganlagebis gamo. es varaudi ki Tavis mxriv sarwmunod

amtkicebs NADPH-is damaCqarebel moqmedebas askorbat-damokidebul

zeJangur Jangvaze da fermentul sistemaSi warmoqmnili zeJangebis

citoqrom P450-ze gaZlierebul damazianebel moqmedebas (cxrili

2.1).

ujeri cximovani mJavebis zeJanguri Jangvis saboloo produqtebs Soris Txelfenovani qromatografiiT 18 aldehidi da ketoni iden-

tificirdeba [19]. yvela isini erTsafexuriani JangviTrmiiReba. karboni-luri naerTebis karboqsilurad Semdgomi daJangva aRar xdeba [35].

mikrosomul sistemaSi in vitro cximovan mJavaTa zeJanguri Jangva ar SeiZleba yvela maRalujeri cximovani mJavebis sruli daJangviT

141

mimdinareobdes. malimitirebel faqtorebad am SemTxvevaSi, upirve-

les yovlisa, reducirebadi eqvivalentebis donorebi (NADPH da

askorbati) unda warmoadgendnen. es savsebiT gasagebicaa: membranis

mkacri orientaciis gamo Jangvis jaWvis sigrZe mcirea da procesi

ar SeiZleba avtokatalizurad warimarTos. membranul preparatebSi

bunebrivi antioqsidantebis Semcvelobac mcirea da amitom maT ar

SeuZliaT zeJanguri Jangvis siCqareze raime arsebiTi gavlenis

moxdena.

cxrili 2.1.

NADPH-isa da askorbatis gavlena lipiduri zeJangebis warmoqmnis

siCqareze (warmoqmnili malonis dialdehidi mg-1 . wT-1) [19]

aRmdgeneli inkubaciis dro

10 wT 20 wT

NADPH, 1.0 mM 36 50

askorbati, 0.8 mM 50 67

NADPH, 1.0 mM + askorbati 0.8 mM 62 88

in vitro pirobebis sawinaaRmdegod ujredSi aRdgenili NADPH da

askorbati nakleb savaraudoa SemzRudveli faqtorebi iyvnen, ramd-

enadac citoplazmaSi maTi realuri koncentraciebi (Sesabamisad

2.5.10-4 da 10-3 M) savsebiT sakmarisia zeJanguri Jangvis reaqciebis

maqsimalur siCqareTa misaRwevad. sainkubacio areSi askorbat-damoki-

debul sistemaze rkinis ionebis mkveTri gamaaqtiurebeli moqmedeba

imaze metyvelebs, rom swored am ionebis deficits SeuZlia

arafermentuli zeJanguri Jangvis siCqaris limitireba. fermentul

sistemaSi mocemul faqtors, savaraudoa, rom didi mniSvneloba ar

unda gaaCndes, radgan rkinasTan misi swrafva maRalia da ujredSi am

ionis realuri koncentracia, rogorc Cans, fermentulad zeJangebis

maqsimaluri siCqariT warmoqmnis uzrunvelsayofad savsebiT sakma-

risia. am SemTxvevaSi wina planze steriuli faqtori, anu radikal-

warmoqmnis centrebisa da Jangvis substratebis (ujer cximovan

mJavaTa) sivrculi ganmxoloeba gamodis. zeJanguri Jangvis sistemis

Jangbadisadmi dabali swrafva am sistemas saSualebas ar aZlevs

efeqturi konkurencia gauwios or sxva _ citoqromoqsidazur da

citoqrom P450-oqsigenazur sistemebs.

142

arsebuli eqsperimentuli monacemebis safuZvelze SeiZleba naT-

lad gamoikveTos yvelaze mniSvnelovani debulebebi [20]:

1. mikrosomul membranebSi damtkicebulia lipidTa zeJanguri Jang-

vis ori sistemis arseboba, romlebic erTmaneTisagan principulad

iniciaciis stadiiT gansxvavdebian. NADPH-specifikur sistemaSi

rkinis ionebi (radikalwarmomqmneli centrebi) eleqtronTa gada-

tanis fermentuli gziT aRdgeba. arafermentul sistemaSi rkinis

aRdgena askorbatiT, an sxva Sesabamisi redoqs-potencialis mqone

aRmdgeneliT xdeba;

2. orive procesis kinetika da steqiometria im mosazrebis sasar-geblod metyvelebs, romlis Tanaxmadac maT mimdinareobas erTi

saerTo _ jaWvuri, radikaluri reaqciebi udevs safuZvlad;

3. iTvleba, rom or- da samvalentiani rkinis ionebi ukavSirdebian

cilebis gansazRvrul funqciur jgufebs, xdebian radikalwar-

momqmneli punqtebi, anu reaqciis iniciaciis centrebi. am mxriv

yvelaze mosalodnelia, rom lipidTa fermentul zeJangur Jang-

vaSi wamyvani roli citoqrom P450-is SH-jgufebs ekuTvnis.

2.2.2 citoqrom P450-is monawileoba cximovani mJavebis

hidroqsilirebasa da epoqsidirebaSi

citoqrom P450-iT katalizebuli cximovani mJavebis Jangvis

reaqciebi SeiZleba hidroqsilirebisa da epoqsidirebis reaqciebad

davajgufoT. hidroqsilirebisas iJangeba rogorc terminaluri meTi-

lis jgufi [39], aseve alifaturi jaWvis Sida naxSirbadatomebi

[40]. cercvis (Vicia faba) mikrosomebis mier palmitinmJavas ω-hidroqsilirebis Seswavlisas [39] gairkva, rom reaqciaSi citoqrom

P450 monawileobs. SidajaWvuri monooqsigenirebisas erTi da igive

mJava SeiZleba sxvadasxva mdgomareobaSi daiJangos, rac mkafiod Cans

miwavaSlas (Helianthus tuberosus) mikrosomebis mier laurinmJavas 8-,

9- da 10-hidroqsilirebis magaliTze (nax. 2.3_II) [40].

unda aRiniSnos, rom laurinmJavas mimarT citoqrom P450 gark-veul stereospecifikurobas amJRavnebs, rac mcenaris saxeobazea da-

mokidebuli. fermentis orive _ ω-hidroqsilazuri da SidajaWvur-

hidroqsilazuri aqtivobebi erTsa da imave mcenareSi arasodes

mJRavndeba [41].

143

COOH COOH

OHlaurinmJava 12-hidroqsi-laurinmJava

I.

COOH

OH

COOH

COOHOH

COOH

OH

laurinmJava

9-hidroqsi-laurinmJava

10-hidroqsi-laurinmJava

8-hidroqsi-laurinmJava

II.

nax. 2.3. laurinmJavas citoqrom P450-damokidebuli ω-(I) da

SidajaWvuri (II) hidroqsilireba [40].

citoqrom P450-damokidebul ω-hidroqsilirebas ganicdis rog-

orc najeri, iseve ujeri mJavebi [42, 43]. ujeri mJavebi SidajaW-

vuri da terminaluri hidroqsilirebis garda, epoqsidirebasac gani-

cdis. es ukanaskneli SeiZleba warimarTos, rogorc ω-hidroqsi-lirebis Semdgomi etapi, an rogorc SidajaWvuri hidroqsilirebis

Tanmdevi reaqcia. cercvis mikrosomebSi 9-dodecinmJavas hidroqsi-

lirebisas adgili aqvs ujeri bmis naxSirbadatomebis epoqsidi-

rebasac, romelic 8-hidroqsilirebis konkurentulad mimdinareobs

(nax. 2.4) [44].

zogierT mkvlevars [45] miaCnia, rom ujeri mJavas epoqsidirebis

reaqcias igive hemoproteini akatalizebs, romelic mis ω- da Sida-jaWvur hidroqsilirebaSi monawileobs. am daskvnamde isini mividnen

citoqrom P450-is stereo- da regio-specifikurobis kvlevis Sedegad, rodesac erTmaneTs adarebdnen ormagi bmis mdebareobiT gansxvavebul

dodecinmJavas izomerebis hidroqsilirebas da epoqsidirebas.

144

COOH

COOHO

COOH

OH

COOHOH

9-dodecinmJava

9,10-epoqsi-laurinmJava

8-hidroqsi-9-dodecinmJava

12-hidroqsi-9-dodecinmJava

nax. 2.4. 9-dodecinmJavas citoqrom P450-damokidebuli epoqsidi-

reba da SidajaWvuri hidroqsilireba [44].

aRmoCnda, rom SidajaWvuri hidroqsilirebisa da epoqsidirebis

reaqciebs Soris kargad gamoxatuli konkurenciaa; amasTan, Tu mJa-

vaSi ormagi bma jaWvis boloSi _ me-11-e da me-12-e naxSirbadatomebs

Sorisaa, maSin SidajaWvuri monooqsigenirebis intensivoba bevrad

sustia epoqsidirebasTan SedarebiT; ormagi bmis gadanacvleba jaWvis

SigniT ki epoqsidirebis saSualebas amcirebs da am dros upira-

tesoba SidajaWvur hidroqsilirebas eniWeba. dodecinmJavas izome-

rebze erTi Tvalis SevlebiTac ki naTeli xdeba, rom rac ufro

jaWvis SigniTaa ormagi bma, miT ufro „miudgomelia“ igi fermenti-

saTvis da am dros epoqsidirebis reaqcia izRudeba.

COOHCOOHCOOH COOH COOH

9-dodecinmJava10-dodecinmJava11-dodecinmJava 8-dodecinmJava 7-dodecinmJava aqve unda aRiniSnos, rom am kanonzomierebas ewinaaRmdegeba erTi

faqti: cis- da trans-izomerebis epoqsidireba erTnairi siCqariT

mimdinareobs.

zeviT ganxilul reaqciebSi citoqrom P450-is monawileoba kla-

sikuri kataliziT _ monooqsigenazuri ciklis mixedviT xorci-

145

eldeba: cximovani mJavas dasaJangad igi molekulur Jangbadsa da

aRdgenili nikotinamiduri kofermentebis aRmdgenel eqvivalentebs

iyenebs. miuxedavad amisa, arsebobs citoqrom P450-is katalizuri

aqtivobis gamovlenis sxva meqanizmic, rodesac es hemoproteini

hidroqsilirebisaTvis organul hidrozeJangebs moixmars da am dros

igi cximovan mJavaTa hidrozeJangebis gardaqmnebs NAD(P)H-isa da

O2-is gareSe akatalizebs [45]. am SemTxvevaSi citoqrom P450-is superojaxis zigierT warmomadgenels cximovani mJavebis hidroze-

Jangebis daJangva zeJanguri Jangvisagan gansxvavebuli ufro „natifi“

meqanizmiT SeuZlia, romlis drosac cximovani mJavas naxSirbadovani

ConCxi malonis dialdehidamde uxeSad ki ar iglijeba, aramed misi

struqturuli fragmentebidan maRali fiziologiuri aqtivobis mqo-

ne molekulebi warmoiqmnebian. amis magaliTia alenoqsidebis warmo-

qmna linolenmJavas hidrozeJangidan (nax. 2.5) [45].

ferment lipoqsigenazas moqmedebis Sedegad formirebuli lino-

lenmJavas hidrozeJangi SeiZleba advilad daiSalos fermentuli

da/an arafermentuli zeJanguri Jangvis Sedegad, magram aRmoCnda,

rom mcenareebSi lipoqsigenazasTan Serwymulia citoqrom P450-is izoforma (CYP74), romelic klasificirdeba, rogorc alenoqsid-

sinTaza [46]. es hemoproteini hidrozeJangidan epoqsids (e.w. aleno-

qsids) warmoqmnis, ris Sedegadac saTave eZleva mcenareuli hormonis _ JasminmJavas biosinTezs. aseve mniSvnelovania citoqrom P450-is kidev erTi fermentuli aqtivobis gamovlena _ cximovani mJavas hidroze-

Jangis alternatiuli daSla 6-naxSirbadatomian ujer aldehidad,

romelic mcenaris paTogenisagan dacvis reaqciebSi monawileobs [47].

alenoqsidsinTaza mniSvnelovan rols asrulebs heveaSi (Hevea brasi-liensis) mimdinare kauCukis formirebis procesSic [48].

JasminmJavas biosinTezi fosfolipaza A-s mier fosfolipidebis

hidroliziT iwyeba, ris Sedegadac linolenmJava miiReba (nax. 2.5).

igi sasignalo molekulaa, romelic or _ principulad gansxvavebul

regulatorul procesSi monawileobs [49]:

1. mcenaris vegetaciuri zrdis SeCereba da mosvenebul mdgoma-

reobaSi gadayvana _ am dros iwyeba specifikuri samarago cilebis

biosinTezi, rasac Tan sdevs nayofebis, Teslebis, tuberebisa da

bolqvebis formireba;

146

OOH

COOH

O2

COOH

COOH

O H

H

O

COOH

O

COOH

O

O

H

O

H

α-linolenmJava

11-hidroperoqsi-linolenmJava

lipoqsigenaza

+

cximovani mJavas

hidrozeJang-liaza

(citoqrom P450 - CYP74)

+

dodec-en-9-al-12-mJava

heqsen-9-ali alenoqsidi

alenoqsidsinTaza

(citoqrom P450 - CYP74)

...

JasminmJava

zeJanguri

Jangva

...

malonis diladehidi

...

nax. 2.5. JasminmJavas winamorbedi alenoqsidis warmoqmna ujeri cxi-

movani mJavas hidrozeJangidan citoqrom P450-is monawi-

leobiT [45].

147

2. abioturi da bioturi stresebis mimarT imunuri sapasuxo reaq-ciebis gaZliereba _ am dros sinTezdeba naerTebi, romlebic

paTogenebis sawinaaRmdegod moqmedeben [50]. esenia:

− eqstensinebi, romelTa moqmedebiTac msxvildeba ujredis ked-

lebi, rac xels uSlis paTogenebis ujredSi SeRwevas;

− Tioneinebi (cisteiniT mdidari cilebi), romlebic ikavSireben

da aneitraleben paTogenebis toqsinebs;

− fitoaleqsinebi _ sesqviterpenoiduli bunebis mcenareuli anti-

biotikebi;

− fenoluri bunebis naerTebi, mag., salicilmJava, romelic fungi-

ciduri moqmedebiT gamoirCeva;

− peptidi sistemini, romelic paTogenebis proteazebis inhibi-

rebas iwvevs da amiT mcenares inficirebisagan icavs.

− fermentebi, romlebic dazianebuli ujredebis daprogramebul

kvdomas _ apoptozs iwveven [51].

JasminmJava iseTi cilebis biosinTezsac iwvevs, romlebic amci-

reben floemuri nakadis siCqares, xuraven bageebs da amiT xels

uwyoben gvalvian amindSi mcenaris wylovani balansis SenarCunebas.

arasaWiroebis SemTxvevaSi JasminmJava samarago glikozidis saxiT

inaxeba da saWiroebis SemTxvevaSi Tavisufldeba.

JasminmJava SeiZleba gamoiyos mcenaris foTlebidan aqroladi

eTeris _ meTilJasminatis saxiT. es naerTi advilad Sedis sxva

foTlebSi, sadac isev JasminmJavad iSleba da zemoTaRniSnuli dac-

viTi naerTebis inducirebas iwvevs. amdenad, meTilJasminatis gamoyo-

fiT mcenaris sagangaSo signalis gamocema da mosalodneli stre-

suli situaciisaTvis dauzianebeli qsovilisTvis an mezoblad mzar-

di mcenareebis winaswari momzadeba SeuZlia.

alenoqsidsinTaza da cximovani mJavas hidroperoqsidliaza sxva

citoqrom P450-ebisagan pirveladi struqturiTac gansxvavdebian.

mag., maT aqtiur centrSi treonini izoleiciniTaa Canacvlebuli

[52]. aseTive Tavisebureba axasiaTebs cxovelebSi prostaglandinebis

sinTezSi monawile citoqrom P450-Semcvel TromboqsansinTazas [53].

unda aRiniSnos, rom es mciredi gansxvaveba principuli xasiaTisaa,

ramdenadac citoqrom P450-is mier Jangbadis aqtivaciis procesSi swored am poziciaSi mdgomi aminomJavuri naSTi monawileobs.

arsebobs mosazreba, rom alenoqsidsinTazas SeuZlia gamoamJRavnos

148

cximovani mJavas hidroperoqsid-liazuri aqtivobac, raSic mas xels

uwyobs aqtiur centrSi histidinis naSTis specifikuri konfi-

guracia [46].

alenoqsidsinTazas apofermentSi N-terminaluri bolos aminomJa-

vuri Tanmimdevroba plastidebis da mitoqondriebis membranebTan

dakavSirebuli specifikuri peptidebis homologiuria [53]. am fer-

mentis aqtivobis gamomJRavnebisaTvis aucilebel pirobas membranul

struqturebTan kavSiri warmoadgens [54]. fermenti aramarto

mikrosomebze, aramed qloroplastebis da vakuolebis membranebzecaa

lokalizebuli [51].

alenoqsidebi prostaglandinebis biosinTezis intermediatebsac

warmoadgenen [55]. aseTi naerTebi identificirebulia bardis (Pisum sativum) mikrosomebis mier araqidonmJavas Jangvis produqtebSi [56]

da selis (Linaria vulgaris) Teslebis cximovani mJavebis hidrozeJan-

gebis citoqrom P450-damokidebuli degradaciiT miRebul naerTebs

Soris.

2.3 citoqrom P450 fenolur naerTTa biogenezSi

dariCinmJava-4-hidroqsilaza (EC 1.14.13.11), romelic trans-

dariCinmJavas p-hidroqsi-dariCinmJavad (p-kumarmJavad) gardaqmnis,

fenilalanin-amiak-liazas (EC 4.3.1.5) Semdeg meore gasaReb ferments warmoadgens mravalricxovani fenoluri bunebis (ligninebis, tani-

nebis, flavonoidebis, fenilkarbomJavebisa da a.S.) naerTTa biosin-

Tezis gzaze (nax. 2.6). am nivTierebaTa umetesobas hidroqsilis

jgufi meoTxe naxSirbadis poziciaSi gaaCnia. fermenti klasifi-

cirebulia mikrosomul Sereuli funqciis oqsidazad, anu monooqsi-

genazad. igi multifermentuli kompleqsia da aqtivobisaTvis mole-

kulur Jangbads da NADPH-s saWiroebs, romelsac igi am kofer-

mentis generirebadi sistemidan (NADP + glukozofosfatdehidroge-

nazidan) iRebs. aRdgenili piridinnukleotididan NADPH-citoqrom

P450-reduqtazas saSualebiT eleqtronebi citoqrom P450-ze gadai-taneba. dariCinmJava, rogorc substrati, I-tipis speqtrul cvlile-

bebs iZleva [57]. fermenti, upirveles yovlisa, NADPH/NADP-fardobiT regulirdeba.

149

naxSirwylebiariTrozo-4-

fosfatiprefenmJava

glikolizifenilalanini

fenil-amiak-liaza

(EN 4.3.1.5)

dariCinmJava-4-hidroqsilaza

(EN 1.14.13.1)dariCinmJava

kumarinebi

ligninebi

fenilkarbon-

mJavebi

taninebi

flavonoidebi (kateqinebi, antocianebi)

acetil-KoA

...

CH3CO~SKoA

pentozuri cikli

nax. 2.6. fenolur naerTTa biogenezis zogadi sqema.

dariCinmJava-4-hidroqsilaza Tavisi substratisadmi absolutu-

rad specifikuria; xolo SesaZlo substratebad fenilalaninis an

4-hidroqsidariCinmJavas gamoyenebisas maTi Sesabamisi hidroqsi-

warmoebulebad _ Tirozinad, an 3,4-hidroqsidariCinmJavad (yavis

mJavad) gardaqmna ar registrirdeba. substratisadmi maRali swrafva

ganapirobebs produqtis mkacr kontrols Tavissave sinTezze, anu am

produqtis koncentracia ujredSi mkacri TviTregulirebis qveS

imyofeba. es imis maCvenebelia, rom dariCinmJavas warmoqmnis stadia

fenolur naerTTa biosinTezis procesSi regulirebis mniSvnelovan

sakontrolo ubans warmoadgens da rom sakuTari produqtiT fenil-

alanin-amiak-liazuri inhibireba regulaciis pirvel ubans qmnis [57]. dariCinmJava-4-hidroqsilaza sruladaa lokalizebuli mikroso-

mul fraqciaSi; mag., bardis apikalur kvirtebSi fermentis aqtivoba

mxolod endoplazmuri retikulumis membranebSia gamovlenili, maSin

rodesac plastidebisa da mitoqondriebis fraqciebi am fermentul

aqtivobas ar avlenen [58]. TviT mikrosomul fraqciaSi ZiriTadi

aqtivoba upiratesad xorklian membranebSia koncentrirebuli. igi

iTrguneba naxSirbadis monooqsidiT, diTiotreitoliT da gansakuT-

rebiT 2-merkaptoeTanoliT [59]. xaoiani retikulumis qvefraqciaSi

magniumis ionebi dariCinmJava-4-hidroqsilazas aqtivobis induci-

rebasa da citoqrom P450-is Semcvelobis zrdas iwvevs. gluvi memb-

150

ranebis qvefraqcia citoqrom P450-s ar Seicavs, magram mniSvne-

lovani 4-hidroqsilazuri aqtivoba gaaCnia. es faqti imaze miuTi-

Tebs, rom dariCinmJavas hidroqsilireba SeiZleba citoqrom P450-is gareSec ganxorcieldes [60]. amasTan dakavSirebiT saintereso

Sedegebi aqvT miRebuli riCsa da lambs [61]. cdebi tardeboda

kartofilis garecxil anaTlebze. am mkvlevarebma aCvenes, rom

citoqrom P450 ikavSirebs trans-dariCinmJavas da es urTierTqmedeba speqtrebis warmoqmnis mixedviT I-tipis substratebis analogiuria.

sxva qsovilebSi hemoproteinis arsebobis pirobebSi am substratis

hidroqsilireba ar xorcieldeba. aqedan gamomdinare, gamoTqmul iqna

mosazreba imis Sesaxeb, rom umaRles mcenareebSi citoqrom P450-s sxva funqciebic gaaCnia da rom misi substratuli specifikuroba

sxvadasxva mcenareebSi farTo varirebas ganicdis.

sorgos eTiolirebuli foTlebidan miRebuli dariCinmJava-4-

hidroqsilazas fermentuli eqstraqtis aqtivoba fermentis mrav-

lobiT formebTan, an sxvadasxva disociaciis xarisxis mqone for-

mebis narevTan aRmoCnda dakavSirebuli [62]. es aqtivoba metad aram-

dgradia, magram misi reaqtivacia SesaZlebelia eqstraqtis 30°C-mde SeTbobiT. garda amisa, in vitro cdebSi fermentis maqsimaluri

aqtivobis misaRwevad aucilebelia disulfiduri bmebi, romlebic am

SemTxvevaSi fermentis msxvili agregatebis warmoqmnas uzrunvel-

yofen. stafordis mier [63] dadgenil iqna, rom sorgos axalgazrda

foTlebis mikrosomuli fraqcia dariCinmJavas-4-hidroqsilazur

aqtivobasTan erTad fenilalanin-amiak-liazur aqtivobebsac flobs.

aqedan gamomdinare, aRniSnulma mkvlevarma misaRebad CaTvala, rom

monofenolis mahidroqsilirebeli fermenti im fermentuli kompleq-

sis nawilia, romelic aminomJavas C6-C3-tipis fenolur mJavebad

gardaqmnis. membranadakavSirebuli fermentuli sistema monawileobs

cianogenuri glukozidebis biosinTezSi da unari aqvs ganaxor-

cielos p-hidroqsifenilacetonitrilis hidroqsilireba [64].

iseve, rogorc yvela sxva membranadakavSirebuli fermenti, dari-

CinmJava-4-hidroqsilazac aucileblad saWiroebs fosfolipidebs,

kerZod lecitins. mag., oxraxuSis (Petroselenum hortense Hoffm.) sus-penziur kulturaSi nanaxi iqna, rom fosfolipaziT fosfolipidis

∼60%-is mocilebis Semdeg hidroqsilazuri aqtivoba mTlianad

qreba [65], xolo Semdgom nedli mikrosomuli lipiduri eqstraqtis

151

damatebiT hidroqsilazuri aqtivoba mtkiced aRdgenas ganicdis.

amJamad, dariCinmJava-4-hidroqsilaza endoplazmuri membranebis

samarkero fermentadaa miRebuli [61].

antocianinebis biosinTezis gzaze intermediatebs 3′,5′-hidroqsi-lazuri (F3′,5′H) reaqciis produqtebi warmoadgenen. es fermenti

flavonon naringeninis eriodiqtiolad da pentahidroqsiflavoninad

gardaqmnas akatalizebs. paralelurad hidroqsilirebis reaqciiT

dihidroflavonoli da dihidrokemferoli dihidrokvercetins da

dihidromiricetins warmoqmnis (nax. 2.7) [66, 67].

O OHOHOH O

O OHOHOH O

OHO OHOH

OH O

OHOH

O OHOHOH O OH O OHOH

OH O OH

OHO OHOH

OH O

OHOHOH

a.

b.

flavonoid-3',5'-

hidroqsilaza

CYP75

flavonoid-3',5'-

hidroqsilaza

CYP75flavonoid-3',5'-hidroqsilaza

flavonoid-3'-hidroqsilaza

CYP75

CYP?

flavonoid-3',5'-hidroqsilaza

flavonoid-3'-hidroqsilaza

CYP75

CYP?

nax. 2.7. naringeninis (a) da dihidrokemperolis (b) gardaqmna

eriodiqtiolad da hidrokvercetinad. reaqciebs Sesaba-

misad akatalizeben flavonoid-3′,5′-hidroqsilaza da fla-

vonoid-3′,5′-hidroqsilaza. reaqciebis produqtebma SeiZ-

leba Semdgom hidroqsilireba ganicadon flavonoid-3′,5′-hidroqsilazas moqmedebiT da pentahidroqsiflavanoni da

dihidromiricetini warmoiqmnan [66, 67].

unda aRiniSnos, rom F3′,5′H-is makodirebeli geni mcenareuli

citoqrom P450-is pirveli geni iyo, romlis klonirebac Petunia hibrida-s yvavilSi bioqimiuri da genetikuri midgomebis kombinirebiT

ganxorcielda [68]. Petunia-s da sxva saxeobebis yvavilebSi di- da

tri-hidroqsilirebuli warmoebulebi _ glukozidebi, cianidini da

delfinidoni muqi wiTeli feris pigmentebs warmoadgenen. F3′,5′H-is

gamoyenebiT yvavilis feris manipulirebis SesaZlebloba gamoyenebul

iqna Sesabamisi genis izolirebisaTvis [69].

152

sayuradReboa, rom F3′,5′H bioqimiurad gansxvavdeba flavonoid-

3′-hidroqsilazasagan (F3′H), romelic naringenins da dihidro-

kumferols eriodiqtiolad da dihidrokvercetinad gardaqmnis (nax.

2.7). F3′,5′H-s SeuZlia aqtiur centrTan miierTos dihidroqsili-

rebuli B-birTvi, xolo F3′H-s amis unari ar gaaCnia.

2.4 citoqrom P450 alkaloidebis biosinTezSi

alkaloidebis umetesoba heterociklSi fuZe Tvisebebis mqone

azotis erTi atomis Secvel naerTebs miekuTvneba. mcenareSi isini

ZiriTadad organuli mJavebis (vaSlmJavas, RvinomJavas, limonmJavas

da sxv.) marilebis saxiTaa. zogierTi alkaloidis molekulaSi sxva-

dasxva azotovani heterociklis kombinacias vxvdebiT. ase maga-

liTad, nikotinis molekulaSi erTmaneTTanaa dakavSirebuli piri-

dini da pirolidini, xolo amidis saxiT zogierT dehidrogenazas

SemadgenlobaSi imyofeba. zogad Tvisebebs rac Seexeba, es maTi

uaRresad maRali fiziologiuri aqtivobaa [70].

alkaloidebis biosinTezis gzebi sxva nivTierebebTan (mag., ste-

rolebTan an terpenebTan) SedarebiT naklebadaa Seswavlili. es

gansakuTrebiT Sualeduri cvlis nebismieri kvlevis im damam-

Tavrebel stadias exeba, romelic individualuri fermentuli sis-

temebis gamocalkevebasa da daxasiaTebas moicavs. mcenareSi alkalo-

idebi gansazRvruli aminomJavebidan iwarmoeba da biogenezis ZiriTa-

di gza amino-, imino-, karbonilis jgufisa da Zvradi naxSirbad-

atomis kondensaciaa [71]. Wvavis rqis (soko Claviceps purpurea-s mozamTre forma, romelic Wvavis marcvalze viTardeba) Sedgeni-

lobaSi myofi alkaloidebis erTi mxriv, warmoqmnasa da meore

mxriv, aminomJavur cvlas Soris pirdapiri kavSiri gamovlinda [70].

amis sasargeblod metyvelebs cdebis monacemebi, romlebSic 14C-iT moniSnuli aminomJavebi gamoiyeneboda. isini mcenareSi foTlis

yunwebidan an vakuum-infiltraciiT, wyalxsnarebis saxiT SeyavdaT.

14C-ornitiniT mcenaris damuSavebisas radioaqtiuri naxSirbadis

mniSvnelovani nawili nikotinis pirolidonis birTvSi jdeboda.

axalgazrda manWkvalaSi 14C-lizinis Seyvanisas radioaqtiuri niSani

gansakuTrebiT intensiurad alkaloid lupaninSi erTveboda. moniS-

nuli liziniT Catarebulma cdebma aCvenes agreTve, rom misi

153

ciklizaciis Sedegad koniini _ Conium maculatum-is mTavari

alkaloidi warmoiqmneba. alkaloid anabazinis piperidinuri birTvic

lizinidan iwarmoeba. erT-erTi Zlier Sxamiani alkaloidi _

hiosciamini, ornitinis, fenilalaninisa da ZmarmJavas ori naSTis

kondensaciiT sinTezdeba; TviT morfinis winamorbedebs Tirozini da

meTionini warmoadgenen; nikotinmJavas piridinuli birTvi asparagin-

mJavasTan gliceraldehid-3-fosfatis kondensaciiT miiReba [70].

amJamad mogvepoveba mTeli rigi monacemebisa citoqrom P450-is mniSvnelovani funqciuri rolis Sesaxeb alkaloidebis biosinTezSi.

ambikasa da Tanaavtorebis mier [72, 73] Seswavlilia L-triftofa-nisa da misi zogierTi analogis unari, daukavSirdnen citoqrom

P450-s da fermentis daJangul formasTan warmoqmnan kompleqsi. C. purpurea-s miceliumis preparatebze triftofanTan fenobarbitalis

an 3-meTilqolantrenis damatebas Tan axlda b5- da P450-citoq-romebisa da ergoalkaloidebis Semcvelobis erTdrouli zrda.

Vinca rosea-s eTiolirebuli nazardebidan gamoyofil iqna

monooqsigenaza, romelic hidroqsilirebis reaqciiT monoterpenul

spirtebs _ geraniolsa da nerols (cis-geraniols) 10-hidroqsiwar-

moebulebad gardaqmnis [74, 75]. geraniolis hidroqsilireba izrdeba

diTiotreitoliT (nakleb efeqturia monoTioli) da inhibirdeba

fosfolipaziT, meTiraponiT an citoqrom P450-is sxva romelime

inhibitoriT. geranioli ufro swrafad hidroqsilirdeba, vidre

neroli, Tumca orives Tanabari Km sidideebi gaaCniaT. membranadakav-

Sirebuli hidroqsilaza qolatiT an rubroliT solubilizebis

Semdeg efeqturad iyenebs NADPH-citoqrom P450-reduqtazas, ci-

toqrom P450-s da lipidebs [76].

10-hidroqsiwarmoebulebi indoluri alkaloidebis biosinTezSi

erTvebian. fermentis aqtivoba NADPH- da O2-damokidebulia. igi

mikrosomebSi lokalizdeba da aRdgenili `P450_CO~ kompleqsisaT-

vis damaxasiaTebel speqtrs iZleva. garda amisa, meTiraponiT damu-

Savebisas kompleqsi Seqcevadad inhibirdeba [76]. yovelive es fermen-

tis monooqsigenazuri bunebis naTeli dadasturebaa. dadginda isic,

rom aRniSnuli hidroqsilaza citoqrom P450-is tips ganekuTvneba. aRniSnul gamokvlevaSi cis-da trans-izomeruli aqtivoba ar SeiniS-

neba, rac imaze miuTiTebs, rom geraniolica da nerolic hidroqsi-

lirebas C_10 meTilis jgufTan ganicdian [77].

154

Catharanthus roseus-Si geraniolis 10-oqsiwarmoebulad mahidro-

qsilirebeli oqsigenaza inhibirdeba indolis jgufis iseTi alaka-

loidebiT, rogorebicaa katarantini, vinblantini da vindolini. in-

hibirebis kinetikis Seswavlam gamoavlina, rom es procesi arakon-

kurentuli da xazovania, anu fermenti alosterulia. vindolini,

romelic monooqsigenazaze SedarebiT dabal inhibitorul zemoq-

medebas axdens, mniSvnelovnad Trgunavs loganmJavas meTiltrans-

ferazas aqtivobas. es nivTiereba erT-erTi winamorbedia sekolo-

ganinis _ mravali indoluri alkaloidis terpenoiduli nawilis

biosinTezSi [78].

mcenareuli citoqrom P450-is superojaxis erT-erT warmomad-genels (CYP80-berbamuninsinTazas) biradikaluri Serwymis meqaniz-

miT ori alkaloiduri monomeris dimerizaciis katalizeba SeuZlia

(nax. 2.8) [79].

H3C NOOHO

OHCH3H

H3C NOOHO

CH3HOH

H3C NOOHO

CH3H

H3C

OHO

NOOHO

CH3HberbamuninsinTaza

P450 (CYP80)

nax. 2.8. berbamunin-sinTaza akatalizebs ori molekula N-meTil-

koklaurinis Serwymas, ris Sedegadac berbamunini an gua-

tegaumerini warmoiqmneba [79].

am SemTxvevaSi ferments unda gaaCndes iseTi aqtiuri centri,

romelsac ori sakmaod mozrdili alkaloiduri monomeris dakavSi-

reba SeuZlia.

155

2.5 citoqrom P450-is monawileoba hiberelinebis

biosinTezSi

mcenareuli organizmis hormonul sistemaze warmodgenebi sadRe-

isod fitofiziologiis damoukidebel mimarTulebadaa Camoyalibe-

buli. mcenaris zrdis regulatorebis mniSvnelovan warmomadgenlebs

miekuTvnebian fitohormonebi, romelTagan SedarebiT detaluradaa

Seswavlili auqsinebi, hiberelinebi, citokininebi, abscisis mJava da

eTileni. isini Zlier mcire dozebiT moixmareba da damaxasiaTebel

morfofiziologiur cvlilebebs iwveven. aqve unda SevniSnoT, rom

mcenareul da cxovelur hormonebs Soris erT-erTi mTavari gansx-

vaveba mcenareuli hormonebis polifunqciurobaa [80].

hiberelinebi qimiuri bunebiT terpenoidebis (tetracikluri

diterpenoidebis) klass miekuTvneba. saxelwodeba momdinareobs

soko-producentis (Gibberella) laTinuri saxelwodebidan. maT sis-

tematizacias axdenen A-birTvis Jangvis, an molekulis hidroqsi-

lirebis xarisxiT [80].

OH CH3CO O

COOH OHHH

CH2

hiberelinebi

(hiberelmJava)

12

34

56

7

8

9

1011

12

1314

15

16

17

ent-kaureni

18 19

mcenareSi hiberelinebis biosinTezi ase gamoiyureba (nax. 2.9).

mevalonmJavidan mcenareSi fermentulad hiberelmJavisa da saer-

Tod yvela hiberelinis winamorbedi intermediati ent-kaureni

iwarmoeba. Tavis mxriv es metaboliti glikolizis gziT acetil-

CoA-dan miiReba da SemdgomSi skvalensa (dihidrotriterpens) da

sxva izoprenoidul nivTierebas aZlevs dasabams. skvalenic xuT nax-

Sirbadatomiani izoprenuli erTeulebisagan Sedgeba.

156

glikolizi

acetil-CoA

mevalonmJava

izopentenilpirofosfati

skvaleni

hiberelinebi sterolebi

nax. 2.9. mcenareSi hiberelinebis biosinTezis zogadi sqema.

C CH3CH2OH CH2 COOHHOH2C

mevalonmJava

skvalenis naxSirbadatomTa ConCxi kaureni Seuqcevadad iJangeba kaurenolad, kaurenalad da kauren-

mJavad (nax. 2.10) reaqciaTa msvleloba alifaturi hidroqsilirebis

gziT mimdinareobs, xolo citoqrom P450-ebis aRmoCenis Semdeg

naCvenebi iqna, rom am tipis fermentebi hiberelinebis biosinTezis

calkeuli safexurebis katalizebas axorcieleben [79, 81-84]. R CH3 R CH2OH R CHOent-kaur-16-eni ent-kaur-16-en-19-oli ent-kaur-16-en-19-aliR COOH OH R COOH

ent-kaur-16-en-19-mJava 7-β-hidroqsi-kaur-16-en-19-mJava nax. 2.10. kaurenis alifaturi hidroqsilireba.

157

hiberelinebis biosinTezSi citoqrom P450-damokidebuli oqsige-

nazebiT mimdinare reaqciebia [81].

aRniSnuli reaqciebi lokalizebulia mikrosomebSi da aRdgenili

piridinnukleotidebis an 105.103 g supernatantis damatebas saWi-

roeben. am SemTxvevaSi NADPH gacilebiT efeqturia, vidre NADH.

verc aerobul da verc anaerobul pirobebSi NADP da NAD kofer-

mentis funqcias ver asruleben. kaurenolis kaurenalad gardaqmna

inhibirdeba NADP-iT, p-qlormerkuribenzoatiT da SKF-525A-iT,

rac imis pirdapiri dasabuTebaa, rom reaqcias Sereuli funqciis

oqsidaza axorcielebs [81].

mevalonatidan kaurenis, skvalenisa da fitoenis warmosaqmnelad

piridinnukleotidebis garda saWiroa ATP-s, Mg2+-isa da Mn2+

-is

Tanamyofoba. NADPH-is gareSe ar xdeba skvalenis sinTezi, xolo

Mn2+ aucilebelia fitoenis misaRebad. sxva produqtebTan SedarebiT

kaurenis miReba SedarebiT dabal pH-ze xdeba da mevalonatis

gardaqmnaSi igi ZiriTad produqts warmoadgens [85].

haronisa da vestis [86, 87] gamokvlevebiT Marah macrocarpus-is umwifari Teslis endospermidan miRebul mikrosomebSi citoqromebis

P450-isa da b5-is garda speqtrulad identificirda NADPH- da

NADH-citoqrom c-reduqtazuli aqtivobebi. hiberelinebis biosin-

Tezis reaqciebi maqsimaluri aRmoCnda mikrosomul preparatebSi,

xolo supernatantSi dadginda faqtoris arseboba, romelic mikro-

somebiT katalizebul Jangvis siCqares dadebiTad aregulirebs.

NADPH-is damateba NADH-ze metad efeqturia. NADH-is, ATP-isa da NADP-is Semcveli xsnari ki bevrad ufro efeqturia, vidre

calke NADH. 2,4-dinitrofenoliTa da sxvadasxva inhibitorebis

gamoyenebiT Catarebuli cdebiT maT mier naCvenebi iqna, rom ATP astimulirebs NADPH-is sinTezs. NADPH-isa da NADP-s dabali

koncentraciebi erTad ufro efeqturia, vidre romelime maTganis eqviva-lenturi koncentraciebi. FAD-is damateba astimulirebs NADPH-

damokidebul Jangvis siCqares. aqedan gamomdinare, aRniSnulma mkvle-

varebma CaTvales, rom mikrosomebSi arsebobs eleqtronis ori _

NADH da NADPH-damokidebuli satransporto gza da flavinnuk-

leotidis roli Cveulebriv JangvasTan dakavSirebul eleqtronis

gadatanaSi mdgomareobs.

umwifari bardis lebnebidan 1.105 g-ze miRebul supernatantSi

158

naCvenebia, rom 2-14C-mevalonatidan warmoqmnili kaureni proteins

ukavSirdeba. es kompleqsi CO-mgrZnobiare JangvisaTvis vargisi subs-

tratia. arakatalizuri „gadamtani~ proteinis fraqcia SeiZleba

endoplazmuri retikulumidan solubilizdes, daikavSiros kaureni

da mas Sereuli funqciis oqsidazebiT Jangvis unari SesZinos [88].

kompleqsi Seicavs skvalensac, romelic igive pirobebSi kaurenis

msgavsad ganicdis fermentul oqsidacias.

Eucalyptus macrocarpa-s endospermis qsovilis homogenatSi

detalurad iqna Seswavlili (_)-kaur-16-enis zrdis hormonebad

JangviTi gardaqmnebis ori sawyisi stadia [89]. orive reaqcia _ (_)-

kaur-16-enis gardaqmna (_)-kaur-16-en-19-olSi da am naerTis Semd-

gomi daJangva (_)-kaur-16-en-19- amJamad saWiroebs molekulur Jang-

bads da NADPH-s. 18O2-is garemoSi pirveli reaqciis msvlelobisas

aldehidis molekulaSi 18O2-is 78% erTveba. orive reaqcia CO-

inhibirebas emorCileba da misi moqmedeba sores ubnis 422 da 453

nm-ebTanaa dakavSirebuli. piki ganpirobebulia citoqromiT, romelic

ZuZumwovarTa citoqrom P450-is msgavsia. es monacemebi imaze

miuTiTebs, rom hiberelin-hormonebis biosinTezi mcenareebSi da

sterol-hormonebis warmoqmna cxovelebSi zogadad analogiuri

procesebiT xorcieldeba.

2.6 steroidTa mahidroqsilirebeli oqsigenazebi

steroidTa hidroqsilirebas TvalsaCino adgili ukavia im

reaqciebs Soris, romlebic citoqrom P450-Semcveli oqsigenazebiT

katalizdeba. aseT reaqciaTa ricxvs miekuTvneba qolesterinidan

gverdiTa jaWvis mocileba pregnenolonis warmoqmniT da Semdgomi

11β, 17-, 18- da 21β-hidroqsilireba. es reaqciebi ZiriTadad

Tirkmelzeda jirkvlis qerqis mitoqondriebsa da mikrosomebSi

mimdinareobs.

sterolTa biologiuri transformacia, garkveuli azriT, SeiZ-

leba ganxilul iqnas, rogorc detoqsikaciisa da ujredidan maTi

mocilebis Taviseburi gamovlena, radgan steroidTa srul fermen-

tul degradacias Tan axlavs ujredis mier maTi naxSirbadatomebis

plastikuri miznebisaTvis gamoyenebac. aqac adgili aqvs nivTierebis

159

molekulaSi Jangbadis atomis Canergvas da aqac aRdgenili cito-

qrom P450 monawileobs. hemoproteinis kvlav aRdgena membranebSi

arsebuli redoqs-jaWviT xorcieldeba, romelic SeuRlebuli araa

protonuli gradientis (∆µH+ membranuli potencialis) aRZvrasTan

da amdenad Tavisufali, aradamfosforebeli Jangvis sistemas ganeku-

Tvneba. igi mitoqondriuli membranis Sida mxaresaa lokalizebuli

da citoqrom P450-s, NADPH-adrenodoqsinreduqtazas (EC 1.6.7.1) da adrenodoqsins Seicavs [90-95]. dagrovili uamravi eqsperi-

mentuli masala saSualebas gvaZlevs sakmaod garkveulad vimsjeloT

mahidroqsilirebeli sistemebis Sedgenilobaze, maTi calkeuli

komponentebis Tvisebebze da moqmedebis meqanizmze.

OH

H3C

CH3

CH3

CH3

12

34

56

7

89

10

11 1213

14 1516

17

18

19

20

21 2422

23 25

26

27

A B

C D

qolesteroli naxSirbadatomebis numeraciiT

H3C

isrebiT naCvenebia qolesterinis

molekulaze fermentTa Setevis ubnebi

oqsigenazebis garkveuli jgufi qolesterinidan naRvlis mJavebis

sinTezSia CarTuli. es procesi 7α-mdgomareobaSi qolesterinis hid-

roqsilirebiT limitirdeba da RviZlis mikrosomebSi xorcieldeba.

rac Seexeba steroiduli hormonebis biosinTezSi qolesterinis

Jangvasa da hidroqsilirebas, masSi monawile citoqrom P450-is 80%

160

mitoqondriuli warmoSobisaa. es sistema da mikrosomebis mahidro-

qsilirebeli sistema arsebiTad gansxvavdebian erTmaneTisagan. pirv-

eli _ arahemur cilas (adrenodoqsins) Seicavs da Zlier waagavs

baqteriul (Pseudomonas putida) mahidroqsilirebel sistemas, maSin

rodesac mikrosomuli sistema Fe,S-cilebs ar Seicavs [90-97].

amJamad dadgenil faqtad iTvleba, rom Tirkmelzeda jirkvlis

qerqis mitoqondriebSi ori sxvadasxva citoqrom P450 figurirebs: erTi maTgani 11β-hidroqsilirebas akatalizebs (aRiniSneba, rogorc

P45011β), xolo meore molekulis gverdiT jaWvs acilebs (aRini-

Sneba, rogorc P450scc). orive citoqromi eleqtronebs adreno-

doqsinidan iRebs, romelic Tavis mxriv NADPH-adrenodoqsinreduq-

taziT regulirdeba [90-94]. Tirkmelzeda jirkvlis qerqis mikro-

somuli sistema steroidTa 21-hidroqsilirebas axorcielebs. ste-

roidTa jaWvSi monawile oqsigenazebi ZiriTadad induqciuri bune-

bisaa da saTanado substratTa gamoyenebisas maTi aqtivobebi maqsima-

lurad izrdeba. aqtivobis daaxloebiT 1-5% konstituciur fermen-

tebs eTmoba da substratTa damatebisas maTi aqtivobebi praqti-

kulad ar icvleba [90-95].

2.6.1 Tirkmelzeda jirkvlis qerqis mitoqondriebis

citoqrom P450

am organos orive citoqrom P450 sadReisod gamoyofili da

srulad daxasiaTebulia [91-95, 98]. orive hemoproteini mWidrod

dakavSirebulia membranasTan da integralur cilebs warmoadgenen

(nax. 2.11), rac ganapirobebs ultrabgeriTa da detergentebiT maT

gamoyofas. P45011β da P450SCC yovel polipeptidur jaWvze erT hems

Seicaven da isini b-tipis citoqromebs miekuTvneba. buferul xsna-

rebSi orive citoqromi gansxvavdeba xsnadobiT da amoniumis sulfa-

tiT fraqcionirebisadmi damokidebulebiT, rac maTi dayofis saSua-

lebas iZleva. mitoqondriuli citoqromebis orive formis maxasia-

Teblebi warmodgenilia cxrilSi 2.2.

sadReisod dadgenilia orive citoqromis aminomJavuri Semadgen-

loba [94, 99, 100] da citoqrom P450scc-is fragmentTa aminomJauri

Tanmimdevroba [95]. cnobilia maTi hemSemcveli peptidebis

161

nax. 2.11. mitoqondriuli membranis citoqrom P450-Semcveli siste-

mis komponentebi. citoqrom P450 mitoqondriuli memb-

ranis integraluri cilaa. NADPH-adrenodoqsinreduq-

taza da adrenodoqsini periferiuli cilebia; isini araa

membranaSi „CamontaJebuli“.

cxrili 2.2

Tirkmelzeda jirkvlis qerqis mitoqondriebis citoqrom P450scc da

P45011β-is Tvisebebi [93, 95, 98]

Tvisebebi citoqromi

P450scc

citoqromi

P45011β

molekuluri masa ∼ 49000 ∼ 43000 maqsimumebi STanTqmis

speqtrebSi:

daJanguli forma 393, 526, 645 394, 510, 645

aRdgenili forma 417, 548 419, 555

CO-sTan kompleqsi 448 448

epr-speqtrSi g-faqtori 1.914, 2.241, 2.415 1, 91, 2.24, 2.42

162

Semadgenlobac [101]. mitoqondriuli citoqrom P450-is orive forma gamoirCeva hidrofobuli aminomJavuri naSTebis maRali (36%) Semc-

velobiT da gogirdSemcveli aminomJavebis toli ricxviT; gaaCniaT

msgavsi fizikur-qimiuri maxasiaTeblebi, Tumca P45011βufro hidro-

fobulia. speqtruli parametrebi damokidebulia cilis gamoyofis

meTodze. zogierT mkvlevars citoqrom P450SCC dabalspinur, xolo

zogierTs _ maRalspinur mdgomareobaSi aqvT miRebuli. hemopro-

teinis gansxvavebul spinur formaTa Tanafardoba mniSvnelovan-

wiladaa damokidebuli misi substratTan (qolesterinTan) dakav-

Sirebis unarze da gasufTavebis xerxze. SeniSnulia, rom P450SCC

advilad asocirdeba 800_850 kD molekuluri masis agregatebad da

am SemTxvevaSi igi 16 suberTeulisagan Sedgeba, romelTagan mxolod

rva Seicavs hemur rkinas [102]. unda aRiniSnos, rom citoqrom

P45011β akatalizebs steroidTa aramxolod 11β- da 18-hidroqsili-

rebis reaqciebs, aramed rekonstruirebul sistemebSi 4-androsten-

3,17-dionis me-19 mdgomareobaSi Jangvasac. aqedan gamomdinare, SeiZ-

leba vivaraudoT, rom P45011β steroidebs ori gansxvavebuli ubniT

ikavSirebs.

mitoqondriul citoqromebTan (iseve rogorc sxva hemoprote-

inebTan) substratebisa da inhibitorebis urTierTqmedebaze mniSvne-

lovan informacias diferenciuli speqtroskopiis meTodi iZleva.

Tirkmelzeda jirkvlis qerqis mitoqondriis citoqrom P450-Tan sxvadasxva nivTierebaTa urTierTqmedebis aseTi speqtrebi pirvelad

naraSimgulum da misma Tanaavtorebma [103] Seiswavles. dadgenil

iqna, rom registrirebadi speqtruli cvlilebebi or did jgufs _

I da II tips ganekuTvnebian. aRmoCnda, rom steroidTa bunebasa da

citoqromTan dakavSirebis tips Soris araviTari korelacia ar

arsebobs.

dakavSirebis pirvel tips Seesabameba substratis urTierTqmedeba

citoqrom P450-is hidrofobul ubanTan, ris Sedegadac izrdeba

meeqvse mdgomareobaSi ligandis polaroba, maSin rodesac dakavSi-

rebis meore tipi xasiaTdeba ligandis uSualo urTierTqmedebiT

citoqrom P450-is hemur rkinasTan ferihemoqromis warmoqmniT. citoqrom P450-is STanTqmis speqtrebisa da epr-is monacemebze

dayrdnobiT sarwmunod unda miviCnioT, rom substratTa urTierT-

qmedebis I da II tipi hemoproteinis maRal- da dabalspinuri forme-

163

biTaa ganpirobebuli. epr-meTodiT citoqrom P450-is detaluri

Seswavla jefkoatis da misi TanamSromlebis [104] mieraa Catare-

buli. maT mier mopovebulia Tirkmelzeda jirkvlis qerqis mito-

qondriebis citoqrom P450-is spinur formaTa raodenobrivi

Tanafardobis Sesaxeb amomwuravi cnobebi. daaxloebiT 80% P450scc

maRal, xolo 90% P45011β _ dabalspinur mdgomareobaSi imyofeba. I-

tipis diferenciuli speqtrebi xasiaTdeba ∼390 nm maqsimumebiT da ∼420 nm minimumebiT, maSin rodesac II-tipis diferenciul speqtrs

maqsimumi ∼420 nm-ze da minimumi ∼390 nm-ze aqvs. citoqrom P450-Tan substratis urTierTqmedebisas STanTqmis diferenciuli speqt-

rebi dakavSirebulia ligandis gavleniT hemoproteinTa spinuri

wonasworobis wanacvlebasTan (cxrili 2.3).

cxrili 2.3

P450SCC da P45011β-citoqromebis Jangva-aRdgeniTi potencialebi[105]

hemoproteini Jangva-aRdgeniTi potenciali (mV)

P450SCC substratis gareSe _412 ± 2

P450SCC + qolesterini _305 ± 1

P450SCC + pregnenoloni _305 ± 30

P45011β substratis gareSe _335 (pH 8.0-ze)

rogorc Cans, substratis citoqrom P450scc-Tan mierTebisas

Zlier izrdeba misi Jangva-aRdgeniTi potenciali, riTac advildeba

ferment-substratis aRdgenis procesi.

citoqrom P450-Si hemis oqtaedruli kompleqsis sqemas rac

Seexeba, arsebobs mtkicebuleba, rom igi citoqrom P450cam-is

analogiuria da rkinis mexuTe aqsialur ligands cisteinili

warmoadgens [106]. citoqrom P450-sa da misi modelebisaTvis epr-

is monacemebi adastureben, rom mexuTe mdgomareobaSi SeiZleba

imyofebodes cisteinis gogirdi, xolo meeqvseSi _ histidinis

azoti. meeqvse ligands wylis molekula avsebs [107]. P45011β-sa da

P450scc-is molekulebis struqturis gamosavlenad gamoyenebul iqna

SezRuduli proteolizis meTodi [108]. naCvenebi iqna, rom orive

164

globaluri fragmentisagan Sedgeba, romlebic trifsinis moqmede-

bisaTvis xelmisawvdomi mcire peptiduri kavSiriT erTdebian. trif-

sinolizze gavlenas ar axdens cilis agregacia, misi adreno-

doqsinTan urTierTqmedeba da membranuli garemocva. avtorTa azriT

[108], katalizuri centri da adrenodoqsinTan kompleqsis

warmoqmnaze pasuxismgebeli ubani lokalizebulia molekulis erT

fragmentSi, romlis molekuluri masa ∼27 kD-s Seadgens. meore

∼22 kD molekuluri masis mqone fragmenti citoqrom P450scc-s

membranasTan urTierTqmedebas uzrunvelyofs.

2.6.2 NADPH-adrenodoqsinreduqtaza (EC 1.6.7.1)

mitoqondriul eleqtronTa satransporto jaWvSi adrenodo-

qsinisa da citoqrom P450-is win NADPH-adrenodoqsinreduqtaza imyofeba. es flavoproteni, romelic erT polipeptidur jaWvze erT

FAD-s Seicavs, ramdenime meTodiTaa gasufTavebuli da krista-

lizebuli. fermenti gamoirCeva hidrofobuli aminomJavebis maRali

SemcvelobiT. Roris Tirkmelzedas qerqis mitoqondriebis reduq-

taza Saqrebs ar Seicavs, maSin rodesac Sesabamisi fermenti xaris

imave organodan Seicavs manozas, fruqtozas, galaqtozas da

glukozas [93]. adrenodoqsinreduqtazas momatebuli swrafva aqvs

naxSirwylebis mimarT da isini cilis molekulis zedapirze Tavs-

debian. gamoyofis meTodisagan damokidebulebiT fermentis moleku-

luri masebi gansxvavebulia (cxrili 2.4).

fermentis mniSvnelovan Taviseburebas is warmoadgens, rom mas

unari aqvs mxolod adrenodoqsinis Tanamyofobisas aRadginos cito-

qrom P450 da citoqrom c. mis gareSe reduqtaza mxolod 2,6-

diqlorfenolindofenols an fericianids aRadgens. Tvlian, rom

adrenodoqsinreduqtazasTan NADPH-is dakavSirebaSi arsebiT rols

histidini da cisteini asruleben. adrenodoqsini reduqtazasTan

warmoqmnis mtkice kompleqss 1 : 1 TanafardobiT. mas disociaciis

dabali konstanta gaaCnia da reduqtazas Jangva-aRdgeniT potencials

ar cvlis. samagierod mniSvnelovnad moqmedebs mis stabilurobaze

da fermentul aqtivobaze. adrenodoqsinreduqtazas kompleqsTan

NADPH-is urTierTqmedebisas ori eleqtroniT aRdgenili sammagi

165

cxrili 2.4

xaris Tirkmelzeda jirkvlis qerqidan gamoyofili mitoqondriuli

NADPH-adrenodoqsinreduqtazas Tvisebebi [93]

parametrebi mniSvnelobebi

prosTetuli jgufi 1 FAD

aminomJavuri naSTebis ricxvi 470

naxSirwylebi 2-9%

STanTqmis maqsimumebi 272, 378, 450

ε450 sm-1.M-1 10 900

izoeleqtruli wertili 5.4

molekuluri masa 50 000_54 0000

Jangva-aRdgeniTi potenciali _274 mV (pH 7.0)

Km NADPH-isadmi 1.82.10-6 moli

kompleqsi (AD_FP_NADP) miiReba. adrenodoqsini da misi reduqtaza intaqturi mitoqondriidan ad-

vilad irecxeba. reduqtaza xsnadi fermentia da mis SedgenilobaSi

aRmoCenili araa membranasTan asocirebisaTvis aucilebeli hidrofo-

buli segmenti. amitom Tvlian, rom igi funqcionirebs rogorc mem-

branasTan ionuri urTierTqmedebiT dakavSirebuli periferiuli ci-

la (nax. 2.11).

2.6.3 adrenodoqsini

ujredSi arsebobs dabalmolekuluri (∼6-14 kDA) cilebi, rom-

lebSic rkinis atomebi dakavSirebulia ara porfirinis birTvTan,

aramed gogirdSemcvel ligandebTan. maT rkina-gogirdproteinebs

uwodeben. aseTi cilebis SemadgenlobaSi SeiZleba Sediodes gogird-

Semcveli kompleqswarmomqmneli ori tipis jgufi: pirvels cistei-

nis sulfhidriluri jgufebi, xolo meores _ e.w. `labiluri go-

166

girdi“ miekuTvneba. am formis gogirdi imiT xasiaTdeba, rom cilis

xsnaris SemJavebisas H2S-is, HS--is an S2-

-is saxiT gamoiyofa.

umartivesi rkina-gogirdSemcveli cilebi rkinis erTidan rvamde

atoms Seicaven, romlebic erTmaneTs polipeptiduri jaWvebis cis-

teinis naSTebiT ukavSirdebian. rogorc irkveva, am naSTebis sivr-

culi ganlageba TiToeuli aseTi tipis cilisaTvis gansazRvrul

mniSvnelobas iZens. cilis molekula rkinis or an met atoms Tu

Seicavs, is Tavisebur `galiismagvar“ klasters warmoqmnis, romlis

SedgenilobaSic `labiluri gogirdi“ (SesaZloa sulfidis saxiT)

da cisteinis naSTi (SeiZleba Tiolatis saxiT) Sedis (nax. 2.12)

[109].

SFeCys

FeS

Fe S

FeSS

CysS

SCys

SCys

_____________

__

_

____

___

_

________________

nax. 2.12. adrenodoqsinis savaraudo aqtiuri centri. dajgufeba,

romelic „galias“ waagavs (Cys _ cisteinis naSTi).

aseT mravalbirTvian klasters SeuZlia miiRos an gasces erTi

eleqtroni. gogirdi, rogorc ligandi, Fe(II)-is umdgradobas apiro-bebs, riTac SeiZleba aixsnas metabolur procesebSi am cilebis

Zlieri aRmdgeneli unari. Fe2+ da Fe3+

ionebs Soris erTeleqtro-

nian gadasvlaSi `galiis“ msgavsi dajgufebis bioqimiuri funqcia

eleqtronTa gadatanaSi ufro mdgomareobs, vidre amaTuim gardaqmnis

katalizebaSi.

adrenodoqsini arahemuri cilaa da feredoqsinebis klass mie-

kuTvneba. igi putidaredoqsinis analogiuria da misi biologiuri

roli NADPH-adrenodoqsinreduqtazadan citoqrom P450-ze eleqtro-

nebis transportSi mdgomareobs, romelTanac igi mtkice kompleqss

167

warmoqmnis [109]. adrenodoqsini gamoyofilia homogenur mdgomareo-

bamde eleqtroforezulad da kristalizebulia. saxelwodeba gamox-

atavs im faqts, rom igi pirvelad miRebulia Tirkmelzeda

jirkvlis qerqidan (Adrenal cortex) rva atomi rkinisa da rva atomi `labiluri gogirdis“ Semcveli cilis aRsaniSnavad. `adrenodoqsi-

nis“ sinonimad gamoiyeneba `feredoqsini“ (Fer _ rkina, redoxin _ eleqtronis gadamtani cila). amis garda gamoiyeneba aRniSvnac:

n_Fe_S*-cilebi, sadac varskvlavi `labiluri gogirdis“ aRmniSvne-

lia, xolo n _ misi atomebis raodenobas gviCvenebs, romelic Cveu-

lebriv rkinis atomTa ricxvis tolia. 2Fe_S*-adrenodoqsini da

testodoqsini gamoyofilia Sesabamisad Tirkmelzeda jirkvlidan da

saTesleebidan. maTi funqcia im ferment-oqsigenazebis eleqtronebiT

uzrunvelyofaa, romlebic steroiduli hormonebis sinTezisas qo-

lesterinis hidroqsilirebas, an naxSirwyalbaduri gverdiTa jaWvis

mocilebas akatalizeben [110]. 2Fe_S*-tipis feredoqsini suqcinalu-

ri warmoebulis saxiT gamoyofilia gulis mitoqondriebis sunTqvi-

Ti jaWvis bc1-kompleqsidan (sxva saxelwodebebi: sleiteris faqto-

ri, riskes cila, britanuli antiluizitis-BAL-is mimarT mgrZno-biare faqtori), romelic mitoqondriuli eleqtronTa satranspor-

to gzis centraluri ubania [110]. am tipis feredoqsinebs gaaCniaT

damaxasiaTebeli optikuri Tvisebebi, romliTac isini gansxvavdebian

sxva rkina-gogirdSemcveli cilebisagan. maT STanTqmis speqtrSi ar-

seboben zolebi 320, 420 da 460 nm-ebze da agreTve mxari 500 nm-is

siaxloves. garda amisa, Fe_S*-tipis cilas Seicavs zogierTi azot-

mafiqsirebeli baqteria, Tumca ucnobia TviT azotis fiqsaciaSi maTi

uSualo monawileoba. mcenareuli tipis feredoqsinebi gamovlenilia

anaerobul (Clostridium pasteurianum) da aerobul (Azotobacter vine-landii) baqteriebSi [111, 112].

Rorisa da xaris Tirkmelzedas mitoqondriebidan gamoyofili

adrenodoqsinebisaTvis dadgenilia aminomJavuri Tanmimdevrobebi. re-

konstruirebuli adrenodoqsini Tavisi speqtruli TvisebebiTa da

katalizuri aqtivobebiT natiurisagan ar gansxvavdeba.

adrenodoqsinis epr- da mesbaueris speqtrebma, agreTve ormagbirT-vulma rezonansma da magnituri amTviseblobis gazomvam saSualeba

Seqmna imis mtkicebisaTvis, rom daJangul diamagnitur cilaSi rki-

nis ori atomi maRalspinur mdgomareobaSi imyofeba da urTierTqme-

168

deben ra rogorc antiferomagnetikebi, iZlevian S=0-is tol jamur

spins. aRdgenil mdgomareobaSi orbirTviani kompleqsi Seicavs erT

atom rkinas samvalentian mdgomareobaSi, xolo meores _ orvalen-

tians maRalspinur mdgomareobaSi. aRdgenili cilis rkinis atomebi

araeqvivalenturebi arian da urTierTqmedebisas iZlevian jamur

S=1/2-is tol spins.

aromatul aminomJavaTa naSTebs eleqtronuli transportis meqa-

nizmSi arsebiTi rolis Sesruleba SeuZliaT. adrenodoqsinis mole-

kula Seicavs erTaderT Tirozins, 4 fenilalanins da saerTod ar

Seicavs triptofans. Tirozin-82-Si nitro-jgufis Seyvana asaxvas

poulobs adrenodoqsinis Tvisebebsa da aqtivobaze. SesaZloa, rom

modificirebuli adrenodoqsinis katalizuri aqtivobis daqveiTebis

mizezi adrenodoqsinreduqtazasTan misi kompleqsis simtkicis Sem-

cireba iyos.

adrenodoqsinis bioqimiuri kvlevis Sedegad arsebuli monaceme-

bis safuZvelze SeiZleba Semdegi zogadi daskvnebis gamotana:

− adrenodoqsini TiTqmis yvela sasicocxlod mniSvnelovan Jangva-

aRdgeniT procesSi (fotosinTezSi, mitoqondriul sunTqvaSi,

azotfiqsaciaSi da sxv.) monawileobs da eleqtronTa gadatanas

axorcielebs;

− calkeul SemTxvevebSi adrenodoqsins ori iseTi eleqtronis ga-

datana SeuZlia, romelTa energia mxolod eleqtronebisadmi ma-

Rali swrafvis mqone wyvilebisTvisaa damaxasiaTebeli;

− hemoproteinebisagan gansxvavebiT, romlebSic kompleqswarmoqmna

porfirinis ukve Camoyalibebul xist dajgufebebTan xdeba, adre-

nodoqsinebis qimiuri Seneba saSualebas iZleva Seiqmnas ligande-

bis aucilebeli ufro plastikuri sivrculi wyoba.

2.6.4 steroidmahidroqsilirebeli sistemis

moqmedebis meqanizmi

Tirkmelzeda jirkvlis mitoqondriebSi (iseve, rogorc sxva ma-

hidroqsilirebel sistemebSi) citoqrom P450-Tan kompleqsSi myofi

Jangbadis gasaaqtiureblad ori eleqtronia saWiro. aqac hemopro-

teinis daJangul formasTan substratis urTierTqmedebis stadia

yvela citoqrom P450-Semcveli mahidroqsilirebeli sistemis ana-

169

logiuria. es imas niSnavs, rom Jangbadis aRdgena mimdinareobs Tan-

mimdevrobiT jer erTi, xolo Semdgom meore eleqtronis mierTebis

gziT, da orive stadiaSi aq adrenodoqsini monawileobs, romlis

rolic qafuris mahidroqsilirebel sistemaSi arsebuli putidare-

doqsinis identuria [90, 113]. uaRresad arsebiTia, rom pirveli

eleqtroniT ferment-substratis kompleqsis aRdgena orive sistema-

Si aRmdgenelis bunebis mimarT araspecifikuria da SeiZleba iseTi

martivi aRmdgeneliT iqnas Secvlili, rogoric natriumis diTioni-

tia. citoqrom P450-substrati-O2 sammagi kompleqsis aRdgena mito-

qondriul sistemaSi adrenodoqsinis, xolo qafur-mahidroqsi-

lirebel sistemaSi _ putidaredoqsinis monawileobas moiTxovs.

miuxedavad amisa, am ori sistemis moqmedebaSi mniSvnelovani sxvao-

bebic arsebobs, rac ferment-substratisa da sammagi kompleqsis

pirvel- da meore eleqtroniT uzrunvelyofis meqanizmSi vlindeba.

Tirkmelzeda jirkvlis qerqis mitoqondriebSi NADPH-dan ci-

toqrom P450-ze eleqtronuli transportis Tanmimdevroba moce-

mulia nax. 2.13-ze. masSi monawileoben reduqtaza (FP), adreno-

doqsini (Ad) da maTi kompleqsebi.

pirvel stadiaze substratis dakavSireba xdeba daJangul citoqrom P450-Tan. NADPH-dan reduqtazas anaerobul aRdgenas Tan sdevs hemo-proteinis Jangva-aRdgeniTi potencialisa da spinuri mdgomareobis cvlilebebi. amave dros STanTqmis speqtris maqsimumi 750 nm-is (an ufro metis) olqSi Cndeba. am procesSi epr-signali araa daregistrirebuli. STanTqmis speqtri or SemTxvevaSia identuri: daJanguli reduqtazas urTierTqmedebisas NADPH-Tan, an diTionitiT aRdgenil reduqtazaze NADP-is damatebisas. FP-is aRdgena SeiZleba ganxorcieldes sxva aRmdgenlebis, kerZod NADH-is an diTionitis gamoyenebiT da am SemTxve-vaSi grZeltalRovan olqSi STanTqmis zolebi ar mJRavndeba. es imis maC-venebelia, rom mxolod NADP warmoqmnis aRdgenil reduqtazasTan kom-pleqss (FP_H2_NADP), romelic or eleqtrons Seicavs da gaaCnia 1.0.10-8 disociaciis konstanta. diTionitiT aRdgenili reduqtaza NADPH-Tan warmoqmnis kompleqss, romelic oTx eleqtrons Seicavs. ganxiluli cvlilebebi aadvileben pirveli eleqtroniT ferment-

substratis aRdgenas, romlis transportSic NADPH-adrenodoq-

sinreduqtaza da adrenodoqsinia CarTuli [90, 93, 98].

170

O2

_

Fe3+

P450_S

Fe2+

P450_S

O2

Fe3+

P450_S

Fe2+

P450_S

Fe3+

O2

_

S NADPH

NADP

FP0

FPB

AdB

Ad0

e1

e2

+

P450_S

SOH + H2O

nax. 2.13. Tirkmelzeda jirkvlis qerqis mitoqondriebSi hidroq-

silirebis katalizuri cikli (ganmarteba ix. teqstSi)

[98].

NADPH-is siWarbisas adrenodoqsinreduqtazas aRdgena pirveli

rigis konstantiT xasiaTdeba da 28 wm-1-is tolia. kofermentis di-

di siWarbisas Cndeba aRdgenis reaqciis kidev erTi faza, rac dakav-

Sirebulia kompleqsSi NADPH-is NADP-iT SecvliT (k=4.25 wm-1).

adrenodoqsinreduqtaza akatalizebs 2,6-diqlorfenolindolfeno-

lis aRdgenas, maSin rodesac citoqrom c aRdgenas mxolod reduq-

tazisa da adrenodoqsinis monawileobiT ganicdis [114]. adreno-

doqsini reduqtazasTan 1 : 1 TanafardobiT mtkice kompleqss warmo-

qmnis, romelic SeiZleba maRali siCqariT (k=300 wm-1) daiSalos da

es damokidebuli araa xsnarSi marilTa koncentraciaze [115].

adrenodoqsinis gavleniT reduqtaza konformaciul cvlilebebs

ganicdis, riTac SeiZleba aixsnas NADPH-is, FP-isa da Ad-is mona-wileobiT c-s aRdgenis procesSi garkveuli induqciuri periodis

arseboba. FP_H2_Ad-is kompleqsis swrafi warmoqmna da misi aseve

swrafi daSla imas mowmobs, rom adrenodoqsini SeiZleba reduqta-

zidan aRsadgen obieqtze (citoqrom c-ze an citoqrom P450-ze) eleqtronebis Zvradi gadamtani iyos [114, 118]. adrenodoqsinis kom-

pleqss flavoproteinTan disociaciis dabali konstanta gaaCnia

[119]. am kompleqsis NADPH-Tan urTierTqmedebisas flavoproteini

aRdgenas ganicdis, xolo adrenodoqsini daJangul mdgomareobaSi

171

rCeba:

NADPH + H+ + FP_Ad NADP_FP_H2_Ad

NADPH-iT citoqrom c-s aRdgenisas FP_Ad-kompleqsis moqmede-

bis meqanizmi detaluradaa Seswavlili [120] da am reaqciis kata-

lizuri cikli mocemulia nax. 2.14-ze.

pirvel stadiaze ori daJanguli cilis mtkice kompleqsi

(FP_Ad) reagirebs NADPH-Tan. A da B reaqciaTa siCqareebi xa-siaTdebian 18 wm

-1 konstantebiT. warmoqmnil sammag kompleqsSi mux-

tis gadatanisas ori eleqtroni reduqtazas flavinur dajgufebazea

lokalizebuli. sammagi kompleqsis disociaciis konstanta 10-8 M-is

tolia. C-stadiaze xdeba erTi eleqtronis gadatana srulad aRdge-

nili reduqtazas flavinuri dajgufebidan adrenodoqsinis Fe,S-klasterze, romelic mxolod erTi eleqtronis aqceptoria. miux-

edavad imisa, rom stacionaluri da swrafi kinetikis meTodiT ••

−− AdFPNADP -nawilakis identificireba ver moxerxda, misi arsebo-

ba savsebiT dasaSvebia, radgan marto reduqtazas ar SeuZlia cito-

qrom c-s aRdgena, xolo aRdgenili adrenodoqsini citoqrom c-sTan swrafad reagirebs (k > 300 wm-1

). garda amisa, NADPH da misi eqvi-

moluri FP da Ad advilad axorcieleben citoqrom c-s aRdgenas [120].

••

−− AdFPNADP nawilakis arseboba absoluturad SesaZlebe-

lia Termodinamikuri TvalsazrisiTac. C- da D-stadiebi mimdina-

reobs procesis malimitirebeli safexuris siCqaris konstantiT

(3.2 wm-1). D-stadiis Sedegad warmoiqmneba erTi eleqtronis Semcve-

li kompleqsi (

•

⋅⋅ ADFPNADP ). am formas 2500-jer naklebi swrafva

aqvs NADPH-is mimarT, vidre kompleqsis oreleqtronian formas.

amiT SeiZleba aixsnas sammagi kompleqsis disociaciiT NADP-is ga-

moTavisufleba (E-stadia). •

⋅ADFP -formas SeuZlia citoqrom c-s Senelebuli aRdgena ( 33.011.0K ÷= wm-1). citoqrom c-s gacilebiT

swrafi aRdgenis reaqcia xorcieldeba nawilakiT, romelic sam

eleqtrons

(•••

⋅⋅ AdPFNADP ) Seicavs da F-stadiaze warmoiqmneba. es nawilaki G-

stadiaze oreleqtronian formad (•••

⋅⋅ AdPFNADP ) gardaiqmneba.

172

NADPH

NADPH-FP-Ad

FP-Ad

citoqrom c (aRdgenili)

citoqrom c (daJanguli)

G

NADP FP-Ad

NADP

E

D

C

B

FP-Ad

NADPHA

F

H

+..

NADP FP-Ad+ ..

NADP FP-Ad+.

+.

NADP FP-Ad+ ...citoqrom c (daJanguli)

citoqrom c (aRdgenili)

citoqrom c (aRdgenili)

citoqrom c (daJanguli)

nax. 2.14. adrenodoqsinreduqtaza _ adrenodoqsinis kompleqsiT ci-

toqrom-c-s NADPH-damokidebuli katalizuri aRdgenis

cikli. FP − adrenodoqsinreduqtaza, Ad − adrenodoqsini (ganmarteba ix. teqstSi) [120].

rogorc vxedavT, citoqrom c-s NADPH-damokidebuli aRdgena

ciklur process warmoadgens, romelSic gaerTianebulia erT- da

sam-eleqtroniani kompleqsebi. srulad daJanguli kompleqsi mxo-

lod katalizuri ciklis dasawyisSi warmoiqmneba. dasaSvebadaa miC-

neuli, rom mitoqondriebSi citoqrom P450 membranaSi citoqrom c-s analogiuri meqanizmiT ganicdis aRdgenas [120]. citoqrom P450-ze eleqtronebis gadatana xdeba sammag kompleqsSi, romelSic reduqta-

za, adrenodoqsini da TviT es hemoproteinia CarTuli. am daSvebis

sasargeblod metyvelebs citoqrom P450scc-isa da adrenodoqsinis

mier kompleqswarmoqmnis faqti, romelic naCvenebia katahirisa da

Tanaavtorebis [108] mier. maT CaTvales, rom es kompleqsi mxolod

qolesterinis Tanamyofobisas warmoiqmneba. mogvianebiT naCvenebi iqna

173

uqolesterinod citoqrom P450scc-isa da adrenodoqsins Soris kom-

pleqswarmoqmnis SesaZlebloba, romelsac disociaciis dabali kons-

tanta (4.10-7 M) gaaCnia da qolesterinTan erTad kidev ufro

qveiTdeba (3.0.10-8 M) [121]. citoqrom P450scc-isa da adrenodoqsinis

kompleqsi temperaturis farTo intervalSi da buferis sxvadasxva

koncentraciaze Zalze umdgradia. ramdenadac adrenodoqsini kom-

pleqss warmoqmnis reduqtazasTan da citoqrom P450scc-Tan, savarau-

do xdeba, rom sammagi kompleqsi samive cilas 1 : 1 : 1 TanafardobiT Seicavs, rac mniSvnelovan rols unda asrulebdes mitoqondriul

hidroqsilirebaSi [94, 122, 123]. am samuSaoebSi gaziarebulia aseTi

Tvalsazrisi. aRniSnuli sakiTxi dResac aqtualuria, radgan igi

dakavSirebulia mitoqondriuli hidroqsilazuri sistemis organi-

zaciis problemasTan da monooqsigenazuri kompleqsis rekonstruq-

ciasTan.

ar SeiZleba gverdi avuaroT zogierTi mkvlevaris [114-120] mo-

nacemebs, romlebic uaryofen mitoqondriuli sistemis funqcionire-

baSi sammagi kompleqsis SesaZlo rols. es Tvalsazrisi Semdeg faq-

tebs emyareba: 1. mitoqondriul membranaSi adrenodoqsinreduqtaza,

adrenodoqsini da P450-citoqromebi jamSi 1 : 10 : 10 fardobiT imyo-febian, anu natiur pirobebSi SeiniSneba adrenodoqsinisa da P450-ebis siWarbe [122]; 2. adrenodoqsinisa da qolesterinis kompleqsi-

reba citoqrom P450scc-Tan SemTxveviT da ara mowesrigebul xasiaTs

atarebs; amave dros adrenodoqsinis urTierTqmedeba citoqrom P450-Tan mciredaa damokidebuli substratis Tanamyofobaze [121]; 3. rekon-struirebul sistemaSi maqsimaluri aqtivoba miiReba adrenodoq-

sin : P450-is 1 : 1 fardobasTan SedarebiT ufro didi Tanafardobisas, anu sistemis maqsimalur brunvaTa ricxvis misaRwevad aucilebelia

adrenodoqsinis siWarbe [116], rac ewinaaRmdegeba samive komponentis 1 : 1 : 1 fardobisaTvis kompleqsis funqciur rols; 4. arsebobs mona-

cemebi imis Sesaxeb, rom mtkiced dakavSirebuli sammagi kompleqsis

(FP_Ad_P450) arseboba saerTod SeuZlebelia. yovel SemTxvevaSi, im

pirobebisaTvis mainc, roca samive komponenti daJangul mdgomareoba-

Si imyofeba [105]. mTel rig SemTxvevaSi monacemebi miuTiTebs imis

Sesaxeb, rom mtkiced dakavSirebuli sammagi kompleqsis FP_Ad-kompleqsi praqtikulad ar ikavSirebs citoqrom P450scc-s [118].

rogor SeiZleba aixsnas mitoqondriuli monooqsigenazas funq-

174

cionirebaSi sammagi kompleqsis arsebobasa da rolze aseTi winaaRm-

degobrivi monacemebi?

rogorc irkveva saTanado pirobebSi aseTi kompleqsi arsebobs.

misi gamovlena damokidebulia sistemis rekonstruqciis xerxze, pH-

is mniSvnelobaze, xsnaris ionur Zalaze da qolesterinis arseboba-

ararsebobaze. ase magaliTad, dimiristoilfosfatidilqoliniT war-

moqmnil fosfolipidur vezukulebSi dabali ionuri Zalisas qo-

lesterinis Tanamyofobis pirobebSi sammagi kompleqsi (FP_Ad_ _P450scc) namdvilad arsebobs [122]. qolesterinis gareSe dabali

ionuri Zalisa da sxva Tanabar pirobebSi sammagi kompleqsi kargavs

citoqrom P450-s da binarul kompleqsad (FP_Ad) gardaiqmneba.

aseve, qolesterinis gareSe maRali ionuri Zalisas samive komponen-

ti disocirebul mdgomareobaSi imyofeba [122].

fosfatidilqolinis vezikulis gamoyenebisas da pirobebis ram-

denadme SecvliT Catarebulma gamokvlevebma aCvenes, rom sammagi kom-

pleqsi ar funqcionirebs [117]. citoqrom P450scc lipidur veziku-

lasTan urTierTqmedebs Tavisi hidrofobuli fragmentiT, xolo

adrenodoqsinTan dakavSirebis centri vezikulis gareT imyofeba da

polaruli bunebisaa. qolesterinis dakavSirebis centri vezikulis

SigniTaa lokalizebuli da hidrofobuli Tvisebebi gaaCnia, anu

gansxvavebul pirobebSi citoqrom P450-Tan kompleqsis komponentTa

dakavSireba sxvadasxvanairad SeiZleba realizdebodes an saerTod ar

xdebodes. arsebobs gafrTxilebac imis Sesaxeb, rom xsnarSi monooq-

sigenazuri kompleqsis konstruirebisas an misi imobilizaciis miRe-

buli monacemebis pirdapirma gadatanam natiur mitoqondriul sis-

temaze SeiZleba mcdar daskvnamde migviyvanos [110-118, 123, 124].

aqamde ar moxerxda intaqtur mitoqondriul sistemaSi sami cilis

steqiometruli Tanafardobis zusti dadgena. amitom mitoqondriul

membranaSi monooqsigenazuri kompleqsis komponentTa urTierTqmede-

bis or tips Soris arCevanis gakeTeba sakmaod Znelia. arsebobs mo-

nacemebi klasteris SesaZlo warmoqmnaze [122, 124]. amis sapiris-

pirod ki arsebobs publikaciebi membranaSi mahidroqsilirebeli kom-

pleqsis komponentTa maRali lateraluri difuziis Sesaxeb [114-

118]. amitom membranaSi komponentTa urTierTqmedebis ori tipis _

lateraluri difuziisa da klasterebis arsebobis Sesaxeb saboloo

daskvnebis gamotana mainc Zneli da naadrevia, miTumetes rom arsebo-

175

ben rogorc erTi, aseve meore modelis damamtkicebeli faqtebi [94,

123, 124].

steroidhidroqsilazuri sistemis molekuluri organizaciis

SecnobaSi udavo interess warmoadgens am sistemis TviTawyoba [94,

123, 124]. meTodologiurad procesis arsi SemdegSi mdgomareobs:

adrenodoqsinreduqtaza daitaneba adrenodoqsin-sefarozas svetze da

Semdgom emateba citoqrom P450. meore varianti gulisxmobs ste-

roidhidroqsilazas komponentTa datanis sxva rigs da am dros aq-

tivobis maqsimumi registrirdeba: maRali ionuri Zalis buferSi

svetze daitaneba citoqrom P450, xdeba sorbirebuli hemoproteinis

dezagregacia da Semdgom svetSi SeaqvT adrenodoqsinreduqtaza. mi-

Rebuli rekonstruirebuli sistema Jangavs Sesabamis substratebs.

miRebuli Sedegebi aCvenebs steroidhidroqsilazuri sistemis Se-

saZlo arsebobas, romelic membranaSi stabilurad mofunqcionire

multifermentuli kompleqsis saxiT imyofeba. steroidhidroqsila-

zuri sistemis TviTawyoba axal perspeqtivas saxavs hormonuli pre-

paratebis fermentul sinTezSi maTi praqtikuli gamoyenebisaTvis.

amgvarad SeiZleba davajamoT arsebuli faqtebi: RviZlis mikro-

somebSi NADPH-dan citoqrom P450-ze eleqtronebis gadatana mim-

dinareobs FAD- da FMN-Semcveli NADPH-citoqrom P450-reduq-taziT da Fe,S-Semcvel cilebs ar saWiroebs. RviZlis fermentuli

sistemis mier citoqrom c-s katalizuri aRdgena mraval saerTos

amJRavnebs amave hemoproteinis adrenodoqsin–adrenodoqsinreduq-

tazas kompleqsiT aRdgenasTan. orive procesSi CarTulia or-ori

Jangva-aRdgeniTi centri: FAD da Fe2,S2-klasteri mitoqondriul

sistemaSi da FAD da FMN mikrosomul sistemaSi. katalizuri aq-

tis ganxorcielebisas orive sistema nawilobriv aRdgenil mdgoma-

reobaSi rCeba. mikrosomuli reduqtaza Tavis mdgomareobas icvlis

2-eleqtron–4-eleqtronSemcvel, an 1-eleqtron–3-eleqtronSemcvel

formamde, xolo mitoqondriuli kompleqsi Tavis mdgomareobas 1-

eleqtron–3-eleqtronSemcvel formamde icvlis.

unda aRiniSnos, rom flavoproteinebis da Fe,S-Semcveli cile-

bis kompleqsebi 1 : 1 TanafardobiT zogierT sxva sistemebSic

gvxvdeba; mag., NADH-rubrerodoqsin – reduqtazasa da rubrero-

doqsinSi [125], feredoqsin : NADP-oqsidoreduqtazasa da feredoq-sinSi [126], putidaredoqsinsa da putidaredoqsinreduqtazaSi.

176

2.6.4.1 qolesterinis gverdiTi alifaturi jaWvis

moxleCvis meqanizmis Sesaxeb

rogorc aRniSnuli iyo, Tirkmelzeda jirkvlis qerqis mitoqon-

driebis citoqrom P450scc akatalizebs qolesterinis gverdiTi jaW-

vis arasrul mowyvetas, ris Sedegadac qolesterinis birTvebTan

dakavSirebuli mxolod ori naxSirbadatomis Semcveli Sualedi

produqti pregnenoloni da izokapronis aldehidi miiReba. es reaq-

cia imiTaa mniSvnelovani, rom igi steroiduli hormonebis biosin-

Tezis gzaze malimitirebel stadias warmoadgens [127]. reaqciis ma-

katalizebel citoqrom P450scc-s xSirad desmolazas uwodeben.

desmolazuri reaqciis ganxilvisas bunebrivad Cndeba kiTxvebi,

kerZod: romeli reaqciebi uswreben qolesterinis gverdiTi jaWvis

moxleCas? romeli Sualeduri naerTebi warmoiqmneba am dros? erTi

Tu ramdenime citoqrom P450scc-s forma akatalizebs am process?

CarTuli arian Tu ara masSi sxva fermentebic? da a.S. mTel rig

samuSaoebSi [128-131] mocemulia mcdelobebi zogierT am kiTxvaze

pasuxis gasacemad da maT safuZvelze SeiZleba warmodgenil iqnas

qolesterinis gverdiTi jaWvis moxleCis gamartivebuli sqema

(nax. 2.15).

gardaqmnis am meqanizmSi arsebiTi isaa, rom qolesterinis mole-

kulas gverdiTi jaWvi sami Tanmimdevruli reaqciis Sedegad scil-

deba. esenia 20-hidroqsilireba, 22-hidroqsilireba da C-20 da C-22-naxSirbadatomebs Soris jaWvis moxleCa (ix. qolesterinis mo-

lekuluri formula). aseTi meqanizmis arsebobis sasargeblod mety-

velebs daxarjuli NADPH-isa da O2-is steqiometria, agreTve war-

moqmnili pregnenoloni. dadginda, rom erTi molekula pregnenolo-

nis warmoqmna sam-sami molekula NADPH-isa da O2-is moxmarebas

iTxovs [132]:

C27H46O + 3O2 + 3NADPH + 3H+ → C21H32O2 + C6H12O + 3NADP + 4H2O

NADPH-is, O2-is da H+-is xarjvis steqiometria gansazRvrulia

ara marto qolesterinis, rogorc substratis, aramed misi Suale-

duri oqsiwarmoebulebisTvisac. cxril 2.5-Si mocemulia gazomvis

Sedegebi.

177

OH

OH

OH

C OCH3 O

INADPH, O2

IIINADPH, O2

+

pregnelononi izokapronis

aldehidi

II

NADPH, O2

qolesteroli

nax. 2.15. qolesterinis (sqemaze mocemulia mxolod misi D-birTvi

da gverdiTi jaWvi) hidroqsilireba alifaturi jaWvis

mocilebiT [132].

cxrili 2.5 [132]

qolesterinis gverdiTi jaWvis mocilebis reaqciis steqiometria

substrati ÍÉÐÒÄÂÍÄÍÏËÏ

NADPH∆

ÍÉÐÒÄÂÍÄÍÏËÏ2O∆

ÍÉÐÒÄÂÍÄÍÏËÏ

+∆H

qolesterini 3 ± 0.2 3.1 ± 0.3 3.0 ± 0.2 20α-HO-qolesterini

1.9 ± 0.3 2.1 ± 0.3 2.1 ± 0.1

20α, 22-(HO)2-qolesterini

1.0 ± 0.3 0.9 ± 0.4 1.1 ± 0.1

miRebuli steqiometria adasturebs, rom samive reaqcia monooq-

sigenazuri meqanizmiT katalizdeba da rom mesame stadiaze gverdiTi

jaWvis mocilebaSi citoqrom P450 monawileobs. dadginda agreTve,

rom pH 6.8-ze mitoqondriebis citoqrom P450-is erTi da igive

fraqcia (P450scc) im SemTxvevaSi uzrunvelyofs maqsimalur fermen-

178

tul aqtivobas, rodesac substratebad gamoiyeneba: qolesterini,

[20S]-20-oqsiqolesterini, [22R]-22-oqsiqolesterini, an [20R, 22R]-20,22-dioqsiqolesterini. qolesterinis yvela oqsiwarmoebulidan

pregnenolonis warmoqmna mniSvnelovnad iTrguneba [20R, 22S]-20,22-qolesterinoqsidiT [130].

calkeuli samuSaoebis [133, 134] safuZvelze gamoTqmulia varau-

di, rom 20,22-qolesterinoqsidi qolesterinidan gverdiTi jaWvis

moxleCis reaqciaSi Sualed produqts warmoadgens. am hipoTezis

Tanaxmad pirvel stadiaze xdeba qolesterinis dehidrireba 22,22-

dehidroqolesterinis warmoqmniT, romelic Semdeg citoqrom P450-is, O2-isa da NADPH-is monawileobiT epoqsidad gardaiqmneba. amis

sapirispirod morisakisa da Tanaavtorebis mier [128] sarwmunod iqna

naCvenebi, rom arc 20,22-dehidroqolesterini da arc 22,22-

qolesterinoqsidi gverdiTi jaWvis moxleCis procesSi ar warmoad-

genen Sualed produqtebs, ramdenadac orive nivTierebis inkubacia

citoqrom P450-Semcveli sistemis yvela komponentTan Jangbadis Ta-

namyofobisas pregnenolons ar iZleva.

morisakim da Tanaavtorebma [131] gamoikvlies C-20 da C-22 ato-mebis stereoqimiuri specifikuroba, romlebic qolesterinis gver-

diTi jaWvis moxleCis procesSi am mdgomareobaSi aRmoCndnen hi-

droqsilirebulebi. am mizniT maT daasinTezes 20-oqsiqolesterinis,

22-oqsiqolesterinisa da 22,22-dioqsiqolesterinis yvela C-20 da C-22-stereoizomeri, romelTa formulebi warmodgenilia qveviT.

R

OH

(20R)

OH

R(20S) R

OH

(22R)R

OH

(22S)

R

OHOH

(20R, 22R)R

OHOH

(20R, 22S)R

OHOH

(20S, 22R)R

OHOH

(20S, 22S)

179

OH

R=sadac

pregnenolonis dagrovebis siCqaris dasadgenad yoveli es ste-

reoizomeri dainkubirda srul mahidroqsilirebel sistemaSi. aR-

moCnda, rom [20R, 22R]- da [20R, 22S]- dioqsiqolesterinebi preg-

nenolonad gardaiqmnebodnen inkubaciis erTi saaTis ganmavlobaSi,

maSin rodesac [20S, 22R]- da [20S, 22S]-izomerebi gardaqmnis gaci-lebiT naklebi siCqariT xasiaTdebodnen. es Sedegebi imis maCvenebe-

lia, rom citoqrom P450scc Zlier mgrZnobiarea qolesterinis mole-

kulaSi C-20- da mcired mgrZnobiarea C-22-atomis steqiometriis mimarT. [20R]-oqsiqolesterini absoluturad araaqtiuria citoqrom

P450-Tan reaqciaSi, maSin roca [20S]-20-oqsiqolesterini didi siC-

qariT gardaiqmneba pregnenolonad. [22R]- da [22S]-22-oqsiqoles-

terinis izomerebi pregnenolonad ufro maRali siCqariT gardaiqmne-

bian, vidre TviT qolesterini. es faqti substratis molekulis C-22-atomis stereospecifikurobis umniSvnelo rolis maCvenebelia.

qolesterinis gverdiTi jaWvis moxleCis procesSi yvelaze saalba-

To Sualedi oqsiwarmoebulebi SeiZleba iyos [20S]-20-oqsiqoles-

terini, [22R]-22-oqsiqolesterini da [20R, 22R]-dioqsiqoles-

terini. am spirtebis pregnenolonad konversiis SedarebiTi siCqaree-

bi Semdegi TanmimdevrobiT mcirdeba:

20,22-dioli > 22-oli > 20-oli ≫ qolesterini.

procesis malimitirebeli stadia SeiZleba iyos C-20- an C-22-hidroqsilireba, radgan 20,22-diolis pregnenolonad gardaqmnis

siCqare gacilebiT maRalia, vidre 20- an 22-olebidan misi miRebis

siCqare. moyvanili monacemebi kidev erTxel adasturebs qolesteri-

nis pregnenolonad gardaqmnis samsafexuriani sqemis samarTlianobas:

Tavdapirvelad xdeba qolesterinis hidroqsilireba C-20- da Semdeg C-22 mdgomareobaSi da Sualed produqtad 20,22-dioqsiqoles-

terini warmoiqmneba. miRebul diols C-22 mdgomareobaSi kidev erT-xel utevs gaaqtiurebuli Jangbadi, rasac Tan axlavs C_H-bmaSi

180

Jangbadis atomis Canergva da gverdiTi jaWvis moxleCa izokapronis

aldehid-hidratis saxiT (nax. 2.15).

citoqrom P450scc-is iseT fraqciebad dasayofad, romlebic C-20- C22-hidroqsilirebas akatalizeben da C-20- C-22-naxSirbadovan kavSirs Slian, gamoyenebul iqna eleqtrofokusirebis meTodi. aR-

moCnda, rom samive reaqcias hemoproteinis erTi da igive fraqcia

axorcielebs [130], Tumca es Sedegi jer kidev ar niSnavs imas, rom

TviT citoqrom P450scc-ebs Soris gansxvaveba ar arsebobdes. dife-

rencialuri speqtroskopiiT Catarebulma kvlevam jefkoati [135] im

daskvnamde miiyvana, rom arsebobs citoqrom P450-is formebi, rom-lebic erTmaneTisagan gansxvavdebian steroidTa mierTebis centrebis

bunebiTa da ricxviT. am mkvlevaris azriT arsebobs P450scc-is ori

forma. pirvels gaaCnia steroidTa dakavSirebis ori centri: L1-

centri ikavSirebs qolesterins da 25-oqsiqolesterins. am SemTxve-

vaSi maRalspinuri kompleqsi warmoiqmneba. dakavSirebis L1-R centri mxolod 20S-oqsiqolesterinTan an 22R-oqsiqolesterinTan, agreTve

pregnenolonTan urTierTqmedebs, rac dabalspinuri kompleqsis miRe-

bas Seesabameba.

citoqrom P450scc-s meore formas dakavSirebis erTaderTi cen-

tri gaaCnia da mxolod 20S, 22R-dioqsiqolesterinTan urTierT-

qmedebs maRalspinuri kompleqsis warmoqmniT. am kompleqsis forma 1

temperaturis daweviT da pH-is gazrdiT dabalspinur mdgomareobaSi

gadadis. process rogorc Cans ar axlavs substratis dakargva. tem-

peraturisa da pH-is SecvliT forma 2-is maRalspinuri kompleqsi

mcire cvlilebebs ganicdis. izolirebul mitoqondriebSi forma 1-

is L1-centri ikavSirebs qolesterins. 20S-oqsiqolesterini ikavebs

forma 1-is L1-R centrs, maSinac ki, Tu am formis L1 centri

mTlianadaa qolesteriniT dakavebuli. 20S-oqsiqolesterinis swraf-

va forma 2-is L2 centrisadmi uaRresad dabalia. 22R-oqsiqoles-

terini maRal swrafvas avlens forma 1-is rogorc L1-aseve L2-

centrebis mimarT. jerjerobiT dasadgenia, warmoadgenen Tu ara es

ori forma citoqrom P450scc- izoformebs an erTi da igive fermen-

tis suberTeulebs, Tu asaxaven fermentze mitoqondriuli membranis

gansxvavebul zemoqmedebas. faqti erTia, rom aseT formaTa arseboba

mniSvnelovan rols asrulebs qolesterinis gverdiTi jaWvis mox-

leCis Tanmimdevruli safexurebis ganxorcielebis rTul procesSi.

181

2.6.5 qolesterini mcenareSi da misi metaboluri gardaqmnebi

xangrZlivi periodis ganmavlobaSi qolesterini tipiur cxove-

lur sterinad iyo miCneuli, radgan misi identifikacia mcenareSi

ar ganxorcielebula. amJamad am sterinis arseboba dadgenilia baq-

teriebSi da maTTan naTesaurad axlos myof lurj-mwvane wyalmcena-

reebSi. umaRlesi mcenareebi mas mcire raodenobiT Seicaven. gamonak-

liss warmoadgenen Teslis zeTebi da yvavilis mtveri [136].

Cvens institutSi n. zambaxiZis [137], agreTve zambaxiZisa da Ta-

naavtorebis mier [138] Seswavlilia umaRles mcenareTa organoebSi

Tavisufali, eTerificirebuli da glikozidirebuli sterinebi da

maTi dinamika. Txelfenovani da air-Txevadi qromatografiis meTodiT

dadgenilia, rom mTeli vegetaciuri periodis ganmavlobaSi Tavisu-

fali sterinebis Sedgeniloba raime sagrZnob cvlilebebs ar ganic-

dis da jamur preparatebSi ZiriTad komponentebs warmoadgenen β-sitosterini, kampasterini, qolesterini da stigmasterini. maTTan

erTad vlindeba ori araidentificirebuli sterinic. qolesterinis

Semcveloba jamur preparatSi sakmaod dabalia (∼4-13%), miuxedavad

imisa, rom mcenareSi is winamorbed nivTierebas warmoadgens karoti-

noidebis, sterinuri sapogeninebis, alkaloidebis da gansakuTrebiT

fitohormonebis biosinTezSi [139, 140]. Tavisufal sterinTa bio-

sinTezs rac Seexeba, naCvenebi iqna, rom es procesi gacilebiT aqti-

urad vegetaciis sawyis fazaSi mimdinareobs [137], rac iseT mniSv-

nelovan fiziologiur process ukavSirdeba, rogoric ujredis mem-

branogenezia.

2.6.5.1 qolesterinis eTerifikacia mcenareSi

qolesterinis araJangviTi gardaqmnisas misi molekulis cikluri

komponenti ucvleli rCeba, xolo gverdiTi alifaturi nawilis oq-

sigenirebisas cxoveluris msgavsad ornaxSirbadatomiani fragmentiT

SeiZleba damokldes da pregnenoloni warmoiqmnas.

mcenareul ujredSi qolesterini upiratesad stearin-, palmi-

tin-, olein- da linolenmJavebTan eTerebis saxiT imyofeba. safiqre-

belia, rom ujredSi egzogenurad moxvedrili qolesterinic JangviT

182

gardaqmnebSi CarTvamde, upirveles yovlisa, eTerifikacias ganicdis.

es procesi, rogorc Cans, misi Warbi raodenobis SeboWvis (deponi-

rebis) umartivesi meqanizmia, romlis Semdgomac saWiroebis SemTxve-

vaSi ujredi mas aqtiuri metabolizmisaTvis iyenebs. am Tvalsazri-

siT gasaTvaliswinebelia ramdenime momenti:

1. qolesterinis transformacia ujredSi SeiZleba iyos, rogorc

~SemTxveviTi~ procesi;

2. qolesterinma SeiZleba monawileoba miiRos endogenur steronu-

li bunebis naerTTa biosinTezSi an Tavisufali saxiT membranaTa

struqturirebaSi;

3. qolesterini SeiZleba gamoyenebul iqnas naxSirbadatomebis wya-

rod;

4. qolesterini SeiZleba gardaiqmnas imgvarad, rom es iyos misi

detoqsikaciis da ujredidan mocilebis Taviseburi reaqcia.

ujredSi gansakuTrebul mniSvnelobas unda iZendes qolesterinis

sruli fermentuli degradacia da plastikuri miznebisaTvis misi

gamoyeneba (punqti 2 da 3).

mcenareSi egzogenuri qolesterinis eTerificirebasa da mis

JangviT gardaqmnebs Soris raodenobrivi Tanafardobis dasadgenad

gamokvleva iqna Catarebuli Cvens laboratoriaSi [141].

vazisa (Vitis vinifera) da iuka didebulis (Yukka gloriosa) foTle-

bis Txeli anaTlebi inkubirda 4-14C-qolesterinTan (1.5 µCi _ 2.10-3 M) da saTanado komponentebTan (palmitinmJava, stearinmJava, ATP, cAMP, glutaTioni, cikloheqsimidi) 4 saaTis ganmavlobaSi fosfa-

tis buferSi (pH 6.2 vazisaTvis da 5.0 iukasaTvis). heqsanoliTa da

eTanoliT eqstraqciis Sedegad miRebul iqna neitraluri lipidebisa

da metabolurad aqtiur nivTierebaTa, anu 80% eTanolSi xsnadi

(organuli mJavebis, aminomJavebis da Saqrebis) jamuri fraqciebi.

Tavisufali da eTerificirebuli sterinebis dayofas vawarmoebdiT

Txelfenovani qromatografiiT (silikageli 5/40 µ, gamxsneli – ben-

zoli : gogirdis eTeri 7 : 3). radioaqtiurobis aTvla xdeboda Txe-

vad-scintilaciur mTvlelze (SL-30). paralelurad dgeboda e.w.

~mkvdari cda~, romlis mixedviTac eqsperimentul monacemebSi Segv-

qonda saTanado Sesworebebi.

Catarebuli eqsperimentebis safuZvelze gamovlinda, rom vazis

183

foTlebSi palmitatTan da stearatTan 4-14C-qolesterinis inkubi-

rebisas mniSvnelovnad izrdeba eTerificirebuli sterinebis jamuri

fraqciis radioaqtiuroba da am SemTxvevaSi stearati ufro efeq-

turia. saintereso is aris, rom eTerificirebis paralelurad

gazrdilia 80%-ian eTanolSi xsnadi fraqciis radioaqtiurobac.

maSasadame, mcenaris foTlebSi, eTerifikacias myisierad mosdevs hi-

drolizi da am dros gamoTavisuflebuli qolesterini Semdgomi

transformaciisaTvis gacilebiT ~labiluria~, vidre ujredSi Tavi-

suflad myofi endogenuri qolesterini.

sainkubacio xsnarSi ATP-s, cAMP-sa da glutaTionis erTdro-

ulad Tanamyofobisas 80%-ian eTanolSi xsnadi fraqciis maqsimalur

zrdas Tan axlavs eTerificirebuli fraqciis radioaqtiurobis

mkveTri vardna. Sedegi kidev erTxel adasturebs im faqts, rom

ujredSi dabalmolekuluri aqtiuri metabolitebis warmoqmnaSi

upiratesad eTerebis hidrolizisas gamoyofili ~labiluri~ qoles-

terini monawileobs. eTerificirebuli sterinebisa da metabolurad

aqtiur naerTTa raodenobrivi Tanafardobebis ganxiluli cvlilebe-

bis fonze Tavisufali sterinebis fraqciis radioaqtiuroba metnak-

lebad mudmivadaa SenarCunebuli.

cikloheqsimidi (50 mkg/ml) mniSvnelovnad Trgunavs eTerebis

sinTezs. SedarebiT naklebi xarisxiT aqveiTebs igi metabolurad

aqtiuri naerTebis warmoqmnas. es ar SeiZleba gamowveuli iyos mi-

toqondriebisaken qolesterinis satransporto gzis blokirebiT, an

mitoqondriul sterolhidroqsilazur sistemaze am inhibitoris

moqmedebiT. Cven davadgineT, rom cikloheqsimidis maRali koncentra-

ciebi (∼100 mkg/ml) sakvlev mcenareTa foTlebSi aerobul sunTqvaze

absoluturad ar moqmedebs, rogorc Cans, mitoqondriul membranaSi

misi ganuvladobis gamo. amitom ufro safiqrebelia, rom cikloheq-

simidiT gamowveuli inhibireba dakavSirebuli iyos ribosomuli ci-

lebiT, kerZod, sterinsinTetazebis warmoqmnis daTrgunvasTan.

analogiuri kanonzomierebebi gamovlinda iuka didebulis foTleb-

Sic: stearati da palmitati calkeulad zrdian qolesterinis radioaq-tiuri niSnis CarTvas eTerificirebuli sterinebisa da metabolurad aqtiuri naerTebis fraqciaSi. ATP, cAMP da glutaTioni zrdian 80%-

ian eTanolSi xsnadi fraqciis radioaqtiurobas, rasac Tan sdevs eTeri-ficirebuli sterinebis fraqciis radioaqtiurobis daqveiTeba.

184

yuradRebas imsaxurebs erTi uaRresad mniSvnelovani garemoeba:

orive sakvlevi mcenaris foTlebSi metabolurad aqtiur naerTTa

fraqciis radioaqtiurobis maqsimaluri mateba mTliani radioaqti-

urobis (igulisxmeba mSrali mcenareuli naSTis, eqstragirebuli

fraqciebisa da 14CO2-is radioaqtiurobaTa jami) ∼25%-s aRwevs, Tu

sainkubacio xsnarSi erTdroulad imyofeba ATP da 3′,5′-AMP (cik-luri adenozinmonofosfati). maSasadame, aTvisebuli qolesterini

biodegradaciulad gardaiqmneba. 4-saaTiani inkubaciis pirobebSi es

sakmaod maRali maCvenebelia da qolesterinis katabolizmSi mona-

wile mJangveli sistemis maRal aqtiurobaze miuTiTebs.

2.6.5.2 qolesterinis Jangva mikroorganizmebsa da

mcenareSi (SedarebiTi analizi)

mikrobebsa da mcenareul qsovilebSi qolesterinis JangviTi de-

gradacia cxovelebTan SedarebiT jer kidev arasaTanadodaa gamokvl-

euli. zogad principebSi msgavseba ki SemdegSi mdgomareobs: 1. hid-

roqsilirebisaTvis samive SemTxvevaSi aucilebelia atmosferuli

Jangbadi; 2. anaerobul pirobebSi hidroqsilireba ar xorcieldeba;

3. hidroqsilireba moiTxovs aRmdgeneli agentis Tanamyofobas da am

mizniT Cveulebriv NADPH an flavinuri fermenti gamoiyeneba;

4. hidroqsilirebas metalSemcveli fermentebi akatalizebs.

mikrobul ujredebSi steronTa JangvaSi monawile fermentebisaT-

vis saWiro eleqtronTa satransporto sistema garkveul msxvil

subujredul nawilakebSia lokalizebuli, romlebic 7500 g-ze srulad

ileqebian, xolo maTSi arsebuli qolesterindehidrogenaza 110000 g-zec ki ar ileqeba. cxovelur qsovilebSi es fermentuli sisteme-

bi endoplazmur membranebSi imyofebian da aRniSnul aCqarebebze

srulad `jdebian” [142]. mikrobuli sterinesTerazas sterindehi-

drogenazasa da sterinhidroqsilazasTvis optimaluria susti fuZe

are (pH 8-9). maTSi hidroqsilirebas axorcielebs flavoproteini,

romelic rkinis, an TuTiis ionebs Seicavs. am fermentis daJanguli

formis regeneracia NADPH-iT xdeba. sterin-1,2-dehidrogenaza

flavinur prosTetul jgufs Seicavs da eleqtronis aqceptorad

K2-vitamins saWiroebs. uujredo preparatebSi misi Secvla qinone-

185

biT, an sxvadasxva saRebavebiT SeiZleba. rogorc gamoirkva, sterinTa

mikrobul transformaciaSi monawile fermentTa aqtivobebi maRali

specifikurobiT (hidrolizuris garda) gamoirCevian. isini moqmede-

ben sterinebTan, rogorc naerTTa klasTan da sxva tipis naerTebs

iSviaTi gamonaklisis garda, mcire siCqariT gardaqmnian [142].

mikroorganizmebSi sterinebis, kerZod qolesterinis JangviTi

degradacia SeiZleba iyos sruli (CO2-mde da H2O-mde) an nawilo-

brivi, romlis drosac metabolizmis saboloo produqtebs SenarCu-

nebuli aqvT sterinebis molekulis ZiriTadi ConCxi da Jangvas

mxolod maTi calkeuli fragmentebi ganicdian. mikroorganizmTa zo-

gierTi saxeoba qolesterins ramdenime Tvis ganmavlobaSi Slis, ma-

Sin roca Aspergilus wentii-s Sereuli kulturebi am nivTierebis

aciklur naerTebad gardaqmnas ramdenime saaTs andomeben. Jangvis

produqtebs mikroorganizmebi gareT gamoyofen. qolesterinis A-

birTvis gardaqmnisas sawyis stadiaze C-4-naxSirbadatomis Camoci-

leba xdeba da ∆43-ketowarmoebuli miiReba. 4-14C-testosterinis fermentaciisas gamoyofili CO2 radioaqtiuri mxolod orsaaTiani

inkubirebis (rogorc Cans induqciis periodis) Semdeg xdeba, maSin

roca sakvebi aredan sterinis ukve mesamedia SeTvisebuli. es faqti

imaze miuTiTebs, rom qolesterinisagan gansxvavebiT testosterinis

A-birTvi ar warmoadgens daSlis pirdapir samiznes. 26-14C da 27-14C-qolesterinis naxSirbadatomebi propionmJavas meTilisa da kar-

boqsilis jgufSi erTvebian [142].

ZuZumwovarTa qsovilebis fermentul sistemebs qolesterinis

alifaturi fragmentis SerCeviTad daJangvis unari aqvT, ris Sede-

gadac naRvlis mJavebi da gansxvavebuli klasebis sterinuri hormo-

nebi miiRebian. mcenares rac Seexeba, maTSi aRniSnuli procesis mi-

marTulebas da siCqares TviT am nivTierebis moqmedebis xangrZlivo-

ba, kvebis reJimi, fizioligiuri mdgomareoba, vegetaciis faza da

sxv. gansazRvravs.

mcenareSi aromatuli da policikluri naerTebis SesaZlo de-

gradaciis sistematuri kvleva dawyebulia Cvens institutSi.

s. durmiSiZis [143], agreTve s. durmiSiZisa da T. beriaSvilis mier

[144] pirvelad iqna naCvenebi qolesterinis cikluri komponentis,

kerZod misi A-birTvis JangviTi degradaciis ganxorcieleba. am miz-

niT maT gamoikvlies sterilur pirobebSi gazrdili stafilosa da

186

simindis nazardebis qsovilebi, romlebic inkubirdebodnen 4-14C-qolesterinis Semcvel xsnarSi. aRmoCnda, rom 7-saaTiani inkubaciis

Sedegad mTlianad micemuli qolesterinis radioaqtiurobis 4-8%

Jangvis produqtebSi [80%-ian eTanolSi xsnad fraqciaSi da CO2-Si)

iyo CarTuli. miRebul naerTTa bunebis gaTvaliswinebiT, aRniSnulma

mkvlevarebma warmoadgines qolesterinis A-birTvis Jangvis savarau-

do Tanmimdevroba: A-birTvis dehidrireba (aromatizacia), hidroq-

silireba me-4 mdgomareobaSi, B-birTvis daSla da seko-naerTis war-

moqmna, karboqsilSi me-4 naxSirbadis daJangva C6-fragmentad (piro-

kateqinad), A-birTvis mocileba da ketomJavas warmoqmna.

eTerifikaciasTan erTad qolesterinis gardaqmnebSi ATP-s da

3′,5′_AMP-s mastimulirebeli moqmedebis Cvens mier gamovlenilma

faqtma [141] saSualeba mogvca misaRebad CagveTvala, rom mcenareSi

am sterinis katabolizmi mTlianobaSi daaxloebiT iseTive Tanmimdev-

ruli meqanizmiT unda xorcieldebodes, rogorc es garinisa da Tan-

aavtorebis mier [145-147] Tirkmelzeda jirkvlis qerqSi iqna dadge-

nili. am mkvlevarTa Tanaxmad procesi sqematurad ase gamoiyureba:

lipiduri

wveTi

gaaqtive-

buli esTe-

raza

esTeraza

32p 32p 32p 32p

receptori

ujredis garsis

membrana

adenilatciklaza

proteinkinaza

akth

gaaqtivebuli

proteinkinaza

pregnenoloni

kortikosteronebi

Tavisufali

qolesterini

cikloheqsimidicilis biosinTezi

+ATP3',5'-AMP

ATP +

mitoqondria

nax. 2.16. Tirkmelzeda jirkvlis qerqSi qolesterinis eTerebis es-

Terazis akth-iT aqtivacia [147] (ganmarteba teqstSi).

sqemis Tanaxmad kortikotropini (adrenokortikotropuli hor-

moni _ akth) urTierTqmedebs ujreduli membranis gare zedapirze

187

myof receptorTan. dakavSirebis Sesaxeb signali ferment adenilat-

ciklazas (EC 4.6.1.1) gadaecema. igi membranis Sida zedapirzea lo-

kalizebuli da akatalizebs ATP-s gardaqmnas ciklur adenozinmo-

nofosfatad (ATP=3′,5′_AMP + pirofosfati). qolesterinis eTere-

bis doneze es naerTi srulad imitirebs kortikotropinis moqmede-

bas, romelic mcenareSi saerTod ar gvxvdeba.

kortikotropinis funqcionirebasi mniSvnelovani momentia misi

gavleniT 3′,5′_AMP-iT citoplazmuri proteinkinazas (EC 2.7.1.37) gaaqtiveba. es fermenti ATP-s daxmarebiT akatalizebs ribosomebSi

cilebis fosforilebas (ATP + proteini → ADP + fosfoproteini). am gziT fermenti romeliRac meqanizmiT aaqtiurebs esTerazas, ro-

melic lipidur wveTSi qolesterinis eTerebis hidrolizs akata-

lizebs da Tavisufal qolesterins warmoqmnis. procesi aramgrZno-

biarea cilis sinTezis inhibitoris _ cikloheqsimidis mimarT, anu

igi ar saWiroebs de novo cilis sinTezs. samagierod, es inhibitori

srulad ablokirebs kortikosterinebis sinTezSi kortikotropinisa

da 3′,5′_AMP-is mastimulirebel moqmedebas. misi Tanamyofobisas

qolesterinis eTerebi Tavisufal qolesterins warmoqmnian, anu aq-

tiuri esTeraza ar iTrguneba, magram cximovani wveTidan mitoqon-

driaSi Tavisufali qolesterinis gadatana aRar xdeba.

proteinkinaza Tirkmelzeda jirkvalSi aaqtiurebs fosforilaza

B-s kinazasac, romelic fosforilebiT aqtiur fosforilaza A-s

iZleva da glikogens glukozo-1-fosfatad Slis. es ukanaskneli

pentozur ciklSi qolesterinis oqsigenirebisaTvis saWiro aRdge-

nil NADPH-s generirebs. amgvarad 3′,5′_AMP-sa da proteinkinazas samive efeqti: esTerazas gaaqtiureba, ribosomebis fosforileba da

fosforilaza B-s stimulireba kortikosteroidebis gaZlierebuli

sinTeziskenaa mimarTuli.

qolesterinis pregnenonolad gardaqmna desmolazuri kompleqsis

(citoqrom P450-Semcveli monooqsigenazas) monawileobiT xorciel-

deba, romelic mitoqondriaSi imyofeba. qolesterinma aucileblad

unda SeaRwios am organelaSi da amitom steroidogenezisaTvis sa-

Wiroa mitoqondriisaken misi uwyveti nakadis arseboba. varaudoben,

rom amisaTvis gamoiyeneba specialuri satransporto cilebi, ro-

melTa biosinTezi gaaqtiurebuli proteinkinaziT stimulirdeba da

cikloheqsimidiT muxruWdeba [147].

188

esTerazis aqtivaciis meqanizmis Sesaxeb garenisa da Tanaavt.

[145-147] warmodgenis samarTlianobam SemdgomSi absoluturi dadas-

tureba hpoves sxva mkvlevarTa eqsperimentul gamokvlevebSi [148,

149], sadac dadgenil iqna sruli korelacia 3′,5′_AMP-s koncen-traciis cvlilebebsa, esTerazasa da proteinkinazis aqtivobebsa da

agreTve lipidur wveTebSi qolesterinis eTerebis klebas Soris.

amgvarad, nax. 2.16-is Tanaxmad, Tirkmelzeda jirkvlis qerqovan

nawilSi qolesterinis alifaturi fragmentis mocileba (pregneno-

lonad gardaqmna) Sidamitoqondriul process warmoadgens. aRniS-

nulidan gamomdinare, Cven SevecadeT mcenareSic gamogvevlina analo-

giuris Tu ara msgavsi suraTis arseboba da gveCvenebina ujredis am

organelis SesaZlo roli qolesterinis A-birTvis JangvaSi da misi

kavSiri mitoqondriebSi mimdinare energetikul procesebTan [150].

kvlevis obieqtad amjeradac aRebuli gvqonda vazisa da iuka di-

debulis axladmoWrili foTlebis Txeli anaTlebi [141]. 4-14C-qolesterinis gardaqmnis intensivobaze mitoqondriuli Jangvisa da

fosforilebis substratis, limonmJavas ciklis inhibitorebis,

agreTve mikrosomuli redoqs-jaWvis stimulatorebisa da inhibito-

rebis gavlenas vafasebdiT gamoyofili 14CO2-is radioaqtiurobis

mixedviT.

Catarebulma gamokvlevebma gviCvenes, rom qolesterinis A-

birTvis Jangva sagrZnoblad stimulirdeba sareaqcio areSi mito-

qondriuli Jangvis substratTa Tanamyofobisas, kerZod, malati da

suqcinati, agreTve aminomJavebi, romelTa JangviTi gardaqmnebi uSu-

alod arian am ciklTan dakavSirebuli (alanini, aspartati da glu-

tamati), mniSvnelovnad zrdian gamoyofili 14CO2-is radioaqtiuro-

bas (cxrili 2.6). Jangvis substratebTan fosforilebis substratis

(ADP-s) Tanamyofoba procesis ufro met stimulirebas iwvevs. sain-

kubacio xsnarSi α-ketoglutaratTan ADP-s Tanamyofoba sterinis A-birTvis Jangvis 60-85%-ian stimulacias iZleva. limonmJavas

ciklis specifikuri inhibitorebi _ malonati da monoftoracetati 14CO2-is radioaqtiurobas vazisa da iukas foTlebSi Sesabamisad

∼35-40% da ∼50%-iT Trgunaven. inhibitorulad moqmedebs procesze

Jangvisa da fosforilebis gamTiSveli _ 2,4-dinitrofenoli da es

misi inhibitoruli moqmedeba srulad kompensirdeba ATP-iT (cxri-li 2.7).

189

cxrili 2.6 [150]

vazisa da iuka didebulis foTlebis anaTlebSi 4-14C-qolesterinis

(4-14C) Jangvisas gamoyofil 14CO2-is radioaqtiurobaze mitoqon-

driuli Jangvisa da fosforilebis substratTa gavlena*

cdis pirobebi

14CO2-is radioaqtiurobebi

imp/wT

vazi iuka didebuli

4-14C-qolesterini (kontroli) 477±17.9 356±8.6 `+ADP 611±24.0 602±7.1 `+suqcinati+malati 614±11.9 628±17.8 `+`+`+ADP 661±14.2 675±4.4 `+α-ketoglutarati 657±4.4 400±6.8 `+`+ADP 1031±26.3 847±17.4 `+alanini+aspartati+glutamati 771.7±20.3 729±12.5 `+`+`+`+ADP 975±14.0 828±20.5

*fosfatis buferi pH 6.0 (vazisaTvis) da pH 5.0 (iukasaTvis); Jangvis substratTa koncentraciebi _ 6.10-3 M TiToeuli; ADP _

1.10-3 M; Mg2+ _ 16 mM; 4-14C-qolesterini _ 2.10-3 M (1.5 µCi) cdis

xangrZlivoba _ 3 sT; cdebis ganmeorebis ricxvi _ xuTjeradi.

cxrili 2.7 [150]

vazisa da iuka didebulis foTlebis anaTlebSi 4-14C-qolesterinis

Jangvisas gamoyofili 14CO2-is radioaqtiurobaze 2,4-

dinitrofenolis (2,4-DNP-s) da ATP-s gavlena*

cdis pirobebi

14CO2-is radioaqtiurobebi

imp/wT

vazi iuka didebuli

4-14C-qolesterini (kontroli) 469.0±21.0 760.0±20.3 `+ANP 350.0±22.7 550.0±21.9 `+ATP 570±19.8 940±21.3 `+ATP+2,4-DNP 430±22.9 670±19.5

* pirobebi igive rac 2.6 cxrilSi 2,4-DNP _ 1.10-3 M; ATP _ 300 µ.

190

mitoqondriebis energetikul mdgomareobasa da qolesterinis A-

birTvis Jangvas Soris pirdapiri kavSiris arseboba kidev ufro

mtkiced aisaxa Cvens cdebSi, sadac sainkubacio xsnaris `energiis

muxtis” mudmiv matebas (adenilaturi akumulatoris energiiT mdi-

dari fosfaturi kavSirebiT Sevsebas) Tan axlda 14CO2-is radioaq-

tiurobis zrdac. rogorc cnobilia am sidides, e.w. atkinsonis [151]

Tanafardobas ([ATP]+1/2[ADP]) : ([ATP]+[ADP]+[AMP]) SeuZlia mii-

Ros 0-dan (sruli deenergizacia) 1-mde (sruli energizacia) mniSv-

nelobebi da mTeli rigi regulatoruli fermentisaTvis gamovleni-

lia am sididesa da enzimur aqtivobebs Soris dadebiTi funqciuri

damokidebuleba [152, 153].

iRebs Tu ara mcenaris mikrosomuli citoqrom P450-Semcveli

redoqs-jaWvi qolesterinis JangvaSi monawileobas? am kiTxvaze pa-

suxis gasacemad Cven mier [150] Catarebul iqna eqsperimentebi, rom-

lebSic aRniSnul sterinTan erTad gamoviyeneT Jangvis am sistemis

tipiuri substrati amidopirini, hemoproteinis specifikuri inhibi-

tori SKF-525A da induqtori fenobarbitali. aRmoCnda, rom amido-

pirini qolesterinis Jangvis mimarT araviTar konkurentul zemoqme-

debas ar avlens. araefeqturia inhibitoris Tanamyofobac, rac am

Sxamis mitoqondriul membranaSi ganuvladobiT SeiZleba aixsnas; rac

Seexeba fenobarbitals, misi induqciuri zemoqmedeba ar gamomJRavnda

maSinac ki, Tu foTlebi winaswar 20-30 sT-iT inkubirdeboda mis

xsnarSi.

amgvarad, mcenareSi ori _ mikrosomuli da mitoqondriuli

NADPH-damokidebuli redoqs-jaWvis arseboba araviTar eWvs ar iw-

vevs. isini funqciurad gansxvavdebian erTmaneTisagan. qolesterinis,

rogorc alifaturi fragmentis, aseve molekulis cikluri kompo-

nentis Jangva mcenareSic mitoqondriis funqciaa. qolesterinis ka-

tabolizmsa da mcenareuli qsovilis aerobul sunTqvas Soris mWi-

dro urTierTkavSiri arsebobs; qolesterinis A-birTvis Jangva ener-

godamokidebuli procesia, ramdenadac masSi monawile fermentebi

ujredis energetikuli potencialis maRal dones saWiroeben.

miRebulma Sedegebma saSualeba mogvca dasaSvebad migveRo qo-

lesterinis A-birTvis Jangvis Tanmimdevruli procesis SedarebiT

martivi sqema: 1. NADPH- da O2-damokidebuli hidroqsilireba meS-

vide mdgomareobaSi; 2. ormagi kavSiris gadanacvleba 4-5-mdgoma-

191

reobaSi da mesame hidroqsilis daJangva; 3. meore stadiaze miRebu-

li produqtis Semdgomi Jangva Sesabamisi karbonmJavasa da CO2-is

warmoqmniT.

cxadia, steroidogenezisa da qolesterinis JangviTi degradaciis

SesaZlo gzis Zieba da dazusteba mcenareSi momaval safuZvlian

kvlevas moiTxovs. erTi ram savsebiT garkveulad gamoiyureba: Se-

daujredul metabolizmsa da gansakuTrebiT energetikul cvlasTan

uaRresad mWidro kavSiri safuZvels iZleva, rom araviTar SemTxve-

vaSi ar gavaigivoT qolesterinis gardaqmnebi detoqsikaciasTan.

2.7 urTierTkavSiri oqsigenazur da imunur sistemebs Soris

gasuli saukunis samocdaaTiani wlebis dasasruls gamoikveTa

citoqrom P450-Semcveli monooqsigenazuri sistemis axali, metad

mniSvnelovani funqcia _ imunur meqanizmSi monawileoba. cocxal

organizmSi am ori sistemis (monooqsigenazuris da imunuris) funq-

ciur aqtivobaTa Sedarebisa da Sejerebis safuZvelze Camoyalibda

Tvalsazrisi imis Sesaxeb, rom isini zogadi universaluri principe-

biT unda iyvnen erTmaneTTan dakavSirebuli da es upirveles yovli-

sa maT adapturobasa da inducibelobaSi mJRavndeba. orive sistema

mimarTulia mravalferovani ucxo da endogenuri naerTis SekavSire-

ba_metabolizirebisaken da induqciis gamomwvevi qimiuri naerTebi-

sadmi gansxvavebulad gamosaxuli `maxsovroba” gaaCniaT. sul ufro

TvalsaCino da sarwmuno gaxda monooqsigenazur da imunur funqcia-

Ta meqanizmebis erTmaneTTan mWidrod Serwymis faqtebi.

saerTod imuniteti ganixileba rogorc genetikurad gansxvave-

buli informaciis matarebeli cocxali sxeulebisa da nivTierebebi-

sagan organizmis dacvis fenomeni. igi ZiriTadad ujredis makrofa-

gur-limfocituri kompleqsiT xorcieldeba, romelic antigenis

(ucxo genetikuri informaciis Semcveli makromolekulebis) `dapy-

robas”, `gadamuSavebas” da antisxeulebis (antigenis SemkavSirebeli

da gamaneitralebeli imunoglobulinebis) producirebas emsaxureba.

garkveul mizezTa gamo qsenobiotikebis ori tipi (dabal- da ma-

Ralmolekuluri) ori mecnieruli mimarTulebis _ bioqimiuri far-

makologiis (dabalmolekuluri agentebisaTvis) da imunologiis (ma-

192

Ralmolekuluri naerTebisaTvis) erTmaneTTan sustad dakavSirebul

kvlevis obieqtebad ganixileboda.

evoluciuri da sistemuri midgomis poziciidan gamomdinare, mo-

lekuluri farmakologiisa da arainfeqciuri imunitetis kvlevaSi mo-povebulma miRwevebma da maTmaranalizma rus mecniers i. kovaliovs [154] saSualeba misca wamoeyenebina axali koncefcia, romlis Tanaxmadac

imunuri sistema gacilebiT farTo (zogad) funqciuri sistemis na-

wils warmoadgens da igi konkretulad qimiur naerTTa Secnobas,

metabolizmsa da organizmidan gamoZevebas emsaxureba. koncefciis Tanax-mad imunologiuri sistema monawileobs homeostazSi aramxolod

ujredul (uaryos ucxo an sakuTari saxeSecvlili ujredebi) da

makromolekulur, aramed dabalmolekulur naerTTa donezec; anu

imunologiuri kontroli totaluria da mravalferovan ucxo da

sakuTar nivTierebaTaTvis saerToa. monooqsigenazuri da imunologi-

uri sistemebis funqciurma SeuRlebam SemosazRvra qimiuri homeos-

tazis metabolur-imunologiuri (imunoqimiuri) sistemis konturebi.

koniugirebuli antigenis sinTezSi, romelic qimiur nivTiere-

baze specifikur pasuxs iZleva, did rols asrulebs citoqrom

P450-Semcveli sistema. masze `ikveTeba” hidrofobuli bunebis mqone

endogenuri da egzogenuri naerTebis mZlavri `nakadebi”. unda aRi-

niSnos isic, rom am fermentuli kompleqsis garda, koniugirebuli

antigenebis sinTezSi monawileoba SeiZleba miiRos sxva fermentebmac

(mag., qsantinoqsidazam, peroqsidazebma, Tirozinazam da sxv.).

barbituratebisa da zogierTi sxva medikamentebis ganmeorebiTi

gamoyenebis Semdeg yalibdeba wamlebis mimarT organizmis toleran-

toba, rasac xSirad metabolizmis daCqarebas da wamlis dezaqtiva-

cias ukavSireben da rac mikrosomuli oqsigenazebis induqciis Sede-

gad miiRweva [155]. wamlebis es unari _ daaCqaron sakuTari metabo-

lizmi, dadgenilia fenobarbitalis, heqsobarbitalis, fenilbutazi-

nis, meprobamatis, tolbutamidis, qlorciklizinisa da sxvaTa mi-

marT. barbitalis SemTxvevaSi, romelic sustad metabolizdeba, to-

lerantoba SeiZleba im receptorTa (ix. 2.6.6.1) mgrZnobiarobis

cvlilebaTa Sedegad ganviTardes, romlebzedac esa Tu is wamali

moqmedebs. meprobamatis mimarT tolerantoba Tavdapirvelad wamlis

mier inducirebuli metabolizmis gazrdis gamo, xolo Semdgom cen-

traluri nervuli sistemisa da wamlis uSualo zemoqmedebis qveS

193

myof sxva organoTa reaqciis cvlilebaTa Sedegad SeiZleba Camoya-

libdes. morfinisa da kodeinisadmi tolerantoba, uwinares yovlisa,

amTvisebel organoTa reaqciis cvlilebebiTaa ganpirobebuli, ramde-

nadac cxovelSi maTi meoradi ineqcia RviZlis im mikrosomuli fer-

mentebis araspecifikur daTrgunvas iwveven, romlebic am wamlebis

metabolizms uzrunvelyofen [155].

cxovelTa imunuri reaqciebis analizisas mxedvelobaSia misaRebi

imunuri pasuxis ori _ specifikurobisa da maxsovrobis parametri,

radgan ganmeorebiT imunizaciaze erTsa da imave antigeniT gaZliere-

bul da daCqarebul pasuxs ganapirobeben. orive sistemas (detoqsi-

kaciurs da imunurs) es ori parametri namdvilad gaaCnia. Zlier

ganviTarebuli maxsovrobis unaris mqone nervuli sistemis mier

gare gaRizianebis miRebis msgavsad, imunuri sistemac Seicavs antige-

nebiT aRqmis saSualebas, anu ujredebSi imyofeba maTi Sesabamisi

receptorebi. citoqrom P450-is sistemas rac Seexeba, igi moqmedebs rogorc srulyofili qemoreceptori da qemoanalizatori, erTnai-

rad reagirebs rogorc Sinagan, aseve garegani qimiuri garemos cvli-

lebebze. es tipiuri kibernetikuli `manqanaa”, romelic aRiqvams da

aanalizebs maxsovrobisa da avtomaturi TviTregulaciis Semcvel

qimiur signals.

saerTod, sisxlis plazmis ciluri sistema SedarebiT martivad

aregulirebs organizmSi qimiur nivTierebaTa doneebs. gancvifrebas

iwvevs bunebis `sibrZne”, romelmac nebismieri mudmivad cvalebadi

viTarebisaTvis yvelaze `gonieri” gadawyvetis avtomaturad misaRebi

meqanizmebi SeimuSava. homeostazis Teoriis Semqmneli amerikeli fi-

ziologi kenoni [156] Tvlida, rom homeostazi `damcveli meqanizme-

bis” didi jgufis is erT-erTi variantia, romelTa zogad Tviseba-

sac funqcionirebis avtomaturi buneba aqvs. am mkvlevaris azriT,

isini Segnebisa da nebisyofisagan damoukideblad moqmedeben, rogorc

`sxeulis sibrZnis” TavisTavadi gamovlena. igi Tvlida, rom es Tvi-

sebebi yvela cocxal sxeuls dasawyisidanve gaaCnia, vinc ki `sibr-

ZniTaa” aRWurvili. 1932 w. kenonma gamosca monografia `sxeulis

sibrZne” [156], romelsac samecniero wreebi gacxovelebuli intere-

siT Sexvdnen. SemdgomSi kenonis am sxva Sromebma safuZveli Cauyares

dRevandel kibernetikas.

eqsperimentSi antisxeulebi advilad inducirdeba aramxolod

194

ucxo warmoSobis dabal- an maRalmolekuluri naerTebisadmi, aramed

sakuTari endogenuri naerTebis mimarTac. es imaze miuTiTebs, rom

antisxeulebi ar warmoadgenen im cilebs, romlebic mxolod makro-

molekuluri qsenobiotikebis neitralizaciasa da maTi organizmidan

gamodevnas emsaxurebian: mikrosomuli fermentebis induqciisa da

qsenobiotikebiT gamowveul imunur pasuxebs Soris garkveuli kavSi-

ri arsebobs. dabalmolekuluri qsenobiotikebi inducireben hidroq-

silazur sistemas, maRalmolekulurebi ki limfocitur imunur

sistemas [157]. am Tvalsazrisidan gamomdinare, hidroqsilazuri da

imunuri sistemebi dabal- da maRalmolekuluri qsenobiotikebisagan

organizmis dacvis erTiani sistemis konteqstSi ganixileba. am miz-

niT Catarebuli gamokvlevebiT naCvenebi iqna, rom cxovelSi Tavisu-

fali fenobarbitalis Seyvana zrdis RviZlSi citoqrom P450-is xvedriT Semcvelobasa da N-demeTilazur aqtivobas. albuminTan ko-

valenturad dakavSirebuli fenobarbitali ki am unars moklebulia.

es faqti dakavSirebuli araa albuminis inhibitorul moqmedebasTan.

eqsperimentebma aCvenes, rom xaris Sratis albuminis cxovelSi Sey-

vana ar moqmedebs citoqrom P450-is Semcvelobasa da mikrosomul

hidroqsilazas aqtivobaze. fenobarbitalisa da albuminis erTdro-

uli ineqcia iseTive mainducirebel efeqts iZleva, rogorsac calke

fenobarbitali. mxolod am ukanasknelis albuminTan kovalenturad

dakavSirebisas mainducirebeli efeqtis dakargva gamowveuli unda

iyos miRebuli koniugatis unariT limfocitebis zedapirze warmoqm-

nas fenobarbitalis damkavSirebeli receptori [157]. makromoleku-

lur matarebelTan, magaliTad, cilasTan kovalenturad mierTebuli

fenobarbitalis cxovelSi Seyvana limfocitebSi imunoglobulinebis

inducirebas iwvevs, romlebic specifikurad ikavSireben fenobarbit-

als da masTan struqturulad axlo mdgom nivTierebebs da maT in-

aqtivacias axdenen.

imunologiuri sistemisaTvis arsebuli `ZiriTadi dgoma”, rom

igi cnobs rogorc `Tavisians”, aseve `ucxos”, magram mxolod `uc-

xos” inaqtivacias axdens, nawilobrivaa samarTliani. sinamdvileSi

imunuri sistema Cveulebriv reagirebs rogorc ucxo, aseve sakuTar

dabal- da maRalmolekulur naerTebTan. ucxo nivTierebebi, romleb-

ic organizmSi adre ar gvxvdebodnen, imunur sistemas de novo ain-ducireben. amitom msjeloba SeiZleba qsenobiotikebis mier induq-

195

ciis rogorc adaptur reaqciebze (mikrosomuli fermentebis sin-

Tezze), aseve imunologiuri meqanizmebis gaZlierebul induqciaze

(antisxeulebis sinTezze). rogorc Cans, organizmSi qsenobiotikebis

garkveuli (araletaluri) dozebiT mudmivi zemoqmedebisas TandaTa-

nobiT viTardebian metabolizebadi adapturi meqanizmebi. qsenobioti-

ki SeiZleba Tavisebur endogenur naerTad iqces da Semdeg imdenad

`Sevides am rolSi”, rom ujredSi misi SeRwevis Sewyvetam sxvadasx-

va funqciuri darRvevebic ki gamoiwvios. prevencia SeiZleba qseno-

biotikis xelaxali miwodebiT ganxorcieldes. amdenad, xangrZlivi

gamoyenebis Sedegad ucxo nivTiereba organizmis romeliRaca meta-

boluri reaqciis substrati xdeba. amis saukeTeso magaliTebia mor-

fini, barbituratebi, alkoholi da sxv. [158].

monooqsigenazur sistemasTan qimiur nivTierebaTa urTierTqmede-

bisas, rogorc wesi maTi SedarebiT polarul da organizmidan ad-

vilad gamoZevebad formebad gardaqmna xdeba. garda amisa, substrat-

Ta Jangva xSirad dakavSirebulia Sualed maRalreaqciisunarian pro-

duqtTa (epoqsidebis, qinonebis, Tavisufali radikalebis) warmoqm-

nasTan. miuxedavad amisa, SemdgomSi isini am reaqciisunarianobas kar-

gaven, ramdenadac maTi generacia optimalurad kontroldeba fermen-

tuli, antioqsidanturi da sxva mravali meqanizmebiT. ujredSi mox-

vedril qimiur nivTierebaTa maRalreaqciisunariani metabolitebis

an Tavisufalradikaluri nawilakebis ,O( 2−• )HO• koncentracia dasaS-

vebs Tu aRemateba, maSin aRZruli disbalansi membranaTa lipiduri

komponentebis, cilis molekulebis da TviT citoqrom P450-is inaq-tivaciaSi aisaxeba [159, 160].

citoqrom P450-is dazianeba qimiur nivTierebaTa metabolizmisa

da, bunebrivia, maTi reaqciisunariani metabolitebis warmoqmnis Se-

sustebas gamoiwvevs. mag., cxovelSi Seyvanili brombenzoli, triq-

loreTileni da vinilqloridi RviZlis mikrosomul cilebs ukav-

Sirdebian. SemdgomSi am toqsikantTa mravaljeradi Seyvanis Sedegad

citoqrom P450-is donis Semcirebis paralelurad dakavSirebis al-

baTobac mcirdeba [161]. ganxiluli movlena damaxasiaTebelia situa-

ciisaTvis, romlis drosac toqsikantTa maRali dozebiT mowamvla

xdeba. yofacxovrebaSi Cveulebriv ganuzomlad xSirad organizmze

nivTierebaTa mcire dozebi zemoqmedeben. amitom igive meqanizmi mo-

nooqsigenazuri sistemis ara totaluri dazianebiT, aramed SerCevi-

196

Tad, _ citoqrom P450-is im calkeuli izoformis specifikuri ga-

moTiSviT gamomJRavndeba, romelic konkretuli nivTierebis reaqcii-

sunariani metabolitebis araadeqvatur raodenobas generirebs. prin-

cipSi mocemuli situaciisaTvis mimdinareobs usargeblo citoqrom

P450-is `wundeba” da `gamoxSirva”. es metabolizmis TviTregulire-

bis saocrad martivi da efeqturi meqanizmia. garda amisa, citoqrom

P450-is dazianebis igive procesi avtomaturad qmnis bunebriv koni-ugirebul antigens: citoqromTan kovalenturad dakavSirebul niv-

Tierebas SeuZlia imunuri pasuxis inducireba da ujredSi mocemuli

tipis nivTierebis moqmedebis damuxruWeba [158].

qimiur naerTTa Jangvis monooqsigenazurma sistemam evoluciis

milioni wlebi gamoiara da am xangrZliv periodSi centraluri po-

zicia SeinarCuna. citoqrom P450-is saxiT bunebam `gamoZerwa” fer-menti, romelic `optimalurad gaxsnil” urTierTobebs amyarebs

usazRvro raodenobis da amave dros sruliad gansxvavebuli qimiuri

bunebis mqone naerTebTan. monooqsigenazur sistemaze gamaval qseno-

biotikebs gamudmebiT moaqvT garemos qimiur Semadgenlobaze da mas-

Si mimdinare cvlilebebis Sesaxeb informacia. es cvlilebebi Tu

xangrZlivi periodisaa, isini Sesabamisi adapturi bunebis reaqciebs

inducireben. qsenobiotikebs SeuZliaT ujredis genetikur aparatTan

urTierTqmedeba (rnm-is sinTezis inducireba) da Tavisive metabo-

lizmSi monawile fermentebis warmoqmna. axal nivTierebaTa gaCena

aiZulebs cocxals saTanado koreqtivebi moaxdinos da ase grZelde-

ba dausruleblad. saxezea TviTganviTarebadi sistema, romelic iseT

principzea agebuli, rom qimiuri evolucia biologiur evolucias

iwvevs. savsebiT samarTlianad aRniSnavs prof. a. arCakovi [20], rom

citoqrom P450-is saxiT arsebobs fermenti, romelsac warmatebiT

SeuZlia daicvas organizmi aramxolod arsebuli sinTezuri toqsi-

kantebisagan, aramed imaTganac, romlebic adamianis mier momavalSi

iwarmoeba. citoqrom P450-is monawileobiT warmoqmnili endogenuri

hidrofobuli nivTierebebi (steroiduli hormonebi, qolesterini,

Tiroqsini, prostaglandinebi, cximovani mJavebi da sxv.) ukavSirde-

bian ra specifikur receptorebs, Tavis regulatorul funqciebs

asruleben. gamoricxuli araa maTi zemoqmedeba citoqrom P450-is Semcvel qemoanalizatorul sistemazec vrceldebodes, romelic

ujredis hormonaluri regulaciis madublirebeli meqanizmi SeiZle-

197

ba iyos. citoqrom P450-is sistema avtomaturad monawileobs plaz-

muri membranebis molekuluri kompoziciis regulaciaSi, romlebic

receptorul da sxva mniSvnelovan funqciebs atareben. naCvenebia

magaliTad, rom fenobarbitalis gamoyenebisas tvinSi sagrZnoblad

izrdeba β-adrenoreceptorebis raodenoba [158]. dasasruls ganxilul problemas biologiuri universalobis

principiT Tu mivudgebiT, Zneli ar iqneba warmovadginoT is ara-

sruli CamonaTvali, romelic monooqsigenazur da imunur sistemaTa

Soris did msgavsebaze miuTiTeben:

1. sxva sistemebisagan gansxvavebiT isini ganusazRvreli wris qimiur

struqturebTan urTierTqmedeben;

2. orive sistema reagirebs rogorc qsenobiotikebTan, aseve endoge-nur naerTebTan;

3. orive sistema induqciuria; 4. arsebobs gansxvavebuli specifikurobis mixedviT substratebze

Semtevi citoqrom P450-is da aseve imunologiuri receptorebisa

da antisxeulebis mravlobiTi formebi;

5. orive sistemas gaaCnia `maxsovrobis” unari; meoradi imunuri max-sovroba didi xnis sayovelTaod cnobili movlenaa, xolo mo-

nooqsigenazuri sistemis induqciis meoradi pasuxis arseboba Ta-

vis eqsperimentul dadasturebas amJamad Rebulobs;

6. orive sistemis zogadi fiziologiuri funqcia qimiuri homeos-

tazis SenarCunebaa;

7. citoqrom P450-is monawileobiT monooqsigenazuri da imunolo-

giuri sistemis SeuRlebuli funqcia ujredSi bunebrivi koni-

ugirebuli antigenebis warmoqmnaSi mdgomareobs, ris Sedegadac

SemdgomSi ama Tu im dabalmolekuluri naerTis mimarT specifi-

kuri imunuri pasuxi inducirdeba;

8. SeuRlebis funqciaSi damatebiT igulisxmeba umniSvnelovanesi

momenti imis Sesaxeb, rom imunuri reaqciebi regulirdeba mo-

nooqsigenazuri sistemiT sinTezirebuli prostaglandinebiT,

kortikosteroidebiT, hiberelinebiTa da sxv.;

9. monooqsigenazuri da imunologiuri sistemebisaTvis saerTo re-

gulatorebi arsebobs.

ujredSi qimiuri nivTierebebis dakavSirebis ori tipi _ qemoa-

nalizatoruli (citoqrom P450 an sxva fermentuli sistemebi) da

198

receptoruli (imunuri) harmoniulad avseben erTmaneTs da organiz-

mis Seuvalobis erTian dauZlevel zRudes qmnian.

2.7.1 molekuluri receptorologiis zogadi principebi

imunur da monooqsigenazur sistemaTa moqmedebasa da regulaciaSi

TvalsaCino adgili receptorebs ukavia. amasTan dakavSirebiT saWiroa maTze

arsebuli warmodgenebis Tanamedrove interpretaciis zogadi codna mainc.

analizuri midgomis saSualebiT Seswavlilia receptorebis qsovilovani da

ujreduli ganawileba, xolo zogierTi maTganisaTvis dadgenilia moleku-

luri aRnagobac [162].

mravalricxovani bunebrivi da sinTezuri molekula_mediatorebis (ago-

nistebisa da antagonistebis) saxiT arsebobs `gasaRebis didi asxma~, ro-

melTa daxmarebiTac mkvlevarebi cdiloben gamoavlinon `boqlomebis~ _

receptorul sistemaTa `aqtiuri centrebis~ struqtura da moqmedebis

principebi. `gasaRebi_boqlomisa~ da `aqtiuri centrebis~ cnebebi enzimo-

logiidanaa nasesxebi. fermentTa Senebis analogiurad maT mimarT peptiduri

bunebis Sesaxeb Sexedulebebma receptorologiaSic SeaRwia. aqve unda aRi-

niSnos, rom arafermentuli receptoruli struqturebis ciluri aRnago-

bis Sesaxeb damkvidrebuli mtkicebulebebi arapirdapir monacemebs eyrdnoba.

receptorTa peptiduri aRnagobis damamtkicebeli Tvalsazrisi [163]

aRiarebs gansazRvruli receptorebisa da gansazRvruli mediatorebis (ago-

nistebisa da antagonistebis) maRalspecifikuri urTierTqmedebis faqts,

rac Tavis mxriv ganpirobebulia Sesabamisi mediatorebis mimarT peptiduri

receptoruli struqturebis komplementarobiT. amis sapirispirod SeiZle-

ba iTqvas is, rom molekuluri biologiis fundamenturi principi – kom-

plementaroba sulac ar aris peptiduri struqturis prerogativa. nebis-

mieri biomolekula, romelic ki meore molekulas wyalbaduri bmis war-

moqmniT ukavSirdeba, am princips emorCileba. amis magaliTia Tundac azo-

tovan fuZeTa komplementaroba.

Tanamedrove receptologiis Teoriulad dasabuTebuli principebi rus-

ma mkvlevarma v. podimovma [163] warmoadgina. am avtoris Tanaxmad recep-

toris cnebaSi igulisxmeba membranis mravalkomponentiani molekuluri

struqtura, romelic Semdegi funqciuri blokebisagan Sedgeba: 1. mediato-

ris (agonistis, antagonistis) `dasajdomi moednisagan,~ anu nawilisagan,

romelic Seicnobs da ikavSirebs mediators; 2. fragmentisagan, romelic

`dasajdomi moednisa~ da mediatoris (agonistis) urTierTqmedebis Sedege-

bis (signalis) receptoruli struqturis efeqtorul ubnebze gadacemis

meqanizmzea pasuxismgebeli; antagonisti urTierTqmedebs ra `dasajdom moe-

199

danTan~, an ar iwvevs mis struqturaSi cvlilebebs, an cvlis mas, magram

ablokirebs signalis gadacemis meqanizms. warmodgenili Teoria Tvlis, rom

receptor-mediatoris urTierTqmedeba sam _ mizidvis, orientaciis da fiq-

saciis _ etapad mimdinareobs. amasTan, urTierTqmedebis sxvadasxva etapze

gansazRvrul rols kavSiris sxvadasxva tipebi asruleben: sakmaod stabi-

luri ionuri da wyalbaduri bmebi mediatoris (agonistis, antagonistis)

asocirebaSi monawileoben; dipol-dipoluri kavSirebi monawileobas Rebu-

loben mediatoris fiqsirebaSi; apolaruli vandervaalsis kavSirebi media-

tor-receptoris kompleqss astabilireben; kovalenturi kavSirebi moqmede-

bas iwyeben receptorebis sxvadasxva saxeebsa da antagonistis calkeul

tipebs Soris; dasaSvebia xelaturi kompleqsebis warmoqmna receptoris

SemadgenlobaSi myof metalis ionTa mediatoris urTierTqmedebisas (mag.,,

α-adrenoreceptorSi rkinis ionebsa da kateqolaminebs Soris).

metalis ionebi mniSvnelovan rols asruleben receptorebis funqcio-

nirebaSi da `dasajdomi moednebis~ struqturirebaSi. isini aramxolod re-

ceptoruli bioqimiuri struqturebis efeqtur rgolebs (mag., Ca), an fer-mentul efeqtorul rgolebze gadacemis fragmentebs warmoadgenen (mag.,

Mn-adenilciklaza, Mg, K, Na-ATP-aza), aramed `dasajdomi moednebis~

komponentTa erTmaneTisadmi orientirebasac uzrunvelyofen.

arsebuli da sakuTari monacemebis safuZvelze v. podimovma [163] re-

ceptorebis struqturisa da funqcionirebaze warmoadgina axleburi kon-

cefcia, romelic Semdegs iTvaliswinebs: receptorebis `dasajdomi moednis~

peptidur bunebaze warmodgenebis uaryofas; receptorebis struqturis

mravalkomponentianobis aRiarebas; komplementarobis principis gafarToebas

mediator_`dasajdomi moednis~ urTierTqmedebaSi da `dasajdomi moednis~

komponentebs Soris urTierTqmedebaSi; receptorebis funqcionirebis prin-

cipis gafarToebul ganxilvas `dasajdomi moednis~ konformaciuli cvli-

lebebidan, `dasajdomi moednis~ pirveladi kompleqsis daSlamde, ris Sede-

gadac mediatorebsa (agonistebsa) da `dasajdomi moednis~ komponentebs

Soris meoradi kompleqsebi warmoiqmnebian; metalTa ionebis (Ca, Mg, Mn) rolis aRiarebas `dasajdomi moednis~ struqturirebasa da funqcio-

nirebaSi.

CaTvala ra, rom yvela receptoruli sistemis `dasajdomi moe-

dani~ erTiani molekulur-arqiteqturuli gegmiTaa naSenebi, v. podimovma

[163] misi Semadgeneli (kandidati) molekulebisadmi Semdegi moTxovnebi

wamoayena:

1. es molekulebi unda imyofebodnen ujredul membranebSi, sadac mudmivad

vlindeba receptorebis arseboba;

2. isini unda flobdnen receptoruli tipis moqmedebis unars da gaaCndeT

maRali biologiuri aqtivoba;

200

3. calkeulad aRebuls ar unda gaaCndes receptoruli moqmedebis maRali

specifikuroba;

4. es molekulebi unda warmoadgendnen naerTTa rigs, romelTa calkeuli

warmomadgenlidan SeiZleba aiwyos aranakleb ramodenime aTeuli sxva-

dasxva “dasajdomi moedani”;

5. naerTTa klasebis calkeuli warmomadgenlebi erTmaneTisadmi komplemen-

taruli unda iyon, anu erTi klasis warmomadgenlisaTvis unda arse-

bobdes sxva klasis komplementaruli molekula;

6. am molekulebs unda gaaCndeT metalis ionebTan urTierTqmedebis mqone

jgufebi;

7. aseTi molekulebisa da ionebisagan Seqmnili `dasajdomi moedani” unda

urTierTqmedebdnen fosfolipidebTan da Seicavdnen damuxtul fragmen-

tebs, romelTa daxmarebiTac xdeba mediatoris (agonistis, antagonis-

tis) mizidva da orientireba;

8. aseTi molekulebisagan nagebi TiToeuli `dasajdomi moedani” komple-

mentaruli unda iyos garkveuli mediatoris mimarT, anu unda arse-

bobdes garkveuli stereoqimiuri SerCeviToba;

9. moqmedebis SerCeviToba unda efuZnebodes agreTve aseTi molekulebisagan

formirebuli `dasajdomi moednis” jgufebs, romlebic garkveuli me-

diatorebis reaqciisunarian jgufebTan iurTierTqmedeben;

10. mediatortTan (agonistTan) urTierTqmedebam `dasajdomi moednis” pir-veladi kompleqsis daSla unda gamoiwvios, ris Sedegadac mediatorisa

da `dasajdomi moednis” erT-erT komponents Soris kompleqsi warmoiqm-

neba. `dasajdom moedanTan” antagonistis urTierTqmedebam misi kompleq-

sis daSla ar unda gamoiwvios;

11. aseTi molekulisagan konstruirebuli `dasajdomi moednebi” unda Sei-

cavdnen fragmentebis mibmis adgils, romlebic receptoruli bioqimiuri

struqturebis efeqtorul elementebze gadacemas ganaxorcielebdnen.

receptoruli meqanizmebis Sesaxeb podimovis mier wamoyenebuli

hipoTeza SemdgomSi SeiZleboda amosaval punqtad qceuliyo molekuluri

informaciis zogadi Teoriis Sesaqmnelad. samwuxarod avtors amis

ganxorcieleba ar dascalda naadrevi sikvdilis gamo (gardaicvala 42

wlis asakSi).

201

2.7.2 oqsidazebi da oqsigenazebi mcenaris mdgradobasa

da fitoimunitetSi

mcenares SeiZleba gaaCndes SeZenili an memkvidrulad miRebuli

mdgradoba (rezistentoba), romelic konstituciur fitoimunitetad

ganixileba. mdgradoba, romelic mas sicocxlis periodSi uyalibde-

ba, SeZenili, anu induqciuri imunitetia. mdgradoba da Semguebloba

paTogenis mier dazianebis xarisxs axasiaTebs. maRali mdgradoba ax-

los dgas imunitetTan. mcenares tolerantuls uwodeben, Tu mas

susti mdgradoba gaaCnia, magram paTogeniT iSviaTad ziandeba. imunu-

ri tolerantoba imunoregulaciis mniSvnelovani fenomenia [164].

daavadebaTa gamomwvevebis mimarT fitopaTologebi fitoimunitetis

or formas ansxvaveben: pasiurs, romelic paTogenis arsebobis miux-

edavad mcenares gaaCnia da aqtiurs, romelic mcenares paTogenis

SeRwevis Semdeg uyalibdeba. mcenareSi SeiZleba SeZenili imunitetic

aRiZras, magram igi cxoveluri imunitetisagan gansxvavdeba Tundac

Tavisi lokaluri bunebis gamo. gamojanmrTelebuli mcenare Cveule-

briv virusis matarebelia, magram daavadebis simptomebs aRar ata-

rebs. garda amisa virusis erTi StamiT daavadebisas mcenare amave

virusis axlo mdgomi StamiT daavadebis mimarT mdgradi xdeba [164].

miuxedavad aRniSnulisa, gamojanmrTelebul mcenareSi araa napovni

cxovelis imunitetisaTvis damaxasiaTebeli antisxeulebi. am ukanask-

nelTa SeyvaniT aRZruli pasiuri imuniteti mcenareze demonstrire-

buli araa. mTel rig SemTxvevebSi mcenareSi organuli qsenobioti-

kis metabolizmi Zlier waagavs ZuZumwovrebsa da mikrobul siste-

mebSi maT metabolizms. mcenareebsac SeuZliaT am nivTierebaTa far-

To speqtris nakleb-, an saerTod aratoqsikur produqtebad trans-

formireba.

iseTi pirobebisaTvis, romlis drosac realurad dgindeba cxo-

velisa da mcenaris imunur reaqciaTa Soris msgavseba, arasrulad da

xSirad araadeqvaturad iyeneben zooimunitetis iseT terminebs, ro-

goricaa aglutinacia (antisxeulebis gavleniT baqteriuli ujrede-

bis an antigenis Sewebeba), precipitacia (antisxeulebis gavleniT

antigenuri nawilakebis gamsxvileba da daleqva) da lizisi (baqte-

riuli ujredebis gaxsna – baqteriolizisi, an eriTrocitis ujre-

debis gaxsna – hemolizi) [165]. antisxeulebis aqtivaciis mqone mce-

nareuli toqsinebi (ricini, abrini, krotini) araciluri dabalmo-

202

lekuluri naerTebia da cxoveluri antisxeulebisagan gansxvavebiT

antigenTan moqmedebis gacilebiT dabali specifikuroba da maRali

Termostabiluroba gaaCniaT. arsebobs monacemebi [165] imis Sesaxeb,

rom kurdRlis sisxlidan momzadebuli antiSrati, romelic baqte-

riaTa aglutinacias iwvevs, mcenares am paTogeniT daavadebisagan

icavs. cnobilia agreTve, rom mTel rig mcenareTa cilur eqstraq-

tebs hemaglutinaciis unaris garda baqteriebis aglutinaciis Tvi-

sebebic gaaCnia. naCvenebia, rom parkosnebis cilur eqstraqtebs sis-

xlis Sratis precipitaciac SeuZliaT [166].

mdgradoba SeiZleba ganxilul iqnas erTi mxriv, rogorc para-

zitis Sxamis detoqsikaciisa da mcenaris mier mis uvnebel formamde

daJangvis, meore mxriv ki parazitis hidrolizur fermentTa aqtivo-

bis daTrgunvis unari. am SemTxvevaSi sunTqvis gaZliereba Canergili

paTogenis da mis mier gamoyofili toqsinebis mimarT mcenaris un-

iversalur damcvel funqciad gvevlineba.

oqsidazuri fermentuli sistemebidan mcenaris mdgradobaSi aq-

tiuradaa CarTuli katalaza, peroqsidaza, glutaTionperoqsidaza,

superoqsiddismutaza, polifenoloqsidaza, askorbatoqsidaza da sxv.

isini sxvadasxva subujredul komponentebSia lokalizebuli. maTi

umravlesoba konstituciurad eqspresirdeba, Tumca toqsikantebs

maTi induqciis eqspresiac SeuZliaT. mcenaris dazianebisas yvelaze

xSirad da efeqturad peroqsidaza aqtiurdeba. misi gavleniT mniSv-

nelovnad matulobs wyalbadis zeJangis, Tavisufali radikalebis da

polimerizaciis produqtTa (kolagenis, ligninis) koncentraciebi.

amis gamo warmatebiT iTrguneba paTogenuri sokos ujredgare fer-

mentebi [167, 168]. toqsinebis mimarT gansakuTrebul mdgradobas

flavoproteinuli oqsidazebi avlenen, romelTa aqtivobebi mcenaris

inficirebisas mkveTrad izrdeba.

polifenoloqsidazuri aqtivobis gazrdiT Cqardeba polifenole-

bis qinonebad gardaqmna, romlebic antisxeulebis msgavsad paTogenis

cilebTan reagireben da maT precipitacias iwveven. am ferments

ukavSireben agreTve e.w. ~zemgrZnobiarobis~ (~hipermgrZnobiarobis~)

imunur reaqcias, rac mcenaris mdgradi jiSebisTvisaa damaxasiaTebe-

li; dazianebul mcenareSi mdgradobis gaZlierebas Tan axlavs pen-

tozuri da limonmJavaciklebis fermentTa gaaqtiveba. glukozo-6-

fosfat- da 6-fosfoglukonatdehidrogenazas aqtivobaTa zrda mdgradi

203

jiSebis qsovilebSi sunTqvis intensivobis gaZlierebiTa da foto-

sinTezis daqveiTebiT mimdinareobs.

aromatul naerTTa biosinTezis regulaciaSi pentozuri ciklis

fermentTa monawileoba mdgradobisa da paTologiur procesTa uSu-

alo kavSiris arsebobaze miuTiTebs. dazianebuli qsovilebis feno-

lebiT, fitoaleqsinebiT, glukozidebiT gajereba, agreTve sunTqvis

da energiis akumulirebis siCqareTa cvlilebebi am kavSiris arsebo-

bis realuri asaxvaa. CamoTvlil procesTa damamTavrebeli etapebic

kontroldeba fermentebiT, romlebic uSualod monawileoben mdgra-

dobis faqtorTa formirebaSi.

piruvat- da malatdehidrogenazebis aqtivobaTa zrda limonmJavas

ciklis brunTa ricxvis gazrdas iwvevs. amis Sedegad adgili aqvs

organul mJavaTa dagrovebas, romlebic xSir SemTxvevaSi paTogenebis

mimarT toqsikurebia. ciklSi warmoqmnili NADH energiis generire-

baSi monawileobs, rac mdgradobisaTvis aucilebeli pirobaa [169].

askorbat- da citoqromoqsidazas aqtivobaTa gaZliereba NADH-isa

da NADPH-is daJangvis maRal xarisxze da mcenaris daavadebaTa

mimarT mdgradobis unaris gamomuSavebaze miuTiTeben.

mcire monacemebi gagvaCnia, romlebic mcenaris mdgradobasa da

energetikul cvlas Soris pirdapir kavSirs exeba. es garemoeba

garkveulad aZnelebs sakiTxis ganzogadebas. fragmentulad naCvenebia,

rom mcenareSi daavadebis mimarT mdgradobis ganviTarebas Tan sdevs

ATP-sa da aRdgenili piridinnukleotidebis dagroveba, cikluri da

aracikluri fotofosforilebisa da mitoqondriuli JangviTi fos-

forilebis gaaqtiureba. sarwmunodaa naCvenebi, rom 2,4-dinitrofe-

noli (sunTqvisa da fosforilebis gamTiSveli) narkotikebis msgav-

sad mcenaris mdgradobis mniSvnelovan daqveiTebas iwvevs [170].

inficirebul ujredSi cilebis funqciuri aqtivobis cvlilebebs

Tu Tan sdevs energetikuli cvlis moSla, maSin daavadebis mimarT

mdgradobaSi energiis warmomqmnel da momxmarebel procesebs Soris

stacionaruli mdgomareobis (homeostazis) SenarCuneba unda vigu-

lisxmoT. am mdgomareobis yovelgvari darRveva infeqciasTan brZo-

lis efeqturobis vardniT aRiniSneba.

mitoqondriebsa da qloroplastebSi sakuTari cilamasinTezire-

beli genetikuri aparatis arseboba aucilebels xdis mcenaris

mdgradobaSi aramarto am organelTa urTierTobis rolis garkvevas,

204

aramed mocemul procesSi qromosomuli da araqromosomuli regu-

latoruli urTierTqmedebis Seswavlasac. cnobilia, rom mcenareebs

SeuZliaT qsenobiotikebis mutagenebamde gaaqtiveba [171]. peroqsida-

zebi da citoqromebi 2-aminofluorenis, benzpirenisa da pentaqlor-

fenolis gardaqmnas axorcieleben reaqciisunarian produqtebad. ori

ukanaskneli qinoidur warmoebulebs iZleva, romelTac Jangbadis

nairgvari radikalebis warmoqmnis unari aqvT. dasaSvebadaa miCneuli,

rom mcenareSi (iseve rogorc cxovelSi) metabolitebis mutagenuri

moqmedebis molekuluri meqanizmi dnm-Tan maT kovalentur dakavSi-

rebaSi an mis fragmentaciaSi mdgomareobs.

gacilebiT mcire da amave dros arapirdapiri monacemebi gagvaCnia

adapturi da fitoimunuri dacvis sistemaSi mcenareuli citoqrom

P450-Semcveli monooqsigenazebis uSualo monawileobisa da am pro-

cesebSi maTi funqcionirebis meqanizmis Sesaxeb.

sandermanis mier [172] SemoTavazebuli e.w. „mwvane RviZlis” hi-

poTezis Tanaxmad monooqsigenazebis sxvadasxva variantebi qsenobi-

otikTa metabolizmSi (gansakuTrebiT funqcionalizaciis stadiaze)

gansxvavebul aqtivobebs amJRavneben da ayalibeben mcenareTa unars,

raTa Tavi gaarTvan maTgan gamowveul qimiur stresebs. ase magali-

Tad, cnobilia, rom sarevela balaxebis populaciebSi am fermentu-

li sistemis aqtivaciis zrda herbicidebisadmi rezistentobasTan

korelirebs [173]. sxvadasxva faqtoris (sinaTlis, meqanikuri dazia-

nebis, paTogenis, qsenobiotikis) gavleniT mcenareul ujredSi ci-

toqrom P450-is SerCeviTi inducirebis SesaZlebloba sakmaod de-

taluradaa gadmocemuli durstisa da TanaavtorTa [174] vrcel

publikaciaSi. am mimarTulebiT kvleviTi samuSaoebi Catarebulia

Cvens laboratoriaSi T. varazaSvilis da Tanaavtorebis mier [175-

177]; kerZod Seswavlil iqna mcenaris (soiasa da simindis) 5-6

dRiani nazardebis fesvebis mikrosomul fraqciaSi monooqsigenazuri

kompleqsis komponentTa cvlilebebi dabaltemperaturuli stresi-

sas, agreTve sokoTi (Fusarium) inficirebisa da qsenobiotikiT (ni-

trobenzoliT) zemoqmedebis pirobebSi. es ukanaskneli SerCeuli iyo

imis gamo, rom igi qimiurad mdgradia, ar axasiaTebs autooqsidacia

da haerze ar iJangeba; garda amisa misi gardaqmna mxolod hidroq-

silirebiT SeiZleba warimarTos (nitro-jgufis amino-jgufamde aRd-

genis alternatiuli gza anaerobul pirobebs moiTxovs da amdenad

205

cdebis variantebSi gamoricxuli iyo).

gamokvlevebma aCvenes, rom dabaltemperaturuli stresi mniSvne-

lovnad zrdis monooqsigenazis sawyisi komponentis NADPH-

citoqrom P450-reduqtazas aqtivobas; paralelurad ki qveiTdeba

citoqrom P450-is katalizuri aqtivoba. Seqmnil situaciaSi eleq-

tronul-mikroskopuli meTodiT daregistrirda endoplazmuri da

mitoqondriuli membranebis gaxSirebuli kontaqtebi [176]. rogorc

cnobilia, cxovelSi aseTi analogiuri struqturuli reorganiza-

ciisas NADPH-is aRmdgeneli eqvivalentebi citoqrom P450-is nac-vlad mitoqondriebSi gadaitaneba (transmembranuli migracia), ris

Sedegadac adgili aqvs araSeuRlebuli Jangvis sistemebis gaaqti-

urebas da eleqtronTa energiis Termoregulaciur procesebSi mox-

marebas [178].

soko fuzariumiT mcenaris inficirebisas mikrosomuli monooq-

sigenaza sapirispiro da amave dros, araerTmniSvnelovan cvlilebebs

ganicdis: izrdeba citoqrom P450-is da misi araaqtiuri formis

citoqrom P420-is raodenobrivi Semcveloba; anu citoqrom P450-is induqcias imavdroulad Tan axlavs misi P420-ad konversia, xolo

NADPH-citoqrom P450-reduqtazas aqtivoba sagrZnoblad iTrgune-

ba. sokos toqsinisadmi hemoproteinisa da misi Sesabamisi reduqta-

zas aseTi gansxvavebuli damokidebuleba infeqciis mimarT maTi

gansxvavebuli mgrZnobiarobiT SeiZleba iqnas axsnili.

nitrobenzolis Semcvel xsnarze gazrdili mcenaris fesvebSi ga-

movlinda monooqsigenazuri sistemis komponentTa aqtivobebis mniSv-

nelovani zrda (induqciis efeqti). garda amisa am SemTxvevaSic kon-

taqtebi xSirdeba or (mitoqondriul da mikrosomul) membranas So-

ris (nax. 2.19). es imis maCvenebelia, rom nitrobenzolis Jangvis

gasaZliereblad (dasaCqareblad) ujredi optimalur pirobebs qmnis

sunTqviTi jaWvidan mikrosomaze aRmdgeneli eqvivalentebis trans-

membranuli migraciis uzrunvelsayofad (mitoqondriuli kontrolis

fenomeni) [177, 179].

nax. 2.19

miRe

vaskvnaT

tiurad

maT mima

9. simindis

struqtu

Semdeg. m

branebiT

ebuli eqsp

T, rom mcen

aa CarTuli

arT aRZrul

fesvis ko

uruli cvl

mitoqondri

×36 000 [

perimentul

naris cito

i Seqmnili

li adaptur

206

ortikalur

lilebebi

iebi garSemo

[177].

i monaceme

oqrom P450stresuli

ri reaqciebi

ujredebSi

nitrobenzo

ortymulia

ebi safuZve

0-Semcveli

situacieb

is realiza

i aRZruli

oliT zemo

endoplazm

els gvaZl

monooqsige

bis aRmofxv

ciaSi.

ultra-

oqmedebis

muri me-

levs da-

enaza aq-

vrisa da

207

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219

Tavi 3

oqsigenazebi qsenobiotikebis detoqsikaciaSi

3.1 citoqrom P450-is roli qsenobiotikebis metabolizmSi

wina TavSi aRvniSneT, rom citoqrom P450-Semcveli monooqsi-

genaza erT-erT sakvanZo ferments warmoadgens mTeli rigi meoreuli

naerTebis biosinTezebSi. aseve didia fermentis mniSvneloba sxva-

dasxva qimiuri struqturis, lipofiluri bunebis mqone organuli

qsenobiotikebis metabolizmSi [1]. SeiZleba ganvazogadoT, rom cito-

qrom P450-is daniSnulebaa lipofiluri bunebis mqone organuli

naerTebis monooqsigenireba da am dros mniSvneloba ara aqvs, subst-

rati endogenuri warmoSobisaa Tu qsenobiotikia.

aq saWirod vTvliT mokled warmovadginoT citoqrom P450-Semc-veli monooqsigenazas roli da adgili qsenobiotikTa detoqsikaciis

procesSi. miRebulia, rom cxovelsa da mcenareSi SeRweulma qseno-

biotikma sami Tanmimdevruli faza unda gaiaros (nax. 3.1) [2-5]:

R R X R X A

Sardi

(cxoveli)

ujredis kedeli,

vakuolebi

(mcenare)

I faza II faza III faza

nax. 3.1. ujredSi qsenobiotikis gardaqmnis sami Tanmimdevruli faza.

pirveli faza warmoadgens funqcionalizacias, anu qsenobio-

tikis molekulaSi axali funqciuri jgufis formirebas. qseno-

biotikebis umravlesoba arapolaruli, wyalSi mcired xsnadi naer-

Tebia da ar Seicavs iseT funqciur jgufebs, romlebic wylovan

fazaSi mofunqcionire ferment–oqsidazebisaTvis xelmisawvdomi iqne-

boda. amisaTvis saWiroa ucxo naerTis winaswari gaaqtiureba pola-

ruli funqciuri jgufis saSualebiT. am mizans ZiriTadad membranaSi

mimdinare Jangva-aRdgeniTi reaqciebi emsaxureba. maTi saSualebiT

xdeba qsenobiotikis molekulaSi axali funqciuri jgufis (hidro-

qsilis, karboqsilis, epoqsidis da sxv.) Seyvana, an arsebulis

garTuleba. am gziT nivTierebis qimiuri inertulobis daZleva xdeba

da miRebuli produqtebis polaruli jgufi Semdgomi fermentuli

gardaqmnebisadmi gacilebiT xelmisawvdomia. I fazis Sedegad ucxo

naerTis molekula iseT pirvelad gardaqmnebs ganicdis, rom is

220

SedarebiT advilad „Seicnos“ fermentma. fuqcionalizaciis fazas

axorcieleben oqsigenazebi, reduqtazebi, esTerazebi, dehalogenazebi

da sxva fermentebi. zog SemTxvevaSi ki es procesi maTi urTierTSe-

nacvlebiT mimdinareobs [6].

funqcionalizaciis yvelaze gavrcelebuli formaa hidroqsili-

reba. es reaqcia upiratesad qsenobiotikis molekulis C_H bmis

gawyvetiT da am atomebs Soris winaswar gaaqtivebuli Jangbadis

atomis CanergviT xorcieldeba. procesi cxovelSi ZiriTadad RviZl-

Si xdeba da citoqrom P450-Semcveli monooqsigenazebiT kataliz-

deba. am mxriv mcenare ufro farTo arCevans akeTebs da hidroqsi-

lirebisaTvis igi citoqrom P450-is garda, peroqsidazebsa da

fenoloqsidazebsac iyenebs. reaqcia maRali aqtivaciis energias

saWiroebs da amis gamo Senelebulad mimdinareobs. aqedan gamom-

dinare, qsenobiotikis sruli, mravalsafexuriani degradaciis

procesisaTvis funqcionalizacia siCqaris malimitirebel stadias

warmoadgens da amdenad, ucxo naerTis gauvneblobis procesis

regulaciac swored am stadiaze xorcieldeba.

meore fazis – koniugaciis dros axlad SeZenili funqciuri

jgufis xarjze gaaqtiurebuli qsenobiotiki Sidaujredul naerTebs

ukavSirdeba. aseTi naerTebi SeiZleba iyos rogorc dabalmoleku-

luri – mono-, di- da trisaqaridebi (xSirad glukoza), amino-

mJavebi, peptidebi (upiratesad glutaTioni da homoglutaTioni),

ise maRalmolekuluri – cilebi, polisaqaridebi (mag., hemicelu-

loza), lignini da sxv. koniugacia ujredisaTvis imitomaa moxerxe-

buli, rom qsenobiotikis koniugats nawilobriv an srulad aqvs

dakarguli toqsikuroba, gaaCnia wyalSi maRali xsnadoba da

Sesabamisad, advildeba ujredSida misi gadaadgileba an ujredidan

gamosvla [7]. koniugaciis faza erTnairad damaxasiaTebelia mcenarisa

da cxovelisaTvis, Tumca mcenareSi es faza gacilebiT moculobiTia

im biopolimerebis mravalgvarobis gamo, romlebic qsenobiotikis

funqcionalizebul metabolits ikavSireben (lignini, celuloza da

hemiceluloza, saxamebeli, peqtinuri naerTebi, polipeptidebi da

sxv.) [8]. sayuradReboa, rom am naerTebTan qsenobiotikis dakavSireba SesaZloa moxdes rogorc arsebul biopolimerTan uSualo mier-

TebiT, ise TanapolimerizaciiT, anu im monomerTan koniugaciiT,

romlidanac Semdgom biopolimeri warmoiqmneba. koniugacia Sesabamisi

fermentebiT (glutaTion-S-transferazebiT, glukozil-transfera-

zebiT, malonil-transferazebiT da sxv.) katalizdeba.

221

mesame, eliminaciis fazaSi ujredi sabolood icilebs qseno-

biotiks. amdros koniugirebuli qsenobiotiki ujredidan gamodis da

organizms tovebs (cxovelis SemTxvevaSi), an garkveul nakveTurebSi

grovdeba (mcenaris SemTxvevaSi) [9]. am fazaSi mcenaresa da cxovels

Soris arsebiTi gansxvavebaa: cxovelis organizmi srulad gamodevnis

ucxo naerTs; am SesaZleblobiT SezRuduli mcenare ki koniugi-

rebuli qsenobiotikis „deponirebas“ axdens da mas vakuolebSi (Tu

koniugati xsnadia) an ujredSoris sivrceSi (Tu koniugati uxsna-

dia) inaxavs [10-12]. miuxedavad aseTi gansxvavebisa, eliminaciis faza

mcenaresa da cxovelSi erTnairia rogorc meqanizmiT, ise arsiT:

orive SemTxvevaSi procesi warimarTeba e.w. ATP-transporterebiT (meoreuli metabolitebis gadatanaSi specializebuli fermentebiT)

[13], da misi daniSnulebaa sasicocxlo procesebisagan toqsikuri

naerTis izolireba.

ganxiluli fazebis xangrZlivoba mraval faqtorzea damokide-

buli, kerZod, qsenobiotikis qimiur bunebaze, mis koncentraciaze da

zemoqmedebis xangrZlivobaze, mcenaris saxeobaze, zrdis pirobebze,

temperaturaze, tenianobaze, ganaTebaze da a.S. garkveul pirobebSi

mcenareuli ujredi masSi SeRweuli qsenobiotikis srul asimila-

cias axdens. aseTi tipis gardaqmnebi ZiriTadad JangviTia da maT

erTobliobas qsenobiotikis Rrma Jangvas, anu JangviT degradacias

vuwodebT. am procesis Sesaxeb sadReisod arsebuli umniSvnelovanesi

monacemebi SeiZleba Semdegi debulebebis saxiT ganvazogadoT [5]:

1. organuli qsenobiotikis JangviTi degradacia mniSvnelovanwilad

ujredis endoplazmuri retikulumis membranebSi (endoplazmur

membranebSi) xorcieldeba;

2. qsenobiotikTa gardamqmnel fermentul kaskadSi centraluri

pozicia membranasTan dakavSirebul monooqsigenazebs ukaviaT; am

multifermentul sistemasa da membranul struqturebs Soris

arsebul cila-lipiduri bunebis urTierTqmedebas gadamwyveti

roli eniWeba;

3. membranuli lipidebi ganapirobeben membranaSi qsenobiotikebis

SeRwevasa da fermentebTan maT dakavSirebas;

4. qsenobiotikTa Jangvis reaqciebis kinetika yvela im Tavise-

burebebs emorCileba, romlebic orfazian (lipidi–wyali) siste-

maSi moqmed yvela ferments axasiaTebs;

5. monooqsigenazebi, erTi mxriv, floben uaRresad dabal substra-

tul specifikurobas, meore mxriv ki aucileblad saWiroeben

222

substratis (qsenobiotikis) molekulaSi arapolaruli jgufebis

Semcvelobas.

uaRresad mniSvnelovania, rom zemoT CamoTvlili xuTive

debuleba Tanabrad exeba rogorc mcenareul, aseve cxovelur

organizmebs.

3.2 citoqrom P450-is mier katalizebuli reaqciebi

baqteriul, mcenareul da cxovelur ujredebSi mimdinare reaqciebi, romlebic endogenuri an egzogenuri substratebis gardaqmnisas citoqrom P450-is monawileobiT katalizdeba, 9 tipad iyofa [1].

1. alifaturi

hidroqsilireba

R CH3 R CH2OH

2. epoqsidireba R CH CH R R CH CH RO

3. aromatuli

hidroqsilireba R R OH

4. JangviTi

dealkilireba:

N-dealkilireba R NH CH3 R NH2 + CH2O

O-dealkilireba O CH3R R OH + CH2O

S-dealkilireba S CH3R R SH + CH2O

5. N-Jangva: pirveladi aminebi R NH2 R NHOH

meoreuli aminebi NH NOHR1R2 R1R2( ) ( )

mesameuli aminebi N N OR1R2R3 R1R2R3( ) ( )

6. S-Jangva S S OR1R2 R1R2( ) ( )

7. dezaminireba C CH3R

O

R CHNH2

CH3

+ NH3

223

8. desulfireba C OC S R1R2R1R2( ) ( )

9. dehalogenireba R CH2Cl R CH2OH

aqve SevniSnoT, rom mocemuli klasifikacia arazustia, radgan,

rogorc SemdgomSi iqneba naCvenebi, N-, O- da S-dealkilireba

alifaturi hidroqsilirebis Semdgom etaps warmoadgens. aseve

mimdinareobs dehalogenirebisa da dezaminirebis reaqciebic. garda

amisa, mniSvnelovani gansxvavebaa alifaturi jaWvis pirvelad da

meoreul naxSirbadatomebis Jangvas Soris, araa warmodgenili

dehidrirebis reaqcia, romelic zogierT SemTxvevaSi aseve cito-

qrom-P450-damokidebuli procesia. amitom Cven SevecdebiT, Seda-

rebiT gansxvavebuli interpretaciiT movaxdinoT monooqsigenazuri

sistemis mier katalizebuli reaqciebis klasifikacia.

3.2.1 hidroqsilireba

hidroqsilireba yvelaze gavrcelebuli procesia, romelSic

citoqrom P450 monawileobs da romelsac adgili aqvs rogorc

endogenuri, aseve egzogenuri naerTebis gardaqmnisas. hidroqsi-

lireba SeiZleba ganicados naxSirbadis, azotis an gogirdis

atomebma, romlebic Sedian rogorc najeri an aromatuli ciklis,

aseve acikluri radikalis SemadgenlobaSi.

nebismieri struqturis mqone qsenobiotikebis molekulaSi hid-

roqsilis jgufis Canergva arsebiTad zrdis mis polarobas da

hidrofilurobas. hidroqsilireba aucileblad saWiroebs gaaqtiure-

buli Jangbadis atoms, amitom reaqcias maRali aqtivaciis energia

aqvs. umravles SemTxvevaSi es procesi mTeli detoqsikaciis pro-

cesis sawyis da amasTanave siCqaris malimitirebel stadias warmo-

adgens. organuli qsenobiotikebis hidroqsilirebas Semdgom maTi

koniugaciis an sruli Jangvis procesebi mosdevs [14, 15].

alifaturi hidroqsilireba citoqrom P450-damokidebuli pro-

cesia, romelic zogierTi meoreuli warmoSobis naerTis biosinTezis

sawyis stadias akatalizebs. mag., mcenaris umwifari Teslebis

mikrosomebSi am gziT mimdinareobs hiberelinebis winamorbedi terpe-

noidis _ ent-kaur-16-enis daJangva 7-β-hidroqsi-kaur-16-en-19-mJava-mde (ix. qveTavi 2.5). Jangvis Sualedur produqtebs spirti, alde-

hidi da mJava warmoadgenen.

224

alifaturi hidroqsilirebiT mimdinareobs agreTve citoqrom

P450-is mier katalizebuli cximovani mJavebis Jangvis reaqciebi.

hidroqsilireba SeiZleba ganicados rogorc terminaluri meTilis

jgufis, ise alifaturi jaWvis Sida naxSirbadatomebma. gairkva, rom

SidajaWvuri monooqsigenirebisas erTi da igive mJava SeiZleba

sxvadasxva mdgomareobaSic daiJangos, rac mkafiod Cans miwavaSlas

mikrosomebis mier laurinmJavas 8-, 9- da 10-poziciebSi hidroqsi-

lirebis magaliTze (ix. qveTavi 2.2.2).

egzogenuri alkanebis da N-alkil-warmoebulebis JangviTi degra-

dacia, Cveulebriv, alkiluri jgufebis hidroqsilirebiT iwyeba.

miuxedavad imisa, rom am naerTebis metabolizmis produqtebs Soris

Sesabamisi hidroqsi-warmoebulebis identifikacia yovelTvis araa

SesaZlebeli, maTi Semdgomi metabolizmis produqtebi erTmniSvne-

lovnad miuTiTeben hidroqsilis jgufebis Semcveli Sualeduri

produqtebis warmoqmnas. eqsperimentulad damtkicebulia, rom foT-

lebiT STanTqmuli dabalmolekuluri [Cl–C5-14C] alkanebi ganicdian

JangviT degradacias 14CO2-mde [16-19]. metabolizmis produqtebis

analizi saSualebas iZleva vivaraudoT, rom es naxSirwyalbadebi

iJangebian monoterminalurad; Sualedur produqtebs warmoadgenen

pirveladi spirtebi, romlebic Semdgom kanbonmJavebamde iJangebian. es

ukanasknelebi krebsis ciklSi erTvebian da naxSirorJangamde iJange-

bian. qvemoT moyvanil reaqciebSi (nax. 3.2-3.4) vxedavT, rom alka-

nebis Jangvis procesis sawyis stadias hidroqsilireba warmoadgens.

dadgenilia, rom mcenaris mier STanTqmuli meTanis radioaqtiuri

niSani ZiriTadad dabalmolekulur naerTebSi – organul mJavebSi

(limonis, qarvis, fumarisa da vaSlis mJavebi) erTveba. eqspoziciis

gaxangrZlivebisas xdeba radioaqtiuri niSnis gadanawileba aminom-

JavebSi, mcire raodenobiT – biopolimerebSi, xolo Saqrebis fraq-

cia araradioaqtiuri rCeba. eqsperimentis msvlelobisas SeiniSneba

mcenareebidan radioaqtiuri naxSirorJangis gamoyofa, rac mcenaris

mier meTanis sruli mineralizaciis maCvenebelia (nax. 3.2):

CH4 CH3OH H CO

HH C

O

OHujreduli

metabolizmi

nax. 3.2. meTanis JangviTi degradacia mcenareul ujredSi.

ufro grZeli jaWvis mqone naxSirwyalbadebis (eTani, propani,

pentani) SemTxvevaSic ZiriTadi radioaqtiuri metabolitebi aseve

225

organuli mJavebia (umetesad – fumarisa da qarvis mJavebi, gvxvdeba

agreTve malonis, limonisa da rZis mJavebi). yvela SemTxvevaSi Sei-

niSneba mcenareebidan 14СО2-is gamoyofa, romelic grZeldeba mcena-

ris garemomcveli atmosferodan alkanebis mocilebis Semdegac, e.i.

mcenareuli ujredi ganagrZobs SeTvisebuli naxSirwyalbadebis Jang-

vis process maT srul gauvneblobamde.

eTanis naxSirbadatomebis CarTva aRniSnul mJavebSi gvaZlevs sa-

fuZvels vivaraudoT, rom es alkani mcenareebSi monoterminalurad

iJangeba. diterminaluri Jangvis SemTxvevaSi unda warmoqmniliyo

mJaunmJava, agreTve glikolisa da glioqsalis mJavebi, rac eqsperi-

mentulad ar dasturdeba. eTanis monoterminalur Jangvis Sedegad

miiReba acetil-CoA, romlis saSualebiTac eTanis naxSirbadatomebi

krebsis ciklSi erTveba. yovelive zemoTqmuli sqematurad ase SeiZ-

leba warmovidginoT (nax. 3.3):

CH3CH3 CH3 CO

HCH3 C

O

CoACH2OHCH3

krebsis

cikli

nax. 3.3. eTanis JangviTi degradacia mcenareul ujredSi.

analogiuri principiT mimdinarebs propanis naxSirbadatomebis

CarTva krebsis ciklSi: propanis monoterminaluri Jangvisas miiReba

propionmJava, romelic, rogorc cnobilia, β-Jangvisas warmoqmnis

malonil-CoA-s; am ukanasknelis dekarboqsilirebisas ki acetil-

CoA warmoiqmneba (nax. 3.4): n-alkanebis hidroqsilirebisas citoqrom P450-Tan substratis

dakavSireba hidrofobuli urTierTqmedebis ZaliT xdeba, amitom

alifaturi jaWvis sigrZis zrdasTan erTad substratisadmi fermen-

tis swrafvac izrdeba. naCvenebia, rom monooqsigenirebisas yvelaze

intensiuri Seteva mimdinareobs mesameul C_H bmaze, ufro naklebad

xdeba Jangbadis CarTva meoreul naxSirbadTan da kidev ufro nakle-

bad _ pirveladTan [20]. zogierTi aromatuli birTvis Semcveli qsenobiotiki, romelic

alifatur gverdiT jaWvs Seicavs, mcenaris ujredSi swored alki-

lis jgufSi hidroqsilirdeba. am dros upiratesad im naxSirbada-

tomebis hidroqsilireba xdeba, romlebic birTvidan daSorebulia.

mag., n-propilbenzolis citoqrom P450-damokidebuli hidroqsilire-

bis ZiriTadi metabolitebia 3-fenil-1-propanoli da 1-fenil-2-

propanoli, xolo 1-fenil-1-propanoli mcire raodenobiT warmoiqm-

neba (nax. 3.5) [4, 5].

226

nax. 3.4. propanis JangviTi degradacia mcenareul ujredSi.

CH2 CH2 CH2OH

CH2 CH CH3

OH

CH CH2 CH3OH

CH2CH2CH3

n-propil-

benzoli

ω-hidroqsilireba

α-hidroqsilireba

(ω−1)-hidroqsilireba

3-fenil-1-propanoli

1-fenil-2-propanoli

1-fenil-1-propanoli nax. 3.5. citoqrom P450-damokidebuli n-propilbenzolis alifaturi

ubnis hidroqsilirebis reaqciebi.

alkiluri jgufebis hidroqsilireba ZiriTadi damaxasiaTebeli

reaqciaa mcenareebSi Sardovanas nawarmi herbicidebis metabolizmis

procesisaTvis. es herbicidebi ganicdian N-alkiljgufebis hidroqsi-

lirebas. N-hidroqsilalkiluri jgufebi, rogorc wesi, umdgradia

CH3CH2CH3 CO

HCH2CH3

CH2OHCH2CH3 CO

OHCH2CH3

CO

CoACH2CH3 C

O

CoACHCH2 C

O

CoACH2HOH2C

CO

OHCH2HOH2C C

O

OHCH2C

O

HC

O

OHCH2C

O

CoA

CH3CO

CoAkrebsis

cikli

227

da swrafad iJangeba, amitom hidroqsilirebis procesis Sedegad

sawyisi qsenobiotikebis N-dealkilireba xdeba (nax. 3.6). mag., herbi-cidi N-meTilfenili metaboluri transformaciis dros swored

aseT N-dealkilirebas ganicdis [21].

zogierT SemTxvevaSi warmoqmnili hidroqsilis jgufebi myisi-

erad koniugireben glukozasTan. mag., bambis foTlebSi [14C] monu-ronisagan jer hidroqsimeTil-warmoebuli, Semdeg ki am intermedi-

atisagan imdenad swrafad warmoiqmneba β-D-glukozidi, rom metabo-

litebs Soris hidroqsilirebuli Sualeduri produqtis identifi-

cireba ver xerxdeba. aseTive β-D-glukozidi formirdeba Saqris

lerwamSi (Saccharum officinarum) diuronis hidroqsimeTil-warmoebu-

lis koniugaciis Sedegad [22].

N

CH3 CH3

H CH

ON

CH3 CH2 OH

N

CH3 H

+

N,N-dimeTilaniliniN-hidroqsimeTil-

N-meTilanilini

(umdgradi intermediati)

N-meTilanilini

formaldehidi

nax. 3.6. herbicid N-meTilfenilis N-demeTilireba N-alkilis jgu-

fis hidroqsilirebis Sedegad.

mcenareebSi meTilis jgufebis hidroqsilirebis produqtebi (hi-

droqsimeTil-warmoebulebi) Sardovanas warmoebuli mravali sxva

herbicidebis pirveladi gardaqmnebis procesebis drosac warmo-

iqmnebian, mag., aseTi saxis transformacias ganicdian: buturoni

xorbalSi [23], monolinuroni ispanaxSi (Spinacia oleracea) [24], ter-butiuroni Saqris lerwamSi [25], qlortoluroni xorbalSi [26].

sainteresoa, rom qlortoluronis hidroqsilireba or poziciaSi

xdeba (nax. 3.7): N-meTilis jgufis hidroqsilireba iwvevs demeTi-

lirebas, xolo aromatul rgolTan dakavSirebuli meTilis jgufis

hidroqsilirebis Sedegad mdgradi hidroqsimeTil-warmoebuli for-

mirdeba, radganac, N-hidroqsimeTil-jgufisagan gansxvavebiT, C-hidroqsimeTil-jgufi bevrad ufro mdgradia. amas adasturebs her-

228

bicidebis moqmedebis mimarT mdgrad da mgrZnobiare xorblis jiSeb-

Si STanTqmuli qlortoluronisagan warmoqmnili metabolitebis

analizi. maT Soris gvxvdeba rogorc N-demeTilirebis produqti,

aseve C-hidroqsimeTil-warmoebuli.

Cl

CH3NHCONCH3

CH3

Cl

CH3NHCONCH3

CH2OH

Cl

CH3NHCONCH3

HCl

CH2NHCONCH3

CH3OH

qlorotoluroni

I pozicia II pozicia

N-hidroqsimeTil-qlorotoluroni

(umdgradi)

- CH2O

N-demeTil-qlortoluroni

(stabiluri)

C-hidroqsimeTil-qlorotoluroni

(stabiluri) nax. 3.7. mcenareSi qlortoluronis pirveladi gardaqmnis gzebi.

mcenareebSi analogiur N-dealkilirebas ganicdian simetriuli

triazinebic. am SemTxvevaSi N-dealkilireba gverdiTi jaWvebis

(alkiluri jgufebis) hidroqsilirebiT mimdinareobs [27]. am

SemTxvevaSic hidroqsilirebuli Sualeduri produqtebi swrafad

koniugirdebian Sidaujredul naerTebTan, amitom mcenareuli bioma-

sidan Sesabamisi hidroqsi-warmoebulebis gamoyofa yovelTvis ar

xerxdeba. mag., atrazini da simazini advilad dealkilirdebian,

Tumca metabolitebs Soris Sesabamisi Sualeduri hidroqsi-warmo-

ebulebi ar identificirdeba [28-30]. saerTod, hidroqsilirebis

produqtebis aRmoCena mxolod erTeul SemTxvevebSia SesaZlebeli,

isic maSin, rodesac isini ZiriTad metabolitebs ar warmoadgenen.

mag., kartofilis (Solanum tuberosum) da simindis suspenziur kul-

turebSi atrazinis metabolitebs Soris hidroqsi-warmoebulebi araa

229

aRmoCenili, magram ionjis mcenareebSi identificirebulia sxva

simetriuli triazinis _ terbutrinis hidroqsilirebis produqti

[31]. zemoTqmulis damadasturebeli kidev erTi magaliTia meTilis

jgufis hidroqsilirebis produqtis aRmoCena [2-14C] terbaciliT

damuSavebul ionjis mcenareebSi [32].

zogierTi qsenobiotikis molekulebSi hidroqsilirebas meTil-

enis (–СН2–) jgufebi ganicdian. mag., qerSi, simindsa da marwyvSi (Fragaria vesca) hidroqsilirdeba karbofuranis mesame lokantis

meTilenuri jgufis naxSirbadatomi, da ara terminaluri meTilis

jgufi [33,34].

[14C] cikloheqsanis metabolizmi miuTiTebs, rom am naxSirw-

yalbadis cikli iSleba Sesabamisi alifaturi produqtebis warmo-

qmniT. dadgenilia, rom cikloheqsanis metabolizmis pirveli etapia

misi hidroqsilirebiT cikloheqsanolis warmoqmna (nax. 3.8) [35].

OH

nax. 3.8. cikloheqsanis hidroqsilireba.

aromatuli hidroqsilireba. hidroqsi-warmoebulebis warmoqmna

metabolizmis sawyis etaps warmoadgens garemos iseTi mniSvnelovani

dambinZureblebis gardaqmnisas, rogorebic aromatuli naxSir-

wyalbadebia. dadgenilia, rom mcenareebSi [l–6-14C] benzolis daSliT

alifaturi produqtebis (mukonisa da fumaris mJavebis) formireba

xdeba. benzolidan analogiuri produqtebi nayofebSic warmoiqmneba.

simindis, bardis da gogras (Cucurbita pepo) sterilur aRmona-

cenebSi, romlebic niSandebuli benzolis xsnarze inkubirdeboda,

niSandebuli fenolia izolirebuli. fenoli qsovilebSi umniSvnelo

raodenobiT identificirdeba, Tumca alifatur produqtebSi

benzolis niSandebuli naxSirbadis CarTvis xarisxi bevrad maRalia.

aqedan gamomdinare, fenoli imdenad swrafad ganicdis Semdgom

gardaqmnebs, rom igi mxolod umniSvnelo raodenobiT, kvalis saxiT

rCeba metabolitebs Soris [35].

aromatuli birTvis hidroqsilireba or stadias moicavs. sava-

raudod, Sualeduri produqtia epoqsidi, romlisganac SemdgomSi

arafermentuli an fermentuli gziT Sesabamisad monofenoli an 1,2-

dihidro-1,2-dioli unda warmoiqmnas (nax. 3.9) [36].

230

O

OHOH

OH

arafermentuli

gadajgufebiT

fenoli

1,2-dihidro-

1,2-dioli

epoqsid-

hidrolaziTepoqsidibenzoli

nax. 3.9. aromatuli birTvis hidroqsilireba benzolis magaliTze.

am tipis reaqciebs adgili aqvs fenoluri naerTebis bio-

sinTezSic, kerZod, ligninis, flavonoidebisa da dariCinmJavas eTe-

rebis formirebis fenilpropanoidul gzaze. maTgan ZiriTadia trans-

dariCinmJavas gardaqmna p-kumarmJavad (nax. 3.10), romlis ganxor-

cielebaSic monawileobs procesis erT-erTi sakvanZo fermenti _

citoqrom P450-damokidebuli dariCinmJava-4-hidroqsilaza [37].

CH CH

COOH

CH CH

OH

COOH

trans-dariCinmJava

4-hidroqsi-trans-dariCinmJava

(p-kumaris mJava)

nax. 3.10. trans-dariCinmJavas gardaqmna aromatuli birTvis hidroq-

silirebiT.

231

dariCinmJava citoqrom P450-is pirveli tipis substrats war-

moadgens [38-40]. produqtiT (p-kumaris mJaviT) inhibirebis gamo da-riCinmJava-4-hidroqsilaza fenilalanin-amiak-liazasTan erTad fe-

nilpropanoiduri gzis erT-erTi maregulirebeli fermentia. ro-

gorc wina TavSi aRvniSneT, am ferments qsenobiotikebis gardaqmnac

SeuZlia (ix. qveTavi 2.7.2)

aromatuli birTvis Semcveli organuli qsenobiotikebis didi

nawili citoqrom P450-damokidebuli aromatuli hidroqsilirebis

gziT gardaiqmneba. aseT transformacias ganicdian aramarto mode-

luri qsenobiotikebi (mag., qlorbenzoli, nitrobenzoli, anilini da

sxv. [41]) (nax. 3.11-13), aramed benzolis birTvis Semcveli mravali

pesticidic (mag., qlorsulfuroni [42], bentazoni [43], diqlofopi

[44] da sxv.) (nax. 3.14_3.16). aqve unda aRiniSnos, rom benzolis

birTvSi nukleofiluri Camnacvleblis SemTxvevaSi hidroqsilireba

ufro aqtiur, me-3 (meta-) mdgomareobaSi xorcieldeba (nax. 3.11 da

3.12), xolo eleqtrofiluri Camnacvlebeli, romelic me-2 da me-4

(orTo- da para-) mdgomareobis wyalbadatomebis Zvradobas zrdis, p-hidroqsilirebul produqtis warmoqmnas uwyobs xels (nax. 3.13).

rogorc Cans, orTo-mdgomareoba steriulad miuwvdomelia fermen-

tis aqtiuri centrisaTvis (ekranirebulia amino jgufiT), amitom

anilinis metabolitebs Soris o-hidroqsilirebis produqti ar

identificirdeba.

umetes SemTxvevaSi, aromatuli birTvis hidroqsilirebis pro-

duqtebi, warmoqmnili hidroqsilis jgufis saSualebiT dauyov-

nebliv glukozilirebas ganicdian. swored amitom hidroqsilirebis

produqtebis gamoyofa yovelTvis ar aris SesaZlebeli. mag., herbi-

cidi bentazoni hidroqsilirdeba 6-hidroqsibentazonis an 8-hidroqsibentazonis warmoqmniT, es intermediatebi SemdgomSi glu-

kozilirebiT koniugirdebian [43]. bentazoniT damuSavebul mcena-

reul qsovilebSi, glukozidebTan erTad herbicidis sawyisi hid-

roqsi-warmoebulebi mxolod umniSvnelo raodenobiTaa aRmoCenili.

es cxadia, miuTiTebs, rom aromatuli birTvis hidroqsilireba

qsenobiotikis transformaciis mTeli Semdgomi procesis malimi-

tirebeli stadiaa.

232

Cl Cl

OHqlorbenzoli m-qlorfenoli

nax. 3.11. qlorbenzolis aromatuli

birTvis m-hidroqsilireba.

NO2

OH

NO2

nitrobenzoli m-nitrofenoli

nax. 3.12. nitrobenzolis aromatuli

birTvis m-hidroqsilireba.

NH2 NH2

OHanilini

p-hidroqsianilini

nax. 3.13. anilinis aromatuli birT-

vis p-hidroqsilireba.

NH

Cl

SO2

NH

CO

N N

NCH3 OCH3

NH

Cl

SO2

NH

CO

N N

NCH3 OCH3

OHqlorsulfuroni

hidroqsi-

qlorsulfuroni

nax. 3.14. pesticid

qlorsulfuronis aro-

matuli birTvis hidroq-

silireba.

233

NSO2

N

OCH CH3

CH3

NSO2

N

OCH CH3

CH3

OH

bentazoni hidroqsil-bentazoni

nax. 3.15. pesticid bentazonis aromatuli birTvis hidroqsilireba.

O

OCH

COOCH3 CH3

Cl

ClO

OCH

COOCH3 CH3

Cl

Cl

OH

diqlofopi hidroqsi-diqlofopi

nax. 3.16. pesticid diqlofopis aromatuli birTvis hidroqsilireba.

fenoqsiZmarmJava da misi warmoebuli herbicidebi umTavresad, aro-

matuli rgolis me-4 mdgomareobaSi hidroqsilirdebian [45]. amave dros, Canergili hidroqsiluri jgufi xSirad glikozilirebis saSualebiT koniugirdeba. aromatul birTvSi halogenirebuli fenoqsiZmarmJavebi hidroqsilirdebian halogenebiT Caunacvlebel benzolis birTvis nax-Sirbadis atomebSi. amave dros, 2,4-diqlorfenoqsi ZmarmJavas (2,4-D) SemTxvevaSi hidroqsilireba xSirad me-4 mdgomareobaSi xdeba da qloris atomi me-3 an me-5 mdgomareobaSi gadainacvlebs (nax.3.17) [46]. mag., 4-hidroqsi-2,5-D warmoadgens 2,4-D-s hidroqsilirebis ZiriTad metabo-lits iseT sarevela balaxebSi, rogorebicaa: yanis Wleqi (Polygonum convolvulus), rZiana (Euphorbia esula), yviTeli Zurwa (Setaria glauca), Svriuka (Avena fatua), veluri mdogvi, RiWa (Sonchnus arvensis) da

234

yviTeli cocxi (Kochia scoparia). herbicidebis mimarT mgrZnobiare da mdgradi soiis jiSebSi 2,4-D-s transformaciam uCvena, rom herbicidis mimarT mdgrad saxeobaSi gansakuTrebiT intensiurad 4-hidroqsi-warmoebuli warmoiqmneba, romelic Semdeg mxolod glukozidis formiT koniugirebs [47].

O CH2 COOHCl

Cl

O CH2 COOHCl

OHCl

4-hidroqsi-2,5-D2,4-D nax. 3.17. 2,4-D-s hidroqsilireba, ris Sedegadac qloris atomi

aromatul birTvSi pozicias icvlis.

citoqrom P450-damokidebuli monooqsigenaza policikluri aro-

matuli naerTebis hidroqsilirebasac akatalizebs. amis magaliTia

naftalinisa da 3,4-benzpirenis transformacia. naftalinis aroma-

tuli birTvi 1 da 2 poziciebSi hidroqsilirdeba, rasac mohyveba

1,2-dihidroqsinaftalinis da Sesabamisi qinonis warmoqmnis stadiebi

(nax. 3. 18) [48, 49].

OH

OH

OHOHO

n O

O

naftalini

α−naftoli

β−naftoli

1,2-dihidroqsi-

naftalini

1,2-dioqsi-

naftalininaftalinis

oqsidi

nax. 3.18. aromatuli birTvis hidroqsilirebiT mimdinare nafta-

linis gardaqmnis reaqciebi.

235

poliaromatul naerTTa Jangvis Semdgomi etapia qinonebis war-

moqmna, rac xSirad aromatuli birTvis gaxleCas iwvevs. unda aRi-

niSnos, rom araCanacvlebul poliaromatul birTvSi yovelTvis ar-

sebobs aqtiuri, e.w. L-ubani, romelic citoqrom P450-damokidebuli

hidroqsilirebis samiznea. mag., 3,4-benzpirenSi aseTi ubania 6-

mdgomareoba, romelic yvela SemTxvevaSi hidroqsilirdeba (nax. 3.19)

[48, 50-53].

OH

OH

OH

OH

OH

OH

3,4-benzpireni

4,6-dihidroqsi-

3,4-benzpireni

2,6-dihidroqsi-

3,4-benzpireni

1,6-dihidroqsi-

3,4-benzpireni

nax. 3.19. aromatuli birTvis hidroqsilirebiT mimdinare 3,4-

benzpirenis gardaqmnis reaqciebi.

N-hidroqsilireba, romelsac citoqrom P450 akatalizebs, L-

Tirozinis gardaqmnis or Tanmimdevrul reaqciaSi gvxvdeba (nax.

3.20) [54]. warmoqmnili N,N-dihidroqsiTirozini SemdgomSi arafer-

mentul dehidrirebas da dekarboqsilirebas ganicdis, ris Sedegadac

cianogenuri glikozidis _ darinis metaboliti miiReba. reaqcia

xorcieldeba rekonstruirebul sistemaSic, romelic lipidur mi-

236

celebze imobilizebuli NADPH-citoqrom P450-reduqtazisa da

citoqrom P450-isagan Sedgeba. qsenobiotikebi, romlebic meoreul aminebs warmoadgenen, degra-

daciis pirvel etapze citoqrom P450-damokidebuli N-hidroqsilirebis gziT gardaiqmnebian (nax. 3.21) [20, 41, 55, 56].

maTi Semdgomi transformacia sxvadasxva gziT mimdinareobs.

CH2 CH

NH2

COOH

OH

CH2 CH

NHOH

COOH

OH

CH2 CH

N(OH)2

COOH

OHL-Tirozini N-hidroqsi-

L-Tirozini

N,N-dihidroqsi-L-Tirozini

nax. 3.20. L-Tirozinis citoqrom P450-damokidebuli N-hidroqsi-lireba cianogenuri glikozidebis biosinTezis procesSi.

NH

N

OH

R1 R2 R1 R2

meoreuli amini meoreuli

hidroqsilamini nax. 3.21. meoreuli aminebis citoqrom P450-damokidebuli N-hidroqsilireba.

C-hidroqsilireba, romelsac aminebisa da halogenidebis meore-

uli naxSirbadatomi ganicdis, aseve P450-damokidebuli procesia. am

reaqciebis Sedegad umdgradi intermediatebi, Sesabamisad – aminos-

pirti da hidroqsilirebuli halogennawarmi warmoiqmnebian, romel-

Ta daSlis saboloo produqti ketonia (nax. 3.22 da 3.23) [55]. am

gziT azotisa da halogenis atomebi wyalbadnaerTis saxiT scildeba

toqsikantis molekulas, rac Semdgom gardaqmnebs bevrad aadvilebs.

237

R CH CH3

NH2

R C CH3

NH2

OH

R C CH3

O

NH3

amini aminospirti (umdgradi)

+

ketoni amoniaki

nax. 3.22. aminebis citoqrom P450-damokidebuli C-hidroqsilireba.

C HalOH

HHalCH Hal C

O

naxSirwyalbadis

meoreuli

halogennawarmi

ketoni

+R1

R2

R1

R2

R1 R2

hidroqsilirebuli

halogennawarmi

(umdgradi)

halogen-

wyalbadmJava

nax. 3.23. halogenalkanebis citoqrom P450-damokidebuli C-hidro-

qsilireba.

am ori ukanaskneli magaliTidan aSkarad Cans, rom citoqrom

P450-damokidebuli dezaminireba da dehalogenireba Tavisi arsiT

hidroqsilirebis reaqciebs warmoadgenen.

3.2.2 demeTilireba

demeTilireba, romelsac mravali qsenobiotiki ganicdis, rogorc wesi, citoqrom P450-iT katalizebuli N-, O- da S-meTilis jgufebis hidroqsilirebis Sedegia [56]. am dros warmoiqmneba Sesabamisi hidroqsimeTilwarmoebulebi, romlebic qimiurad arastabilur radika-lebs warmoadgenen da formaldehidis saxiT advilad scildebian molekulas. Sesabamisad, arCeven N-, O- da S-demeTilirebis reaqciebs.

N-demeTilirebis TvalsaCino magaliTia N-meTilfenilis (nax. 3.6), N,N-dimeTilanilinisa da Sardovanas warmoebuli herbicidis – qlortoluronis gardaqmna (nax. 3.7), romlebic zeviT ganvixileT. es herbicidi N- da C-meTilis jgufebs Seicavs. hidroqsilirebas ganicdis orive tipis meTilis jgufi, magram N-hidroqsimeTilis jgufi umdgradia da igi formaldehidis saxiT exliCeba azotis atoms (xdeba N-

238

demeTilireba). amitom metabolitebs Soris identificirdeba ara uSualod N-hidroqsimeTilqlorotoluroni, aramed N-demeTilire-

buli qlorotoluroni. aromatul birTvTan mierTebuli C-hidroqsi-meTilis jgufi gacilebiT mdgradia da igi demeTilirebas ar ganicdis.

N-demeTilirebas eqvemdebareba herbicidi izoproturonic (nax.

3.24) [57]. sainteresoa aRiniSnos, rom es naerTi fitotoqsikuria,

N-demeTilirebis pirveli stadiis Sedegad misi fitotoqsikuroba

sagrZnoblad mcirdeba, xolo orive N-meTilis mocilebis Semdeg

mTlianad qreba.

CH

NHCO

NCH3 CH3

CH3 CH3

CH

NHCO

NH CH3

CH3 CH3

CH

NHCO

NH H

CH3 CH3

izoproturoni

CH2O CH2O

demeTilirebuli

izoproturoni

didemeTil-

izoproturoni

_ _

nax. 3.24. herbicid izoproturonis orjeradi N-demeTilirebis

reaqciebi.

dimeTilanilinis N-demeTilirebis analogiuri meqanizmiT wari-

marTeba mikrosomebSi citoqrom P450-damokidebuli aminopirinis

Jangva (nax. 3.25) [20, 41, 58]:

NN

N

CH3

CH3

CH3

CH3

NN

N

CH3

CH3

H

CH3

- CH2O

nax. 3.25. aminopirinis N-demeTilirebis reaqcia.

239

O-demeTilireba eTerebis tipis substratebidan meToqsijgufe-

bis mocilebis farTod gavrcelebul reaqcias warmoadgens. reaqciis

meqanizmi N-demeTilirebis analogiuria da produqtebs Sesabamisi

hidroqsiwarmoebuli da formaldehidi warmoadgenen, rac mkafiod

Cans p-nitroanizolis gardaqmnis magaliTiT (nax. 3.26) [20, 55].

O CH3

NO2

H CH

O

O CH2

NO2

OH

OH

NO2

+

p-nitro-anizoli

form-

aldehidi

p-nitrofenoliO-hidroqsimeTil-

p-nitroanizoli

(umdgradi) nax. 3.26. p-nitroanizolis O-demeTilirebis reaqcia.

O-demeTilirebis gziT mniSvnelovnad kargavs Tavis toqsiku-

robas garemos damabinZurebeli naerTi – mesameuli buTil-meTil

eTeri anu meTil-tert-buTil eTeri (MTBE) (nax. 3.27) [59-61],

romelic tetrameTil-tyviis nacvlad gamoiyeneba, rogorc sawvavis

antidetonatoruli danamati.

O

C

CH3

CH3

CH3

CH3 H CH

OO

C

CH2

CH3

CH3

CH3

OH

OH

C CH3

CH3

CH3 +

mesameuli buTil-

meTil eTeri mesameuli buToqsi-

meTanoli (umdgradi)

form-

aldehidimesameuli

buTilis spirti

nax. 3.27. MTBE-s O-demeTilirebis reaqcia.

citoqrom P450-is mier katalizebul biosinTezur procesebs

rac Seexeba, aq O-demeTilirebis reaqciaa ligninis biosinTezSi

eToqsikumarinis O-demeTilireba [62, 63]. S-demeTilirebas, rogorc ukve aRvniSneT, win uZRvis citoqrom

P450-is mier katalizebuli S-meTilis jgufis hidroqsilireba,

romlis produqti – S-hidroqsimeTilis jgufi arastabilurobis

240

gamo Sidamolekulur gadajgufebas ganicdis da formaldehidis ge-

nerirebiT iSleba (nax. 3.28) [55].

NH

NN

N

S CH3

NH

NN

N

S CH2 OH

NH

NN

N

SH

H CH

O

form-

aldehidi

+

6-merkaptopuriniS-hidroqsimeTil-

6-merkaptopurini

(umdgradi)

6-meTilmer-

kaptopurini

nax. 3.28. 6-meTilmerkaptopurinis S-demeTilirebis reaqcia.

3.2.3 N- da S-Jangva

N-Jangva, romlis drosac citoqrom P450 azotis atomis

uSualo monooqsigenirebas axdens, arastabilur produqts – N-oqsids warmoqmnis. N-Jangvas aminebis hidroqsilirebis erT-erT

SesaZlo gzad miiCneven. citoqrom P450 mxolod pirveladi aminebis

N-JangvaSi monawileobs, meoreuli da mesameuli aminebi ki

mikrosomuli NADPH-specifikuri flavoproteiniT iJangebian. N-oqsidi xSirad sawyis qsenobiotikze ufro toqsikuria. citoqrom

P450-s SeuZlia N-oqsidebTan Semdgomi urTierTqmedebac da

sabolood N-demeTilirebuli produqti miiReba (nax. 3.29) [41, 55].

CH3 NCH3

CH3

CH3 NCH3

CH3

O

H CH

OCH3 N

H

CH3

trimeTilamini trimeTilaminis

N-oqsidi

+

form-

aldehidi

dimeTilamini

nax. 3.29. trimeTilaminis N-Jangvis da Semdgomi N-demeTilirebis

reaqcia.

N-Jangvis arseboba araerT magaliTzea naCvenebi da yvelaze Tval-

saCinos klasikuri mikrosomuli substrati _ N,N-dimeTilanilini

241

warmoadgens [20]. Tumca isic unda aRiniSnos, rom es qsenobiotiki

ramdenime gziT SeiZleba daiJangos. es, rogorc wesi, TviT naerTis

qimiur aRnagobaze, kerZod, arapolaruli jgufebis ganlagebazea

damokidebuli. N,N-dimeTilanilinis molekula or hidrofobul

ubans Seicavs: erT mxares meTilis jgufebs, meore mxares ki

fenilis radikals. amis gamo mikrosomuli NADPH-damokidebuli

monooqsigenaza dimeTilanilins sami alternatiuli gziT: N-JangviT, N-demeTilirebiT an p-hidroqsilirebiT Jangavs (nax. 3.30).

NCH3 CH3

NCH3 CH3

NCH2 CH3

OH

NCH3 CH3

OH

NH CH3

NH H

NH CH3

OH

NH H

OH

O

CH2O CH2O

CH2O CH2O

N,N-dimeTil-

anilini

N,N-dimeTilanilinis

N-oqsidi

N-monomeTilanilini anilini

p-hidroqsi-N,N-dimeTilanilini

p-hidroqsi-N-monomeTilanilini

p-aminofenoli

(umdgradi)

_ _

__

nax. 3.30. N,N-dimeTilanilinis citoqrom P450-damokidebuli Jangvis

gzebi. msxvili SriftiT mocemulia Jangvis ZiriTadi iden-

tificirebuli produqtebi, xolo wyvetili isrebiT naCve-

nebia Teoriulad SesaZlebeli reaqciebi.

N-Jangvis Sedegad formirebuli N,N-dimeTilanilinis N-oqsidi SemdgomSi citoqrom P450-is moqmedebiT umdgrad N-hidroqsimeTil-

warmoebulad gardaiqmneba, romelsac advilad scildeba formal-

dehidi da N-monomeTilanilini miiReba.

N-hidroqsimeTil-warmoebuli SesaZloa N-Jangvis Sualeduri

242

rgolis gareSec, kerZod, uSualod N,N-dimeTilanilinis hidroqsi-

lirebis saSualebiT warmoiqmnas. am SemTxvevaSic Jangvis tipi N-demeTilirebaa da produqts N-monomeTilanilini warmoadgens.

citoqrom P450-iT N,N-dimeTilanilinis Jangvis mesame gzaa aro-

matuli birTvis hidroqsilireba, romelic p-mdgomareobaSi xorci-eldeba da kidev erT produqts – p-hidroqsi-N,N-dimeTilanilins

generirebs.

unda SevniSnoT, rom mikrosomuli NADPH-damokidebuli Jangvis

Sedegad sareaqcio areSi swored es sami produqti – N-oqsidi, monomeTilanilini da p-hidroqsi-N,N-dimeTilanilini identificir-

deba, Tumca Teoriulad SesaZlebelia, rom dimeTilanilinis mono-

oqsigenirebis produqtebs citoqrom P450 Semdgomi Jangvis substra-tebad iyenebdes, ris Sedegadac saboloo jamSi p-aminofenoli unda

warmoiqmnas. praqtikulad es momdevno reaqciebi ufro naklebad

unda iyos damokidebuli citoqrom P450-ze, radgan hemoproteini

specifikuria hidrofobuli substratebis mimarT, ganxilul SemTx-

vevaSi ki yoveli reaqciis Semdeg miRebuli produqtis polaroba

sagrZnoblad izrdeba. citoqrom P450-damokidebuli N-Jangva

mimdinareobs anilinis biotransformaciis SemTxvevaSic (nax. 3.31)

[20, 41].

HN

H

HN

HO

OHN

H

HN

H

OH

nax. 3.31. anilinis citoqrom P450-damokidebuli Jangvis gzebi.

243

anilinis N-Jangvaze gacilebiT ufro maRali siCqariT mimdi-

nareobs p-hidroqsilirebis reaqcia. am dros warmoqmnili p-amino-fenolis polaroba ufro metia, vidre N-hidroqsianilinisa, amitom

ujredisTvis, detoqsikaciis TvalsazrisiT, p-hidroqsilireba ufro

xelsayreli unda iyos.

S-Jangva, romlis saSualebiTac mikrosomuli monooqsigenazebi

gogirdSemcvel hidrofobul naerTebs gardaqmnian, N-Jangvisagan

gansxvavebiT, yovelTvis citoqrom P450-damokidebulia. unda

aRiniSnos, rom am dros miRebuli produqti ufro maRali

inseqticiduri aqtivobiT gamoirCeva, vidre sawyisi naerTi.

warmoqmnili S-oqsidi SemdgomSi sulfojgufamde iJangeba.

mcenareuli citoqrom P450-damokidebuli S-Jangvis magaliTebia

qlorpromazinisa (nax. 3.32) da meTiokarbis gardaqmna Sesabamis

sulfoqsidSi [55].

CH2CH2

S

NCH2 N

CH3

CH3

ClCH2CH2

S

NCH2 N

CH3

CH3

Cl

O

qlorpromazini

qlorpromazinis

sulfoqsidi

nax. 3.32. qlorpromazinis S-Jangva.

citoqrom P450-damokidebul procesebs Soris yvelaze iSviaTad

dehidrirebis tipiuri reaqcia gvxvdeba. citoqrom P450-Semcveli

monooqsigenaza aseTi tipis aqtivobas amJRavnebs flavonoidebis

biosinTezSi, rodesac igi C6_C3_C6 naxSirbadovani ConCxis mqone

naerTebidan flavonoidebis sinTezs akatalizebs. am dros citoqrom

P450 funqcionirebs, rogorc izoflavosinTaza, romelic awarmoebs

(2S)-naringeninidan izoflavonebis – apigeninis da genisteinis

warmoqmnas (nax. 3.33) [62, 63].

244

O

O

OH

OH

HOH

OHO

O

OH

OH

OH

O

O

OH

OH

(2S)-naringenini

apigenini

genisteini nax. 3.33. citoqrom P450-damokidebuli dehidrirebis reaqciebi

izoflavonebis biosinTezSi.

zemoTganxiluli masalidan Cans erTi mxriv, mcenareuli cito-

qrom P450-Semcveli monooqsigenazuri sistemis polifunqciuroba,

meore mxriv ki misi specifikuroba hidrofobuli bunebis mqone

naerTebis mimarT. SeiZleba davaskvnaT, rom rogorc biosinTezur,

aseve detoqsikaciur procesebSi citoqrom P450-is monawileoba

gamoixateba endogenuri an egzogenuri naerTis erTi konkretuli

ubnis specifikur gaaqtiurebaSi, riTac xSir SemTxvevaSi metabo-

luri gzis mimarTulebis gansazRvra xdeba.

245

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249

Tavi 4. monooqsigenazuri sistemis regulacia

citoqrom P450-Semcveli monooqsigenazuri sistemis fuqcioni-

rebis TaviseburebaTa gaTvaliswinebiT SeiZleba gamoiyos misi regu-

laciis Semdegi doneebi:

− regulacia aRmdgeneli eqvivalentebis doneze – katalizuri

ciklis Sesrulebisas citoqrom P450 substratSi Casanerg Jang-bads aRmdgeneli eqvivalentebis saSualebiT aaqtiurebs, amitom

qsenobiotikTa Jangva TavisTavad damokidebuli unda iyos yvela

im eleqtronis satransporto sistemaze, romelTac citoqrom

P450-is saTanado eleqtronebiT uzrunvelyofa SeuZliaT;

− regulacia substratul doneze – citoqrom P450 specifikuria hidrofobuli substratebis mimarT, amitom misi Tvisoba rome-

lime konkretuli qsenobiotikis mimarT am ukanasknelis hidro-

fobulobis xarisxzea damokidebuli;

− regulacia induqciis doneze – citoqrom P450 inducirebadi cilaa, ujredSi misi Semcveloba mkveTrad izrdeba sxvadasxva

faqtorTa (mag., qsenobiotikebis an paTogenebis moqmedebiT, gana-

Tebis reJimiT da sxv.) gavleniT;

_ regulacia membranul doneze – citoqrom P450-is, rogorc WeS-maritad membranuli fermentis mier katalizuri aqtivobis gamom-

JRavneba mniSvnelovnadaa damokidebuli mikrosomebis membranul

organizaciasa da TviT maTSi mimdinare fizikur-qimiur proce-

sebze;

_ regulacia citoqrom P450-is qimiuri modifikaciis doneze –

induqciis sapirispirod, ujredSi SeiZleba adgili hqondes ci-

toqrom P450-is inaqtivacias an transformacias, rac mis mier

generirebuli Tavisufali radikalebis fermentze zemoqmedebis

Sedegia.

4.1 regulacia aRmdgeneli eqvivalentebis doneze

4.1.1 urTierTqmedeba eleqtron-satransporto gzebs Soris

mikrosomul NADH- da NADPH-specifikur redoqs-jaWvebis

urTierTqmedebaSi ZiriTadad maT Soris aRmdgeneli eqvivalentebis

cvla da hidroqsilirebis aqtis ganxorcielebaSi TiToeuli maTga-

250

nis Tanamonawileobis donis gansazRvra igulisxmeba. gansakuTrebiT

mniSvnelovania eleqtronTa gadatanis meqanizmisa da am procesSi

citoqrom b5-is rolis garkveva. amTaviTve aRvniSnavT, rom [1] uamra-

vi gamokvlevebis Sedegad dagrovili faqtebi imis sasargelod mety-

veleben, rom:

1. NADH-is Jangvis jaWvidan NADPH-is Jangvis jaWvze adgili aqvs

aRmdgeneli eqvivalentebis gadatanas;

2. NADH-citoqrom b5-reduqtaza uSualod monawileobs qsenobio-

tikTa hidroqsilirebaSi.

pirveli argumentis eqsperimentuli dadastureba aRdgenili

NADH-iT dimeTilanilinis NADPH-damokidebuli hidroqsilirebis

(N-demeTilirebis) stimulaciaa [1]. sainkubacio xsnarSi NADPH-is

dabali (KM-is rigis) koncentraciebisas, NADH-is damateba mniSv-

nelovnad aCqarebs dimeTilanilinisa da eTilmorfinis N-deme-

Tilirebis reaqciebs. es Sedegi saSualebas iZleva miRebul iqnas,

rom NADH-is Jangvis procesSi misi reducirebadi eqvivalentebi

SeiZleba moxvdnen NADPH-is redoqs-jaWvSi da rodesac es ukanask-

neli sruladaa gajerebuli Tavisi substratiT, mastimulirebeli

gavlena moaxdinos hidroqsilirebis reaqciaze. Semdgom dadgenil

iqna, rom NADH-is mastimulirebeli efeqti vlindeba NADPH-is

rogorc dabali, aseve maRali koncentraciebisas da agreTve

NADPH-generirebadi sistemis (glukozisa da glukozo-6-fosfatdehidrogenazis narevis) damatebisas. am faqtebze dayrdnobiT

estabrukma da TanamSromlebma (1971 w.) [2, 3], xolo cota mogviane-

biT arCakovma da TanamSromlebma (1974 w.) [1] warmoadgines

NAD(P)H-sistemaTa monawileobis sqemebi citoqrom P450-is mier

ganxorcielebul hidroqsilirebis reaqciebSi. saWirod vTvliT

TvalsaCinoebisaTvis moviyvanoT orive sqema (nax. 4.1 da 4.2). rogorc vxedavT, am or sqemas Soris principuli gansxvavebaa.

nax. 4.1-ze mocemul sqemaze araa dakonkretebuli flavoproteinisa

da citoqrom P450-s Soris arsebuli Sualeduri gadamtani da igi

daudgenel X-komponentad iTvleba. nax. 4.2-ze warmodgenili sqemis

Tanaxmad ki am ubanze funqcionirebs citoqrom b5-is qvefraqcia, ro-

melic mikrosomebSi am hemoproteinis saerTo Semcvelobis 20_30%-s

ar aRemateba.

estabrukis sqemis mixedviT eleqtronTa gadatanis NADPH-isa

da NADH-is redoqs-jaWvebi damoukideblad funqcionireben.

NADPH-is Jangvis sistema citoqrom P450-is aRdgenisaTvis saWiro

251

pirvel eleqtrons, xolo NADH-is Jangvis gza citoqrom b5-is

gavliT Jangbadis gaaqtivebisaTvis saWiro meore eleqtrons iZleva.

arCakovis sqemis mixedviT ki eleqtronTa gadatanis orive gzas

saerTo ubani gaaCnia da yovel jaWvs hidroqsilirebisaTvis aucile-

beli orive eleqtroniT uzrunvelyofs.

e_

e_

PCO

SS

S

O2

PS

NADPH FP1citoqrom P450

NADH FP2 citoqrom

b5

S

O2

H2O

SOH

R ROOH

X

citoqrom P450

+ +

+ +

CO450++

hγ

O2

_

450+ + +

P450 + + +

nax. 4.1. eleqtronebis gadatana endoplazmuri retikulumis membra-

nebSi (estabrukis mixedviT) [2, 3]. R _ ujeri cximovani mJavaa, S – hidroqsilirebis substratia.

NADPH FP1citoqrom b5 citoqrom P450

NADH FP2citoqrom b5

S O2

H2OSOH

nax. 4.2. redoqs-jaWvebs Soris reducirebadi eqvivalentebis cvlis

SesaZlo gzebi (arCakovis mixedviT)[4]. sruli isrebiT aRniSnulia

eleqtronTa yvelaze savaraudo cvlis gzebi.

252

arsebuli faqtebi jerjerobiT ar iZlevian romelime sqemis

utyuarobis srul garantias.

nikotinamiduri kofermentebis Tanamyofobisas hemoproteinis

aRdgenis siCqaris gazomva saSualebas iZleva, dadgindes redoqs-

jaWvebis SigniT eleqtronTa gadatanis siCqareebis konstantebi,

agreTve NADH-is Jangvis jaWvidan NADPH-is Jangvis jaWvze eleq-

tronTa gadatanis reaqciis siCqareTa konstantebi. am SemTxvevaSi

SeuZlebelia gaizomos mxolod NADPH-specifikuri redoqs-

jaWvidan NADH-is Jangvis jaWvzeQ eleqtronis gadatanis siCqare.

miuxedavad amisa, NADPH-iT citoqrom b5-is aRdgenis mrudi gviCve-

nebs, rom es siCqare sakmaod maRalia da NADPH-is Jangvis jaWvSi

citoqrom b5-is aRdgenis siCqaresTan savsebiT Sesadarebeli sididea

[1]. am mrudze ar Cndeba „gadatexis wertilebi”, romelTac SeiZle-

boda NADPH-sadmi specifikuri flavoprotein-reduqtaziT cito-

qrom b5-is ori qvefraqciis gansxvavebuli siCqariT aRdgena eCvenebi-

naT. safiqrebelia, rom NADPH-specifikuri flavoproteiniT orive

citoqromi erTnairi siCqariT aRdgeba, anu NADPH-is Jangvis jaW-

vidan NADH-is Jangvis jaWvze eleqtronTa gadatana mimdinareobs

imave, an ufro meti siCqariT, rac TviT NADPH-is Jangvis jaWvSi

citoqrom b5-is aRdgenas gaaCnia. vinaidan NADH-is Jangvis jaWvSi

citoqrom b5-is Semcveloba NADPH-is Jangvis jaWvTan SedarebiT

gacilebiT maRalia, amitom NADPH-is Jangvis jaWvidan NADH-is

Jangvis JaWvze eleqtronTa gadatanis reaqciis siCqaris konstanta

ar SeiZleba NADPH-citoqrom b5-reduqtazuli reaqciis siCqaris

konstantaze naklebi iyos.

redoqs-jaWvebs Soris eleqtronTa gadanawilebis zusti ubnebis

dasadgenad Seswavlil iqna ferment pronazas efeqturoba [1]: dabali

koncentraciebisas mas NADPH-specifikuri flavoproteinis SerCe-

viTi solubilizebisa da amdenad, citoqromebis b5-isa da P450-is aRdgenis siCqareze zemoqmedebis unari gaaCnia. sainkubacio narevSi

pronazis damatebisas mniSvnelovnad qveiTdeba NADPH-iT citoqrom

b5-is aRdgenis reaqciis siCqare, magram praqtikulad ar icvleba

NADH-iT misi aRdgena. amgvarad, Tu NADH-is Tanamyofobisas ci-

toqrom P450-is aRdgena NADH-redoqs-jaWvidan NADPH-is Jangvis

jaWvze eleqtronebis gadasvlis Sedegia, maSin mikrosomebis prona-

ziT damuSavebas am reaqciis iseTive inhibireba unda gamoewvia, ro-

gorsac eleqtronTa NADPH-specifikuri gadatanis sxva reaqciebi

ganicdian. amis sapirispirod aRmoCnda, rom mikrosomebis proteazu-

253

li damuSaveba NADH-citoqrom P450-reduqtazul reaqciaze ar moq-

medebs [1]. es Sedegi TavisTavad gamoricxavs NADPH-specifikuri

flavoproteinis monawileobas eleqtronTa saboloo gadatanaSi

NADH-is Jangvis jaWvidan NADPH-is Jangvis jaWvze. sxvagvarad

rom iTqvas, redoqs-jaWvebs Soris eleqtronTa gadanawileba war-

moebs ara sawyis _ flavoproteinul ubanze, aramed momdevno – ci-

toqromebis doneze. amis sasargeblod metyvelebs is faqtic, rom

mikrosomebSi araa identificirebuli NADH + NADP → NAD + + NADPH reaqciis makatalizebeli fermenti _ transdehidrogenaza.

analogiuri Sedegebi iqna miRebuli dimeTilanilinis N-

demeTilirebis reaqciis siCqareze propilgalatis mainhibirebeli

moqmedebis SeswavliTac, rodesac kosubstratebad NADPH da

NADH gamoiyeneboda [1]. es nivTiereba urTierTqmedebs da Trgunavs

NADPH-specifikur flavoproteins. NADH-is Jangvis jaWvidan

NADPH-is Jangvis jaWvSi eleqtronTa gadatana sawyis flavopro-

teinul ubanze rom xdebodes, aRniSnul inhibitors unda moexsna

NADPH-is TanamyofobiT inducirebul demeTilirebis reaqciaze

NADH-is mastimulirebeli moqmedeba. garda amisa, propilgalats

unda daeTrguna NADH-is Tanamyofobisas citoqromebis _ b5-isa da

P450-is aRdgenis siCqare. Catarebulma gamokvlevebma aCvenes, rom es

inhibitori absoluturad araefeqturia NADH-damokidebuli reaq-

ciebis mimarT.

mikrosomebis or sunTqviT jaWvs Soris eleqtronTa cvlis Se-

saZlo punqti, rogorc Cans, citoqrom b5-is donea. amasTan, yovelT-

vis ar xdeba eleqtronTa gadatana FP2-dan citoqrom P450-ze [5, 6]. mZlavri NADH-citoqrom b5-reduqtazuli sistemis arsebobisas

NADH-is Jangvis jaWvSi citoqrom b5 mTlianad aRdgenil mdgoma-

reobaSia da mxolod NADH-is siWarbisas SeiZleba eleqtronis ga-

datanis es gza CairTos: NADH → FP2 → citoqrom P450. arsebobs eqsperimentuli monacemebi, romlebic mikrosomebSi ase-

Ti fermentuli kompleqsis funqcionirebas adastureben [7]. Tumca

NADH-citoqrom P450-reduqtazis gamoyofili preparati citoqrom

b5-s mxolod kvalis saxiT Seicavs.

ganxiluli faqtebidan gamomdinare, unda aRiniSnos, rom NADPH-is

Jangvis jaWvidan NADH-is Jangvis jaWvze reducirebadi eqviva-

lentebis gadatana SeiZleba xdebodes an am ukanasknelSi myofi ci-

toqrom b5-is NADPH-specifikuri flavoproteiniT uSualo aRdge-

nis gziT, an am citoqromze eleqtronTa gadataniT NADPH-damokide-

254

buli gzis citoqrom b5-dan. am reaqciis siCqare NADPH-is Jangvis

jaWvSi citoqrom b5-is aRdgenis siCqaris tolia.

NADH-specifikuri jaWvidan NADPH-is Jangvis jaWvze eleq-

tronTa gadasatanad SeiZleba gamoirCes sami yvelaze SesaZlo gza:

1. uSualod NADH-specifikuri flavoproteinidan citoqrom P450-ze; 2. uSualod NADH-damokidebuli gzis citoqrom b5-dan citoqrom

P450-ze; 3. NADH-is Jangvis jaWvis citoqrom b5-dan NADPH-is Jangvis

gziT citoqrom b5-ze.

am reaqciaTa siCqareebi Zlier mcirea.

warmodgenil sqemebs saerTo is gaaCniaT, rom oriveSi daSvebu-

lia citoqrom b5-dan citoqrom P450-ze eleqtronTa uSualo gada-

tanis SesaZlebloba. NADH-redoqs-jaWvidan citoqrom P450-hidroqsilazur sistemaze citoqrom b5-is saSualebiT eleqtronTa

gadatana amJamad dadasturebul faqtad SeiZleba miviCnioT.

amgvarad, SegviZlia sarwmunod miviRoT, rom NADH-is JangviT

jaWvSi ar arsebobs eleqtronTa terminaluri autooqsidabeluri

aqceptori. am jaWvSi citoqrom b5 mudmivad aRdgenil mdgomareobaSi

imyofeba da eleqtronTa Taviseburi koleqtoris rols asrulebs,

saidanac aRmdgeneli eqvivalentebis gadineba SeiZleba ganxor-

cieldes an mikrosomebis NADPH-damokidebuli redoqs-jaWviT, an

mitoqondriis citoqromoqsidazuli sistemiT, romelzec momdevno

paragrafSi gveqneba saubari.

4.1.2 eleqtronTa transmembranuli cvla mikrosomasa

da mitoqondrias Soris

eleqtronTa transmembranuli migracia sayovelTaod cnobili

faqtia [8, 9]. konkretul SemTxvevebSi reducirebadi eqvivalentebis

membranaTa Soris gadatana SeiZleba ganxorcieldes or mezobel mi-

krosomul membranas Soris, or mitoqondriul membranas Soris, an

mikrosomul da mitoqondriul membranebs Soris. membranuli qseno-

bioqimiisaTvis gansakuTrebiT sayuradRebosa da sainteresos mesame

SemTxveva warmoadgens, radgan:

1. mitoqondriidan mikrosomaze, kerZod, NADPH-specifikur redoqs-

255

jaWvze eleqtronTa gadasvlas Tan axlavs am ukanasknelis JangviTi

unaris gaZliereba da Sesabamisad, qsenobiotikTa mTliani biodegra-

daciis procesis sawyisi, siCqaremalimitirebeli stadiis _ hidroq-

silirebis reaqciis siCqaris mniSvnelovani zrda. maSasadame, am

SemTxvevaSi adgili aqvs mitoqondriis mxridan qsenobiotikTa Jang-

viTi gardaqmnebis regulacias aRmdgeneli eqvivalentebis doneze.

literaturaSi es fenomeni „mitoqondriuli kontrolis” saxelwo-

debiT moixsenieba. am regulatoruli meqanizmis kidev erTi biolo-

giuri mniSvneloba imaSi mdgomareobs, rom endoplazmur membranebSi

qsenobiotikTa Jangvis gaZlierebiT, mitoqondria Tavs izRvevs maRa-

li koncentraciebiT masSi ucxo nivTierebebis moxvedrisagan, rom-

lebic sunTqviTi jaWvis (citoqromoqsidazuri sistemis, gansakuTre-

biT pirveli kompleqsis _ NADH-CoQ-reduqtazis) Zlier inhibire-

basa da energetikuli cvlidan mitoqondriis gamoTiSvas iwvevs.

2. mikrosomuli NADPH-specifikuri Jangvis jaWvidan mitoqon-

driaze redoqs-eqvivalentebis gadataniT aqtiurdeba citoqromoqsi-

dazuri sistema, rac energetikuli SeuRlebisa da mitoqondriis

(mTlianad ujredis) energetikuli statusis zrdaSi mdgomareobs.

aseTi funqciis SeZeniT mikrosomuli Jangvis sistema, sxva metabo-

luri gzebis analogiurad, „srul uflebebSia aRdgenili” energeti-

kuli da plastiuri cvlis TvalsazrisiT. aq mniSvnelovania meore

garemoebac: mitoqondriis Sida membrana eqstramitoqondriuli

NADPH-isaTvis ganuvladia; amitom energetikuli saWiroebebisaTvis

misi aRdgenili eqvivalentebis aseTi uSualo da pirdapiri gamoyene-

ba SeuZlebelia. im SemTxvevaSi, rodesac mikrosomuli NADPH-

specifikuri jaWvi qsenobiotikis hidroqsilirebiT araa dakavebuli

(misi citoqrom b5 „moclilia”), mas am nikotinamiduri kofermentis

Tavisufali, hidroqsilirebasTan araSeuRlebuli Jangva da misi

aRmdgeneli eqvivalentebis mitoqondriaze gadacema ZaluZs. maSasa-

dame, NADPH-is Tavisufalma Jangvam SeiZleba mikrosomisa da mito-

qondriis energetikuli SeuRleba ganaxorcielos. cxadia, am

SemTxvevaSi saxeze gveqneba NADPH-is eleqtronebis mitoqondriuli

realizaciis kidev erTi alternatiuli gza.

4.1.2.1 mitoqondriuli kontroli

pirvelad igi RviZlis hepatocitebSi iqna gamovlenili [10-16]

(sinti, Senkmani da sxv., 1972-1973 w.w.) da misi arsi mitoqondriuli

Jangvis substratebiT citoqrom P450-is I-tipis qsenobiotikTa hi-

256

droqsilirebis stimulaciaSi mdgomareobs. sxvagvarad rom vTqvaT,

mitoqondriebi endoplazmur membranebSi ucxo naerTTa biotrans-

formaciis ujreduli kontrolis adgils warmoadgenen. mitoqon-

driebisa da mikrosomebis kombinirebul sistemaSi am kontrolis

gansaxorcieleblad ori yvelaze SesaZlo meqanizmi ikveTeba:

1. mitoqondriidan mikrosomaze reducirebadi eqvivalentebis gada-

tana ori (mitoqondriuli da mikrosomuli) citoqrom b5-is sa-

SualebiT, risTvisac aucilebeli pirobaa orive tipis membranul

struqturaTa uSualo kontaqti;

2. piridinnukleotidebis gaSualebuli gadatana malat-aspartatuli

da izocitratuli SuntirebiT (maqoseburi meqanizmiT), rodesac

Sidamitoqondriul NADH-s mikrosomul hidroqsilirebaSi mona-

wileobis miReba SeuZlia citoqrom P450-substratis kompleqsze

eleqtronis gadatanis gziT.

maSasadame, mitoqondriuli kontrolis ganxorcielebisas mikro-

somis eleqtronTa satransporto sistema qsenobiotikTa JangviTi

degradaciisaTvis saWiro energias mitoqondriebidan iRebs ara ATP-s qimiuri energiis, aramed saTanado energiis mqone eleqtronebis

saxiT.

mitoqondriul kontrols struqturuli aspeqtebic gaaCnia.

eleqtronuli mikroskopiiT naCvenebia, rom kombinirebul sistemaSi

piruvatis damatebisas endoplazmuri retikulumis membranebi vezi-

kulebad gardaiqmnebian da mitoqondriis garSemo orientirdebian

[10]. am dros, rogorc Cans, aucilebeli xdeba „moclili” citoqrom

b5-is didi raodenobiT gamoyeneba, raTa moZebnil iqnas membranul

struqturaTa sakontaqto adgilebi am ubnebze redoqs-potenciale-

bis swrafad gasaTanabreblad. eleqtronTa transmembranuli gadatana

mitoqondriuli filamentis erTganzomilebian da endoplazmuri ba-

dis cisternaTa organzomilebian sivrceSi difuziis gaZlierebis

pirobebs qmnis. safiqrebelia, rom ujredis sxvadasxva ubnebSi re-

doqs-potencialebi am gziT ufro swrafad Tanabrdeba, vidre cito-

zolis samganzomilebian sivrceSi metabolitebisa da kofaqtorebis

Tavisufali difuziiT.

cxovelur qsovilSi mitoqondriuli kontrolis realuri arse-

boba momdevno wlebSi sxva avtorTa mierac iqna dadasturebuli [14-

17]. procesis regulatorul meqanizms dRes Semdegi axsna aqvs: qse-

nobiotikis maRali koncentracia, romelmac citoqrom P450 srulad

gaajera, mitoqondriaSi SeiRweva da sunTqviTi jaWvis inhibirebas

257

iwvevs. am pirobebSi adgili aqvs mitoqondriidan mikrosomaze cito-

qrom P450-is maqsimaluri funqcionirebisaTvis saWiro aRmdgeneli

eqvivalentebis damatebiTi nakadis warmoqmnas. amis Sedegad izrdeba

qsenobiotikis Jangvis siCqare, ris Sedegadac mitoqondriul

sunTqvaze misi mablokirebeli moqmedeba moixsneba. safiqrebelia, rom

mitoqondriis NADH-dehidrogenazidan (sunTqviTi jaWvis I ansam-

blidan) eleqtronTa transmembranul gadatanaSi CarTulia cito-

qrom b5-is orive (mitoqondriis gare membranisa da mikrosomis) qve-

fraqcia. am varauds adasturebs erTi mxriv, mitoqondriul kon-

trolSi suqcinatis (sunTqviTi jaWvis II ansamblis substratis)

absoluturi araefeqturoba, xolo meore mxriv, qsenobiotikis Jang-

vis gaZliereba I kompleqsis inhibitorebiT _ rotenoniTa da amita-

liT. qsenobiotikTa JangvaSi aseTi daxvewili regulatoruli meqa-

nizmis arsebobis miuxedavad, mainc iTvleba, rom eleqtronTa mito-

qondriuli wyaro hidroqsilirebis procesisaTvis mainc damxmarea

da im wyarosTan SedarebiT, romelsac mikrosomuli NADPH-

damokidebuli eleqtronTa satransporto gza warmoadgens, meore-

xarisxovania.

yovelive zemoTqmuli SegviZlia warmovidginoT sqematurad (nax.

4.3), romelSic gaTvaliswinebulia mitoqondriul kontrolSi izo-

citratuli da malat-aspartatuli maqoseburi meqanizmebis monawi-

leobac.

ra viTareba gvaqvs am mimarTebiT mcenareul ujredSi? Cvens la-

boratoriaSi, upirveles yovlisa, Seiswavleboda qsenobiotikis (di-

meTilanilinis) gavlena mitoqondriebSi suqcinatisa da izocit-

ratis Jangvaze [16]. aRmoCnda, rom pirvelis Jangvis siCqare 1/3-iT qveiTdeba, magram sunTqviTi kontroli ucvleli rCeba. ufro Zlier

inhibirebas ganicdis izocitratis Jangva. am procesis siCqare nax-

evrdeba, xolo sunTqviTi kontrolis mniSvneloba 3-dan 2.5-mde mcirdeba. maSasadame, dimeTilanilini udavod inhibitorulad moqme-

debs sunTqviTi jaWvis, da gansakuTrebiT, misi I kompleqsis funq-

cionirebaze.

mcenaris mikrosomebisa da mitoqondriebis kombinirebul siste-

maSi dimeTilanilinis N-demeTilireba Zlierdeba malatis, izoci-

tratis da suqcinatis damatebiT [17], e.i. dimeTilanilinis mikroso-

muli Jangva mitoqondriuli kontrolis qveS imyofeba. sayuradRe-

boa, rom es efeqti vlindeba kombinirebul sistemaSi, magram ara

mTlian qsovilSi.

258

O2

FP5

NADPH

RH O2

ROH

NADH

2+Ca + NADH

2+Ca+NADHe-

e-

I kompleqsi III kompleqsi

II kompleqsi

IV kompleqsie- e-

e-

citoqrom b5

e-e-

e-citoqrom b5

citoqrom P-450FP1

e- e-FP2

mikrosomuli

membrana

e-

mitoqondriis

gare membrana

mitoqondriis

Sida membrana

perimitoqondriuli

sivrcee-

e-

s u n T q v i T i j a W v i

izocitrati

izocitrati

aspartati

aspartati

oqsaloacetati

malati

malati

oqsaloacetati

2+Ca

c

nax. 4.3. qsenobiotikebis hidroqsilirebis „mitoqondriuli kontro-

lis” savaraudo sqema. punqtiriT mocemuli qsenobiotikis mainhibi-

rebeli moqmedeba sunTqviT jaWvze.

limonmJavas ciklis intermediatebis mastimulirebeli moqmedeba

CamorCeba NADPH-is efeqts. amasTan, NADPH-iT miRweuli qsenobio-

tikis Jangvis stimulacia mitoqondriis substratTa damatebisas

aRar izrdeba, e.i. sainkubacio suspenziaSi egzogenurad damatebuli

NADPH-is Tanamyofobisas, mitoqondriuli kontroli azrs kargavs

da ixsneba. mniSvnelovania isic, rom qsenobiotikis dasaJangad saWi-

ro eleqtronebi citoqrom P450-ma SeiZleba calkeulad miiRos an

mxolod NADPH-dan, an mxolod mitoqondriidan, magram upirateso-

ba pirvels eniWeba.

cdebis calke seriaSi kombinirebul sistemas citoqromoqsidazis

dasablokad natriumis azids vamatebdiT [17]. am dros mitoqond-

riidan mikrosomaze eleqtronTa migraciisaTvis optimaluri pirobe-

bi iqmneba. procesis intensivobas amjerad citoqromebis P450-isa da b5-is aRdgenis kinetikiT vafasebdiT. mitoqondriul Jangvis subst-

ratebTan SedarebiT, NADPH-is moqmedebis efeqti am SemTxvevaSic

ufro maRali aRmoCnda. unda aRiniSnos, rom orive citoqromis aRd-

genis doneebi yvela variantSi erTmaneTTan korelaciaSia, rac mito-

qondriul kontrolSi citoqrom b5-is uSualo monawileobaze mi-

259

uTiTebs.

miRebuli Sedegebidan yuradRebas imsaxurebs N-demeTilirebaze

suqcinatis mastimulirebeli moqmedeba, rac procesSi sunTqviTi

jaWvis II kompleqsis (suqcinatdehidrogenazis) monawileobis maCve-

nebelia. rogorc Cans, am kompleqsidan eleqtronebi pirdapir xvde-

bian mikrosomebis NADPH-specifikur JangviT jaWvze. am faqts

adasturebs suqcinatdehidrogenazis inhibitorebis _ malonatisa da

oqsaloacetatis damTrgunveli moqmedeba mikrosomuli citoqromebis

aRdgenaze [17]. garda amisa, suqcinatis efeqti aramgrZnobiare aR-

moCnda rotenonis moqmedebis mimarT, rac imis maCvenebelia, rom am

dros migraciaSi sunTqviTi jaWvis I kompleqsi ar monawileobs.

mitoqondriebidan mikrosomebze eleqtronebis gadatanis intensi-

voba damokidebulia Jangvisa da fosforilebis SeuRlebis xarisxze.

Cven SevZeliT gveCvenebina [17], rom citoqrom P450-is aRdgenis ma-Rali siCqare dabali energetikuli SeuRlebis, an Jangvisa da fos-

forilebis 2,4-dinitrofenoliT gaTiSvisas miiReba. ADP-s damate-biT SeuRlebis gazrdisas piriqiT, hemoproteinis aRdgenis siCqare

muxruWdeba. maSasadame, mitoqondriidan mikrosomaze eleqtronebis

migraciis erT-erT aucilebel pirobas sunTqviT jaWvSi eleqtrone-

bis Tavisufali nakadis arseboba warmoadgens.

savsebiT misaRebia, rom mitoqondriidan mikrosomaze eleqtrone-

bis gadatanaSi erTdroulad sunTqviTi jaWvis I, II da III kompleq-

sebi monawileoben, magram energetikuli TvalsazrisiT ufro xel-

sayrelia, rom eleqtronTa migracia II da III kompleqsidan xde-

bodes, radgan aq eleqtrons SedarebiT dabali energia aqvs. rac

Seexeba I kompleqsidan mikrosomebze eleqtronTa pirdapir gadata-

nas, igi iZulebiTi gza unda iyos, romelic III kompleqsis blokire-

bisas iwyebs funqcionirebas. mitoqondriuli kontrolis ganxor-

cielebaSi cianid-rezistentuli sunTqvis sistemebic SeiZleba mona-

wileobdnen, radgan maT bc1-kompleqsze eleqtronTa uSualo Semota-

nis unari aqvT.

4.1.2.2 NADPH-is Tavisufali mikrosomuli Jangva

cxoveluri qsovilisa da baqteriebis monooqsigenazebisagan

gansxvavebiT, RviZlis citoqrom P450 arc erT substrats ar Jan-gavs sruli SeuRlebiT da NADPH-is redoqs-eqvivalentebis xarjva

yovelTvis araa dakavSirebuli qsenobiotikis hidroqsilirebasTan

260

[18]. misi garkveuli nawili sxva oqsidazur reaqciebs xmardeba. ni-

kotinamiduri kofermentis aseT Jangvas Tavisufali ewodeba.

mTel rig kvleviT centrebSi, da maT Soris Cvens laboratoria-

Sic [19], naCvenebi iqna, rom reaqciaSi _ SH2 + NADPH + H+ + O2 → SHOH + NADP + H2O, eqsperimentulad miRebuli monacemebi ara-

sodes pasuxoben steqiometrias: NADPH : SH2 : O2 = 1 : 1 : 1. aRmoCnda, rom eleqtronuli donoris molekulaTa nawili im Jangbadis aRdge-

nas xmardeba, romlis substratSi Canergvac ar xdeba. am movlenas

gaTiSvas, xolo substratebs, romelTa Tanamyofobis drosac es

movlena SeiniSneba, gamTiSvelebs uwodeben.

gaTiSvis efeqts Cveulebriv ukavSireben sxvadasxva substratuli

specifikurobis mqone citoqrom P450-is mravlobiTi formebis arse-

bobas, radgan mraval oqsidazur reaqciaSi, romlebic Tavisufali

Jangvis sistemebs miekuTvnebian, O2-is aRdgena am hemoproteiniT ka-

talizdeba. am dros NADPH-is redoqs-eqvivalentebi sxvadasxva oq-

sidazur reaqciebze nawildeba, sadac eleqtronul donors O2 war-

moadgens. am ukanasknelis nawili aqtiur formebad (superoqsidad da

peroqsidad) aRdgeba, nawili ki endogenuri substratis Jangvas

xmardeba.

egzogenuri substratis gareSe mikrosomebSi NADPH-is Tavisu-

fali Jangva oTxi sxvadasxva gziT SeiZleba warimarTos [18]:

pirveli sami _ oqsidazuri reaqciaa, meoTxe _ monooqsigenazuri

meqanizmiT endogenuri substratis (XH) Jangvaa. pirvel reaqciaSi 2 moli •−2O -is warmoqmnaze 1 moli NADPH ixarjeba. H2O2-is war-

moqmnisas NADPH eqvimolurad iJangeba, xolo misi siWarbe, romel-

ic •−2O -isa da H2O2-is warmoqmnaze ar ixarjeba, wylis warmoqmnas

xmardeba. egzogenuri substratebis Tanamyofobisas citoqrom P450-s sami tipis oqsidazuri reaqciis katalizeba SeuZlia. amocana aq im-

aSi mdgomareobs, rom gairkves am reaqciebidan romeli mimdinareobs

namdvilad mikrosomebSi da rogoria TiToeuli maTganis wili Jang-

badis aqtivaciis saerTo reaqciaSi.

NADPH + 2O2

NADPH + H + O2

2NADPH + 2H + O2

NADPH + H + O2 + XH

NADP + H + 2O2

_+

NADP + H2O2+

+ 2NADP + 2H2O+ NADP + XOH + H2O

(1)

(2)

(3)

(4)

.

261

Cvens laboratoriaSi inhibitoruli analiziT sarwmunod iqna

naCvenebi [20-22], rom mcenaris mikrosomebSi NADPH-is Tavisufali

Jangva ramdenime oqsidazuri reaqciiT xorcieldeba. maTi daTrgunva

mxolod naxSirbadis monooqsidis, natriumis azidis da p-qlor-

merkuribenzoatis erToblivi moqmedebiT miiReba. maSasadame, mikro-

somebSi NADPH specifikuri (citoqrom P450-damokidebuli) da

araspecifikuri (Fe3+-isa da SH-jgufebis Semcvel fermentebze damo-

kidebuli) gzebiT iJangeba. azidiT araspecifikuri gzis inhibireba

NADPH-citoqrom P450-reduqtazis aqtivobis zrdas iwvevs. amasTan, qsenobiotikis monooqsigenirebasTan SeuRlebuli NADPH-is Jangva

yovelTvis Tavisufal Jangvaze ufro maRali siCqariT mimdinareobs.

es Sedegebi imaze miuTiTeben, rom NADPH-is momxmarebel specifi-

kur da araspecifikur oqsidazebs Soris martivi konkurencia arse-

bobs, rac eleqtronuli aqceptorebis gansxvavebuli redoqs-

potencialebiT unda iyos ganpirobebuli. qsenobiotikis Tanamyofo-

bisas NADPH-is moxmarebis siCqaris zrda imis maCvenebelia, rom

citoqrom P450-is substratTan dakavSireba specifikuri Jangvis

gzis gaZlierebas iwvevs, sadac nikotinamiduri kofermentis mTeli

aRmdgeneli potenciali qsenobiotikis Jangvas xmardeba.

mcenaris mikrosomul fraqciaSi NADPH-is mzardi koncentra-

ciebis Setanisas jer gajerebis momenti, Semdeg ki Jangvis siCqaris

daqveiTeba davafiqsireT [20, 21]. gansazRvrul koncentraciamde

NADPH-is Tavisufali Jangva mixaelis-menTenis gantolebas emorCi-

leba (KM=0.2 mM), anu klasikur fermentul reaqcias warmoadgens.

NADPH-is gamjerebeli koncentraciebisas TviT kofermentiT miRe-

buli inhibireba misi daJanguli formiT an Tavisufali Jangvis ro-

melime produqtiT unda iyos gamowveuli. es movlena citoqrom

P450-is inaqtivaciis Sedegi araa, radgan Seqmnil situaciaSi hemo-

proteini monooqsigenazur aqtivobas inarCunebs (fraqcia maRali

siCqariT Jangavs dimeTilanilins). safiqrebelia, rom NADPH-is si-

Warbisas moqmedebaSi modis garkveuli regulatoruli meqanizmi,

romlis saSualebiTac NADPH-is aRmdgeneli eqvivalentebis ufro

racionaluri realizacia xdeba. aseTi meqanizmis arsebobas sxva mo-

nacemebic adastureben [21, 23]: anaerobul pirobebSi NADPH-is Ta-

visufali Jangvis Sewyvetisas, Jangbadis deficitis dadgomisTanave,

nikotinamiduri kofermentis daJanguli formis aRdgena SeiniSneba.

NADPH-is amgvari „ukuaRdgena” gansakuTrebiT mkafiod sareaqcio

areSi NADP+-is damatebisas SeiniSneba. aseT suraTs adgili ar aqvs

262

citoqrom c-s an K3[Fe(CN)6]-is saSualebiT mikrosomuli redoqs-

jaWvis Suntirebisas da naxSirbadis monooqsidiT an meTiraponiT

citoqrom P450-is blokirebisas. es Sedegebi Tavis mxriv imaze mi-

uTiTeben, rom regulirebis aRniSnuli meqanizmis amoqmedebaSi mo-

nooqsigenazuri sistemis yvela komponenti monawileobs.

gamoricxuli araa, rom NADPH-is racionalur xarjvas eleq-

tronebis ucnobi, lipidis an qinonis tipis aqceptori warmoad-

gendes, romlis funqciac NADPH-is eleqtronebis dasaJang subst-

ratze gadatanaSi mdgomareobs. es hipoTeza sqematurad SeiZleba

Semdegnairad warmovidginoT (nax. 4.4).

NADPH

aRdgenili

aqceptori

daJanguli

aqceptori NADP+

O2 H2O

aRdgenili

aqceptori

daJanguli

aqceptorisubstrati

daJanguli

substrati+ + +

+

+

+

nax. 4.4. NADPH-is aRmdgeneli eqvivalentebis xarjvis regulaciis

savaraudo meqanizmi.

sqemaze Seqcevadi reaqciis wonasworobis erT romelime mxares

gadaxras aqceptoris, substratis an Jangbadis koncentraciuli

cvlilebebi unda ganapirobebdes. mag., Jangbadis deficitisas kofer-

mentsa da aqceptors Soris reaqcia NADPH-is aRdgenis mxares

(marjvnidan marcxniv) unda mimdinareobdes. substratis dabali kon-

centraciisas aqceptoris aRdgenis Semdeg wonasworoba myardeba,

rac substratiT inhibirebis msgavs kinetikur suraTs iZleva. amgvarad, mcenareul mikrosomebSi unda moqmedebdes regulato-

ruli meqanizmi, romelic NADPH-is Tavisufali Jangvisas misi

aRmdgeneli eqvivalentebis gansazRvruli fondis SenarCunebas

uzrunvelyofs. am SemTxvevaSi qsenobiotikis ujredSi SeRweva im

regulatorul signals warmoadgens, romlis saSualebiTac NADPH-

is aRdgeniTi potenciali monooqsigenazuri sistemis mier qsenobio-

tikebis SeuRlebul JangvaSi moixmareba.

263

4.2 monooqsigenazuri sistemis aqtivobis

substratuli regulacia

uaRresad farTo substratuli specifikurobis mqone citoqrom

P450-is katalizuri aqtivobis „samiznes” lipofiluri (hidrofo-

buli) bunebis mqone naerTebi warmoadgenen, romlebic SesaZloa iyv-

nen rogorc Sidaujreduli warmoSobis, aseve qsenobiotikebi. aqedan

gamomdinare, citoqrom P450-is saerTo fondi SeiZleba gaiyos „en-

dogenur” (fiziologiur) da „egzogenur” (detoqsikaciur) reaq-

ciebSi monawileobis mixedviT. adre arsebobda Sexeduleba [24, 25],

romlis mixedviTac ujredSi funqcionirebs citoqrom P450-is sxva-dasxva _ specifikuri (mag., fenoluri naerTebis biosinTezSi mona-

wile) da araspecifikuri (qsenobiotikebis detoqsikaciaSi mona-

wile) formebi. aqve unda SevniSnoT, rom am mosazrebis avtorTa

mcdelobas, gamoevlinaT da amgvarad erTmaneTisagan gaemijnaT cito-

qrom P450-is formebi, romlebic calkeuli procesebis mimarT spe-

cifikurobas gamoamJRavnebdnen, sasurveli Sedegi ar mohyolia.

Cveni TvalsazrisiT, principulad warmoudgenelia, rom ujredSi

winaswar, cal-calke arsebobdes specialurad qsenobiotikis Jang-

visaTvis da „endogenuri” cvlisaTvis gankuTvnili hemoproteinis

formebi. es, upirveles yovlisa, ewinaaRmdegeba cocxali ujredisaT-

vis damaxasiaTebel fundamentur, ekonomiurobis princips. amasTan

dakavSirebiT, saWirod CavTvaleT dagvedgina, fiziologiur da de-

toqsikaciur procesebSi citoqrom P450-is gansxvavebuli formebi

monawileoben, Tu arsebobs hemoproteinis saerTo fondi, romelsac

orive reJimSi funqcionirebis unari gaaCnia.

Cvens laboratoriaSi [24-26] naCvenebi iqna, rom qsenobiotikTa

detoqsikaciaSi da endogenuri cvlis procesebSi SeiZleba citoqrom

P450-is erTi da igive izoforma monawileobdes. amis dasadgenad

soias (Glycine hispida) 10-dRiani nazardebis fesvebidan miRebul mi-

krosomul fraqciaSi SeviswavleT fenoluri naerTebis biosinTezsa

(endogenuri cvlis modeli) da dimeTilanilinis Jangvis (detoqsi-

kaciis modeli) reaqciebs Soris citoqrom P450-is raodenobrivi

ganawilebebi. aRmoCnda, rom mikrosomul suspenziaSi am ori proce-

sis erTdrouli msvlelobisas adgili aqvs orive procesis damu-

xruWebas. amasTan, fenolebis biosiTezis sawyisi nivTierebis – trans-dariCinmJavas citoqrom P450-damokidebuli hidroqsilireba 75-80%-

iT, xolo dimeTilanilinis aseve citoqrom P450-damokidebuli N-

264

demeTilireba mxolod 15-25%-iT iTrguneba. NADPH-damokidebuli Jangvis Km-sidideebis Sedarebam da kinetikurma analizma gviCvena, rom dariCinmJavasagan gansxvavebiT dimeTilanilinis mimarT hemoproteins 3.5-jer maRali swrafva gaaCnia da dimeTilanilini konkurentulad ain-hibirebs dariCinmJava-4-hidroqsilazas (nax. 4.5).

DMA(Km=0.7.10-6 M)

CA(Km=2.5.10-6 M)

1/VO2

1/[S]

nax. 4.5. mikrosomul fraqciaSi dimeTilanilinisa (DMA) da trans-dariCinmJavas (CA) NADPH-damokidebuli Jangvis kinetika lainuiver-

berkis koordinatebSi [24]. (sainkubacio xsnari: 3 ml; 2 mg/ml mikroso-muli cila, 1/15-fosfatis buferi pH 7.4, NADPH _ 6 µM).

fenilalaninisa da trans-dariCinmJavas radioaqtiuri preparatebis gamoyenebiT dadgenil iqna, rom dimetilanilini erTnairi intensivobiT Trgunavs aramarto biosinTezis am sawyisi substratebis gardaqmnas,

aramed fenolur naerTTa jamur reaqciaSi 4-3H-fenilalaninisa da 4-14C-trans-dariCinmJavas radioaqtiuri niSnis CarTvasac [25]. dime-

Tilanilinis garda citoqrom P450-is biosinTezur (dariCinmJava-4-hidroqsilazur) aqtivobaze gavlenas axdenen sxva qsenobiotikebic

(nax. 4.6). sayuradReboa, rom qsenobiotikis mainhibirebeli moqmedeba

korelaciaSi imyofeba fermentis substratisadmi Tvisobisa da am

ukanasknelTa hidrofobulobis xarisxis sidideebTan [26].

amgvarad, sarwmunod SeiZleba CaiTvalos, rom qsenibiotikis

Jangva organuladaa dakavSirebuli citoqrom P450-damokidebul Si-

daujredul procesebTan. am urTierTobis arsi imaSi mdgomareobs,

265

rom `endogenuri” naerTis gardaqmnaSi monawile hemoproteins ucxo

naerTis detoqsikaciis katalizebac SeuZlia.

nax. 4.6. eqsperimentuli monacemebi citoqrom P450-is “endogenuridan” “egzogenur” cvlaze gadarTvis Sesaxeb. wriuli diagramebiT (marcxniv) naCvenebia, rogor Trgunavs TiToeuli qsenobiotiki “endogenur” process – dariCinmJavas hidroqsilirebas. aqve diagramebze (marjvniv) moyvanilia am qsenobiotikebis hidrofobulobis xarisxis (heqsansa da wyals Soris ganawilebis koeficientis) da citoqrom P450-is mimarT maTi Tvisobis (1/Km) sidideebi. suraTidan vxedavT, rom rac ufro hi-drofobulia substrati da metia misi fermentTan Tvisoba, miT ufro meti citoqrom P450 gadaerTveba saerTo Sidaujreduli fondidan qse-nobiotikis hidroqsilirebaze [26].

qsenobiotikis ujredSi SeRweva im adaptur-regulatorul sig-

nals warmoadgens, romelic monooqsigenazas `endogenuridan” `egzo-

genur” cvlaze gadarTvas apirobebs.

ra bunebisaa es signali? rogor xdeba qsenobiotikis `Secnoba”?

cxadia, am kiTxvaze pasuxebi TviT qsenobiotikis fizikur-qimiur

TvisebebSi unda veZioT. amis Sesamowmeblad SeviswavleT gansxvave-

buli qimiuri bunebis substratebis NADPH-damokidebuli Jangvis

dariCinmJava

aminopirini

anilini

p-nitro-

anizoli

N,N-dimeTi-

lanilini

3,4-benzpireni

hidrofobulobis xarisxi

Tvisoba fermentis mimarTdariCinmJavas

hidroqsilirebaSi

qsenobiotikis

hidroqsilirebaSi

citoqrom P450-is saerTo fondis monawileoba

dariCinmJava

aminopirini

anilini

p-nitro-

anizoli

N,N-dimeTi-

lanilini

3,4-benzpireni

hidrofobulobis xarisxi

Tvisoba fermentis mimarTdariCinmJavas

hidroqsilirebaSi

dariCinmJavas

hidroqsilirebaSi

qsenobiotikis

hidroqsilirebaSi

qsenobiotikis

hidroqsilirebaSi

citoqrom P450-is saerTo fondis monawileoba

266

kinetika. sakvlev nivTierebad gamoviyeneT citoqrom P450-damoki-debuli dealkilirebis da aromatuli birTvis hidroqsilirebis

substratebi – dimeTilanilini da aminopirini (N-demeTilireba), p-nitroanizoli (O-demeTilireba) da aromatuli hidroqsilirebisaT-

vis SerCeul iqna CanacvlebulbirTviani (dariCinmJava, anilini) da

kondensirebul birTviani (benzpireni) naerTebi [26].

miRebuli Sedegebidan upirveles yovlisa yuradRebas imsaxurebs

is faqti, rom TiToeuli substrati Jangvis maqsimalur siCqares

mxolod misTvis damaxasiaTebel koncentraciaze aRwevs. amaSi

mJRavndeba substratTa individualoba. Vmax-is mniSvnelobebis mixed-

viT ki maT Soris didi sxvaoba ar aris. JangviTi procesebis grafi-

kebis lainuiver-berkis koordinatebSi gamosaxvisas konkurentuli

inhibirebis klasikuri suraTi miiReba. es ki imas niSnavs, rom qi-

miur Tvisebebsa da JangviTi reaqciis tipis gansxvavebis miuxedavad

substratebs Soris fermentis aqtiuri centrisaTvis martivi kon-

kurencia arsebobs. es konkurencia Tavis mxriv substratebis fizi-

kur Tvisebebze, kerZod maT hidrofobulobis xarisxze aRmoCnda da-

mokidebuli.

hidrofobulobis xarisxisa da fermentisadmi swrafvis zrdis

mixedviT sakvlevi substratebi Semdegi TanmimdevrobiT ganlagdnen:

NHH

CH CHCOOH

O CH3 NCH3CH3N

N

NCH3

CH3CH3

CH3

p-nitro-anizoli

N,N-dimeTil-

anilini

amino-

pirini

anilini dariCin-

mJava

3,4-benzpireni

< < < < <

sayuradReboa, rom am or sidides Soris mxolod martivi kore-

lacia ki ar vlindeba, aramed fermentis substratisadmi Tvisobis

sidide wrfiv damokidebulebaSia substratis hidrofobulobis xa-

risxidan kuburi fesvis mniSvnelobasTan. es ki imaze miuTiTebs, rom

molekulis polaroba warmoadgens erT-erT (da ara erTaderT) Zi-

riTad parametrs im Tvisebebidan, romlebic citoqrom P450-Semcveli monooqsigenazas mier nivTierebis Jangvas ganapirobeben.

sxva malimitirebel faqtorebs unda miekuTvnebodes substratSi is

qimiuri bma, romelic hidroqsilirebisas iSleba; lipidebTan da

267

fermentis aqtiur centrebTan ganlagebul aminomJavas naSTebTan sub-

stratis molekulis urTierTqmedeba da a.S.

angariSgasawevia is faqtic, rom zemoTaRniSnul kanonzomierebas

erTnairad emorCileba rogorc qsenobiotikebi, aseve mcenareuli

citoqrom P450-is bunebrivi endogenuri substratebi. rogorc Cans hemoproteinis mier nivTierebis `substratad Secnoba~ xdeba misi

fizikur-qimiuri Tvisebebis da ara misi endogenuri Tu egzogenuri

warmoSobis mixedviT.

citoqrom P450-is substratuli specifikurobis fenomeni mniSv-

nelovnad ganisazRvreba TviT fermentis aqtiuri centris Taviseburi

aRnagobiT. sadReisod dadgenilia, rom citoqrom P450-is hemi kra-teris magvari struqturis mqone apofermentis siRrmeSi mdebareobs

da aqtiuri centris Fe2+-dan wylis fazamde manZili 1.4 nm-s aRwevs [27, 28]. hemi wylis fazisagan daSorebulia hidrofobuli jibiT,

romelic daviwroebasTan erTad hidrofobulobis mzard gradients

qmnis. aseTi gradienti SeiZleba warmoqmnan fosfolipidebma, an apo-

citoqromis SemadgenlobaSi myofma hidrofobuli aminomJavebis naS-

Tebma. gradientis daniSnulebaa substratis gadatana wylidan fer-

mentis aqtiuri centris mimarTulebiT. lipofiluri substrati, cito-qrom P450-Tan miaxloebisTanave, magnitiviT miizideba am gradientis

mier, advilad moswydeba wylis fazas da hemoproteinis aqtiuri

centrisaken gadaitaneba. amasTan, rac metad hidrofobulia sub-

strati, miT ufro swrafad miaRwevs igi aqtiur centrs da Sesaba-

misad, maRali iqneba misi Tvisoba fermentis mimarT. monooqsigenazu-

ri ciklis ganxorcielebis Semdeg Jangvis produqti, sawyis sub-

stratTan SedarebiT, ukve polarul molekulas warmoadgens, amitom

misi molekula igive gradientis arsebobis gamo gamoidevneba cito-

qrom P450-is aqtiuri centridan. xatovani Sedareba rom vixmaroT, polaruli produqti TiTebSua moqceuli alublis kurkis msgavsad

dasxlteba aqtiuri centridan da wylovan fazaSi gadainacvlebs.

4.3 citoqrom P450-is regulacia induqciis doneze

citoqrom P450-is raodenobis regulacia SesaZlebelia misi in-

duqciiT. mcenareul ujredSi citoqrom P450-is induqciis faqtore-bia monooqsigenazas substratebi, paTogenebi, dazianeba-dabereba da

ganaTeba [29, 30]. qsenobiotikebi mcenareuli citoqrom P450-is Zlier induqtorebs

268

warmoadgenen. miwavaSlas tuberebis anaTlebSi naCvenebia eTanolis,

fenobarbitalis, sxvadasxva herbicidebis moqmedebiT gamowveuli ci-

toqrom P450-is Semcvelobis zrda [31, 32]. unda aRiniSnos, rom

fenobarbitali, rogorc induqtori, gansxvavebulad moqmedebs cito-

qrom P450-is substratul specifiurobaze. magaliTad, Tu dariCinm-

Java-4-hidroqsilazuri aqtivoba 1.5-jer izrdeba, laurinmJavas Si-

dajaWvuri hidroqsilirebis intensivoba 26-jer matulobs [33].

sainteresoa agreTve, rom ZuZumwovrebis laurinmJava-ω-hidroqsi-lazas specifiuri induqtori _ qlofibrati mcenareebSi igive mJa-

vas SidajaWvur hidroqsilazas inducirebs. qsenobiotikiT induqcia

induqtoris qimiuri bunebisa da koncentraciis garda, damokidebu-

lia preinkubaciis periodze, mcenaris saxeobaze, mis organoze,

asakze, ganaTebaze da sxva faqtorebze. Cvens laboratoriaSi miRebuli eqsperimentuli Sedegebis mixed-

viT [34], citoqrom P450-is induqtorebs warmoadgenen rogorc en-dogenuri (dariCinmJava), ise egzogenuri (dimeTilanilini, 3,4-benz-

pireni) substratebi. qsenobiotikebis induqciuri efeqti gacilebiT

aRemateba dariCinmJavaTi miRebul Sedegs. es endogenuri substrati

72-sT-iani preinkubaciis Semdeg citoqromebis P450-is da P420-is matebas mxolod 1.3_1.4-jer iwvevs. drois igive monakveTSi dimeTi-

lanilinis moqmedebiT citoqrom P450-is Semcveloba sakontrolo

variantTan SedarebiT 3_4-jer izrdeba.

gansxvavebul efeqts amJRavnebs 3,4-benzpireni. misi moqmedebiT

citoqrom P450-is zrda TiTqmis ar SeiniSneba, maSin rodesac

citoqrom P420-is mkveTr matebas aqvs adgili. miuxedavad amisa,

induqtorze gazrdil mcenareebSi citoqromebis P450-is da P420-is jamuri Semcveloba erTnairad izrdeba. safiqrebelia, rom Tavdapir-

velad adgili aqvs qsenobiotikis moqmedebiT citoqrom P450-is induqcias, xolo Semdeg inducirebuli hemoproteinis P420-ad kon-versia xdeba. aRniSnuli mosazrebis sasargeblod metyvelebs dime-

Tilanilinisa da 3,4-benzpirenis induqciuri efeqtebisa da maTi

Jangvis Sedegad warmoqmnili metabolitebis reaqciisunarianobis

Sedareba. aRmoCnda, rom 3,4-benzpirenis NADPH-damokidebuli mikro-

somuli Jangvis Sedegad formirdeba Sesabamisi diolebi da qinonebi. Tavis mxriv, cnobilia, rom qinonebs nairgvari Jangbadovani radi-

kalebis generirebis unari aqvT. dimeTilanilinis Jangvis arc erTi

SesaZlo produqti (monomeTilanilini, anilini, p-hidroqsi-dimeTilanilini da sxv.) ar iZleva aseT „agresiul“ produqtebs.

269

igives adasturebs 3,4-benzpirenisa da dimeTilanilinis hidroqsili-

rebis paralelurad mimdinare lipidTa zeJanguri Jangvis inten-

sivoba, romelic 3,4-benzpirenis Jangvisas 3-jer ufro metad stimu-

lirdeba, vidre dimeTilanilinis SemTxvevaSi [34]. amrigad, 3,4-benzpirenis Jangvis dros warmoqmnili reaqciisuna-

riani intermediatebi inducirebuli citoqrom P450-is P420-ad gardaqmnas iwveven da citoqrom P450-is mimarT 3,4-benzpirenis

induqciuri aqtivoba mkafiod imitom ar vlindeba, rom efeqti

fermentis inaqtivaciiTaa ekranirebuli.

sxvagvarad rom iTqvas, qsenobiotikebis induqciuri efeqti mniSv-

nelovnadaa damokidebuli maTi Jangvis produqtebis qimiur bunebaze.

rac ufro naklebia radikalwarmoqnis unari, miT metad gamoiwveven

isini citoqrom P450-is Semcvelobis zrdas.

mcenareuli citoqrom P450-is induqtorebia misi endogenuri

substratebic, rasac mowmobs maSa-lobios aRmonacenebSi induqciis

Seswavla [34]. naCvenebia, rom dariCinmJava 1,8-jer, xolo geranioli

2,7-jer iwvevs citoqrom P450-is matebas. induqciis efeqti Zlier

blokirdeba cikloheqsimidis an levulinatis Tanaobisas cilis da

hemis sinTezis daTrgunvis gamo. induqtoris rolis Sesruleba SeuZlia zogierTi liTonis

ionsac. miwavaSlas tuberebis anaTlebSi miRebulia citoqrom P450-is induqcia Mn2+- da Fe3+

-ionebiT [31]. aseTive efeqtia miRebuli

kartofilis norC fesvebze HgCl2 da CdSO4-is moqmedebiT: hemo-

proteinis raodenoba Cd2+-ionebiT 50%-iT, xolo Hg2+

-ionebiT -

100%-iT izrdeboda [36]. paTogenuri sokoebi an elicitorebi (paTogenuri an arapaTo-

genuri sokoebidan miRebuli sxvadasxva fraqciebi) dasnebovnebul

mcenareSi citoqrom P450-is matebas iwveven. citoqrom P450-Semcveli monooqsigenazebi akatalizeben flavonoiduri da terpe-

noiduli fitoaleqsinebis biosinTezs. amdenad, citoqrom P450-is induqcia mocemul SemTxvevaSi paTogenebis toqsinebis moqmedebaze

mcenaris sapasuxo reaqcias warmoadgens. paTogenebiT an elici-

torebiT induqcia specifiuria. magaliTad, Phytophtora megaspermas-is elicitori gansxvavebulad moqmedebs oxraxuSis ujredul

kulturaSi fenilpropanoiduri gzis citoqrom P450-damokidebul

aqtivobebze. miRebulia, rom inducirebadia am biosinTezis mTavari

fermenti _ dariCinmJava 4-hidroqsilaza, agreTve Sikimat-3'-hidroqsilaza, xolo flavonoid-3'-hidroqsilaza ar inducirdeba

270

[37-39]. induqciuri efeqtebi aRwerilia sxva SemTxvevebSic. dazi-

aneba an "dabereba", e.i. mcenareuli qsovilebis anaTlebis inkubacia

tenian atmosferoSi an kargad aerirebul xsnarebSi, mraval-

ricxovani procesebis, maT Soris JangviTi fermentebis biosinTezis

aqtivacias iwvevs. am dros inducirdeba citoqrom P450-ic, rasac cxadyofs dariCinmJava-4-hidroqsilazas mateba miwavaSlas anaTlebis

"daberebisas" [38]. sainteresoa, rom qsenobiotikiT induqciisas,

"daberebis" SemTxvevaSic dariCinmJava-4-hidroqsilazasTan erTad,

laurinmJavas SidajaWvuri hidroqsilazac inducirdeba [40]. citoqrom P450-is sinaTliT induqcia pirvelad naCvenebi iqna

bardis eTiolirebuli nazardebis mokletalRiani TeTri sinaTliT

dasxivebis Sedegad, dariCinmJava-4-hidroqsilazas aqtivobis 5-jera-

di matebis magaliTze [41]. SemdgomSi igive efeqti aRmoaCnda wiTeli

da grZeltalRiani wiTeli sinaTlis impulsebsac [42], amasTanave

aRmoCnda, rom dariCinmJava-4-hidroqsilazas zrdis fonze citoqrom

P450-is jamuri Semcveloba mxolod umniSvnelod imatebs. analo-

giuri efeqti SeiniSneba cximovan mJavaTa hidroqsilazebis mimarTac:

mikrosomuli hidroqsilazebis mTliani raodenoba mcirdeba kidevac, Tumca am dros ω-hidroqsilazuri avtivoba gazrdilia [43, 44].

unda aRiniSnos, rom hemoproteinis sinaTliT induqciasTan dakav-

SirebiT erTmaneTisagan radikalurad gansxvavebuli eqsperimentuli

monacemebi arsebobs. magaliTad, sibneleSi gazrdili oxraxuSis

ujredul kulturaSi citoqrom P450-is raodenoba araregis-

trirebadia, xolo 24 sT-iani dasxivebis Semdeg maqsimums aRwevs

[45]. amis sapirispirod naCvenebia, rom maSa-lobios samdRiani

nazardebis sinaTleze gadatanisas citoqrom P450-is raodenoba

swrafad klebulobs, rac sinaTleze hemis ngreviTaa gamowveuli

[46]. naTesebis sibneleSi dabruneba kidev erTxel iwvevs citoqrom

P450-is dagrovebas. aseTi urTierTgamomricxavi monacemebis arseboba imiT aixsneba, rom citoqrom P450-is yvela izoforma araa sinaTliT

inducirebadi [29, 30]. Cveni monacemebis mixedviT [47, 48], sinaTleze gazrdili mcenaris

mikrosomebis STanTqmis speqtrSi, 434_438 nm-is ubanSi, intensiuri

STanTqmis maqsimumi Cndeba. es piki iwvevs 420 da 450 nm-ebze

maqsimumebis gadafarvas, rac citoqromebis raodenobriv gansazRvras

aZnelebs. savaraudoa, rom aseTi eqstinqcia swored im nawilakebs

axasiaTebT, romlebic sinaTleze citoqrom P450-is hemis daSlis

Sedegad warmoiqmnebian. eTiolirebul mcenareebSi, romelTa fesvebi

271

bunebrivTan ufro miaxloebul pirobebSi viTardebian, citoqromuli

komponentebis Semcveloba yovelTvis metia, vidre sinaTleze

gazrdil imave asakis mcenareebSi.

4.4 monooqsigenazuri sistemis aqtivobis regulacia

membranul doneze

4.4.1 membranuli fosfolipidebis gavlena citoqrom P450-is

meoreul struqturaze da aqtivobaze

citoqrom P450-Semcveli monooqsigenazuri sistemis mniSvnelo-

vani Tavisebureba hidrofobuli Tvisebebis flobaa, rac membranaSi

misi CaSenebis saSualebas iZleva. amasTan dakavSirebiT bunebrivad

ibadeba kiTxvebi: rogoria membranis roli hemoproteinis funqcioni-

rebaSi da raSi mdgomareobs am evoluciuri gamogonebis biologiuri

azri? ra doniT SeiZleba am ferments mivusadagoT sxva fermentebi-

sadmi (mag., ATP-azebisadmi an dehidrogenazebisadmi) sayovelTaod

aRiarebuli warmodgenebi, romelTa Tanaxmadac membranis fosfoli-

pidebs wylovan fazaSi araaqtiuri formiT myofi fermentebi kon-

formaciulad aqtiur mdgomareobaSi gadahyavT?

mocemul kiTxvebze sapasuxod Seswavlil iqna α-spiralis Semc-

veloba maRali sisufTavis izolirebul da fosfolipidur biSreSi

CaSenebul hemoproteinSi [49]. paralelurad isazRvreboda cito-

qrom P450-is hidroqsilazuri aqtivoba reaqciebSi, romlebSic aq-

tiuri Jangbadis donorad kumolis hidrozeJangi gamoiyeneboda. aR-

moCnda, rom izolirebul citoqrom P450-s xsnarSi gacilebiT nak-

lebi hidroqsilazuri aqtivoba gaaCnia, vidre gasufTavebis Semdeg

xelovnur liposomur membranaSi CaSenebul mikrosomul hemoprote-

ins. am dros fermentis reaqtivireba da sawyisi hidroqsilazuri

aqtivobis sruli aRdgena xdeba. amasTan, misi konformaciis arsebiT

cvlilebebs adgili ar aqvs. korelacia ar vlindeba cilis mole-

kulaSi α-spiralis Semcvelobis cvlilebasa da liposomur membra-

naSi CaSenebisas misi aqtivobis zrdas Soris. es monacemebi im war-

modgenis sawinaaRmdegod metyveleben, romlis Tanaxmadac am membra-

nuli cilis konformaciaSi unda arsebobdes mniSvnelovani gansxvave-

bebi solubilizebul da membranadakavSirebul mdgomareobaSi. aqedan

gamomdinare, fosfolipidis reaqtivaciul unars Sesabamisad sxva

axsna unda miscemoda.

272

hemoproteinis Tvisebebze fosfolipiduri biSris gavlenis sis-

tematurma kvlevam [49] aCvena, rom membranaSi citoqrom P450-is Ca-Senebisas cilis molekulis sixiste izrdeba. xsnarTan SedarebiT

xelovnur membranaSi CaSenebuli hemoproteini aRdgenisas, an II-tipis

substratTan dakavSirebisas Tavis konformaciul mdgomareobas ga-

cilebiT naklebad icvlis.

mkveTrad izrdeba lipidur biSreSi CaSenebuli hemoproteinis

Termostabilurobac. proteoliposomebSi TiTqmis sruladaa gamq-

rali xsnarSi arsebuli, fermentisaTvis damaxasiaTebeli fazuri

denaturaciuli gadasvla; e.i. membranaSi fermentis CaSenebas Tan

axlavs hemoproteinis molekulis stabilizacia da misi konforma-

ciuli Zvradobis Semcireba.

hemoproteinze fosfolipiduri biSris mastabilizebeli moqmede-

bis efeqti naTlad uCvenebs fermentis xsnadi formidan membranulze

gadasvlis uaRresad did mniSvnelobas.

citoqrom P450 labilur, swrafad inaqtivirebad fermentebs ga-

nekuTvneba. izolirebul hemoproteins xsnarSi aRdgenil mdgomareo-

baSi inaqtivaciis umaRlesi siCqare gaaCnia [48]. substratis damate-

biT inaqtivaciis siCqare umniSvnelod mcirdeba. membranis Semadgen-

lobaSi hemoproteini Zlier stabiluria rogorc aRdgenil, aseve

daJangul mdgomareobaSi. mastabilizebeli moqmedebis modelireba

advilad SeiZleba izolirebul hemoproteinze fosfolipidebis da-

matebiT. amasTan, naTlad Cndeba maTi specifikuroba. damcveli efeq-

ti gaaCnia mikrosomuli fosfolipidebis narevs da fosfatidileTa-

nolamins. aseT cdebSi fosfatidilqolins damcveli unari ar gaaC-

nia, magram Tu hemoproteini winaswaraa CaSenebuli fosfatidilqo-

linur liposomaSi, maSin am fosfolipidsac eqneba gamosaxuli

damcveli moqmedeba. am Sedegebis axsna SeiZleba, Tu davuSvebT, rom

fosfolipidis muxti moqmedebs mxolod citoqromTan misi ur-

TierTqmedebis siCqareze. uaryofiTad damuxtuli fosfolipidebi

cilasTan ufro swrafad urTierTqmedeben. fosfatidileTanol-

amin_citoqrom P450-is kompleqsis warmoqmna swrafad xdeba da ami-

tom am fosfolipidis mastabilizebeli moqmedebac ucbad, citoqrom

P450-is Semcvel xsnarSi misi CamatebisTanave vlindeba. fosfati-

dilqolinis SemTxvevaSi aucilebelia winaswari inkubacia, romlis

drosac cila unda CaSendes fosfolipidur biSreSi da mxolod

amis Semdeg lecitinic gamoamJRavnebs Tavis damcvel moqmedebas.

eukariotul ujredebSi hemoproteinis membranuli forma SeiZ-

273

leba arsebobdes substratis gareSe. prokariotebSi xsnadi cito-

qrom P450 substratis gareSe swraf inaqtivacias ganicdis, da Sesa-bamisad, substratis koncentraciis Semcirebisas iSleba. euka-

riotebSi arsebobs am sistemis aqtivobis regulirebis sxva SesaZ-

leblobebic. ujredSi hemoproteinis Semcveloba ar Semcirdeba mas-

Si hidroqsilirebis substratis koncentraciis daqveiTebisas, anu

fosfolipidis daxmarebiT citoqromi „gamodis” substratis mxridan

kontrolidan [49].

4.4.2 membranuli fosfolipidebis roli NADPH-citoqrom

P450-reduqtazisa da citoqrom P450-is urTierTqmedebaSi

endoplazmur membranebSi arsebuli ZiriTadi redoqs-jaWvi, ro-

melic NADPH-is mimarT mkacr specifikurobas amJRavnebs, fosfo-

lipidur matriqsTanaa dakavSirebuli. jaWvis sawyisi komponentis _

NADPH-damokidebuli flavoproteinis aqtivobas Cveulebriv afase-

ben an egzogenurad damatebuli eleqtronTa aqceptorebis, an endo-

genuri aqceptoris _ citoqrom P450-is aRdgenis siCqareebis mixed-viT. meore kriteriumi yvelaze sarwmunod asaxavs monooqsigenazuri

tipis reaqciebSi flavoproteinis monawileobas.

anaerobul pirobebSi citoqrom P450-is aRdgenis siCqaris gazom-va im faqts emyareba, romlis Tanaxmadac hemoproteinis mier eleq-

tronTa aqceptirebis siCqare (konstanta _ 10_20 wT-1) gacilebiT

naklebia aRdgenili citoqromis CO-sTan urTierTqmedebis siCqareze

(konstanta – 1.7.104 wT-1). erTi katalizuri ciklis Sesasruleblad

monooqsigenaza or eleqtrons saWiroebs da pirveli eleqtronis

aqceptireba mowmdeba anaerobul pirobebSi citoqrom P450-is CO-sTan kompleqsis warmoqmniT [49].

mikrosomul suspenziaSi NADPH-is damateba citoqrom P450-is srul aRdgenas iwvevs. amasTan, reduqtazuli reaqcia orfazianobiT

xasiaTdeba. pirveli (swrafi) faza 1_2 wm-s ikavebs da anaerobiozis siRrmeze damokidebuli araa. am stadiaze citoqrom P450-is aRdge-nis siCqare oTaxis temperaturaze 2_8 nmoli.wm-1 mg cilaze far-

glebSia.

meore stadiaze citoqrom P450-is aRdgenis siCqare Zlier daba-

lia. misi dafiqsireba ramdenime wuTis ganmavlobaSi SeiZleba. es

stadia mikrosomul suspenziaSi Jangbadis Semcvelobazea damoki-

274

debuli, romelsac hemoproteinis kvlav daJangva da reaqciaSi mona-

wile citoqrom P450-is molekulaTa raodenobis Semcireba SeuZlia.

NADPH-citoqrom P450-reduqtazuli reaqciis orfazianoba SeiZ-

leba aixsnas ori mizeziT:

1. substratTan dakavSirebuli da Tavisufali citoqrom P450-is aRdgenis gansxvavebuli siCqareebiT;

2. mikrosomul fraqciaSi hemoproteinis sxvadasxva formebis arse-

bobiT, romelTac substratisa da reduqtazis mimarT gansxvave-

buli Tvisoba gaaCniaT.

I-tipis substratebis dakavSireba reduqtazuli reaqciis sawyisi

fazis siCqaris mniSvnelovan zrdas iwvevs. amasTan, reaqciis swraf

fazaSi aRdgenili citoqrom P450-is raodenoba korelaciaSia hemo-

protein_substratis kompleqsis SemcvelobasTan. varaudoben, rom

citoqromTan I-tipis substratTa dakavSirebis Sedegad hemoprotei-

nis eleqtronuli struqtura iseT cvlilebebs ganicdis, romelic

aumjobesebs mis mier eleqtronis aqceptorebis unars da aCqarebs

reduqtazuli reaqciis msvlelobas.

substratis Tanamyofobisas NADPH-reduqtazuli reaqciis aCqa-

reba SeiZleba aixsnas citoqrom P450-is redoqs-potencialis mate-

biT, an reduqtazis aqtivaciis energiis daqveiTebiT. es imiT unda

iyos gamowveuli, rom hemoproteini da misi reduqtaza „kom-

partmentalizebulia” membranis fosfolipiduri matriqsis izolire-

bul zonaSi, romlisTvisac damaxasiaTebelia temperaturuli fazuri

gadasvlebi. amitom am zonas reduqtazis aqtivaciis energiis Semci-

rebis unari unda gaaCndes. Tavis mxriv, I-tipis substratebic mem-

branuli fosfolipidebis temperaturuli fazuri gadasvlebis Sem-

cirebis unars floben. amitom reduqtazuli reaqciis sawyis siCqa-

reze substratTa mastimulirebeli moqmedeba eWvs ar unda iwvevdes.

amis sasargeblod mowmobs monacemebi fosfolipidur matriqsSi sub-

stratebis mier inducirebuli konformaciuli cvlilebebis Sesaxeb,

romelTa Sedegadac izrdeba citoqromisa da misi reduqtazis efeq-

tur SejaxebaTa ricxvi [48].

I-tipis substratebiT NADPH-reduqtazuli reaqciis stabili-

zaciis fenomenis gadauWarbeblad Sefaseba Zalian Znelia, radgan

monooqsigenazuri sistemiT mravali qsenobiotikis (pirvel rigSi

barbituratebisa da pirazolinebis warmoebulebis) hidroqsilirebis

intensivoba arsebiTad damokidebulia citoqrom P450_substratis kompleqsis NADPH-reduqtaziT aRdgenaze.

275

hidroqsilirebis reaqciebze mastimulirebel gavlenas axdens

Mg2+. aRmoCnda, rom am efeqtis arsi NADPH-reduqtazis gaaqtiu-

rebaSi mdgomareobs. garda amisa, SeniSnulia D2O-s mainhibirebeli

moqmedeba reduqtazuli reaqciis siCqareze da eTilmorfinis N-

demeTilirebaze.

xanmokle moqmedebis barbituratebis SemTxvevaSi, romelTa kon-

centraciebic mkurnalobis dros 5.10-5 M-s ar aRemateba, reduqta-zuli reaqciis aqtivoba mniSvnelovan faqtors warmoadgens maTi

metabolizmis regulaciaSi. RviZlSi wamlebis metabolizmis Seswav-

liT dadginda, rom aminopirinis N-demeTilireba mWidro korela-

ciaSia NADPH-citoqrom P450-reduqtazuli reaqciis aqtivobasTan

da ara TviT citoqrom P450-is raodenobasTan. korelaciis gamovle-

nis mizniT Catarebulia SedarebiTi analizi NADPH-reduqtazuli

reaqciis siCqares, sxvadasxva substratTa hidroqsilirebasa da am

procesTa aqtivaciis energiebs Soris. aRmoCnda, rom aminopirinis N-

demeTilirebis maqsimaluri siCqare (7 nmoli.wT-1 mg cilaze 37°C-ze) da am procesis aqtivaciis energia (53.6 kj/moli), NADPH-

reduqtazuli reaqciis imave maCveneblebs utoldeba (Vmax=7.26 nmo-li.wT-1 mg cilaze, aqtivaciis energia _ 53.3 kj/moli). es monacemebi sarwmuno mtkicebulebaa imis Sesaxeb, rom reduqtazuli reaqcia amino-pirinis metabolizmis siCqaremalimitirebeli rgolia [48].

ganxiluli faqtebi gansakuTrebul azrs SeiZenen, Tu reduq-

tazis funqcionirebaSi fosfolipiduri komponentis rols gaviT-

valiswinebT. fermenti maRal mgrZnobiarobas amJRavnebs mikrosomebis

fosfolipiduri biSris da maT mier warmoqmnili hidrofobuli zo-

nebis yovelgvari dazianebis mimarT. SeniSnulia reduqtazuli aqti-

vobis mkveTri daTrgunva fosfolipazebis C-sa da D-s, triton X-100-is da dimeTilsulfoqsidis dabali koncentraciebis gamoyenebi-

sas. cnobilia reduqtazaze sawinaaRmdego efeqtis magaliTebic,

romlebic gamowveulia mikrosomis fosfolipiduri komponentis hi-

drofobulobis zrdiT Mg2+-is Tanamyofobisas.

rekonstruirebuli monooqsigenazuri sistemis funqcionirebaSi

fosfatidilqolinis rolis Seswavlam aCvena misi absoluturi au-

cilebloba citoqrom P450–CO kompleqsze eleqtronebis gadatanaSi.

lecitinze damokidebuli aRmoCnda NADPH-reduqtazuli reaqciis

swrafi (sawyisi) faza, romelic izolirebuli citoqrom P450-is maqsimalur aqtivobasTanaa SeuRlebuli. safiqrebelia, rom es damo-

kidebuleba dakavSirebulia citoqromis mier eleqtronTa aqceptire-

276

bis unarTan, radgan aRwerilia citoqrom P450-is aRdgenis SesaZ-lebloba qsantin/qsantinoqsidazuri sistemiT, mxolod fosfoti-

dilqolinis Tanamyofobisas. TavisTavad substratis misaerTeblad

citoqrom P450 fosfolipids uSualod namdvilad ar saWiroebs,

magram misi gavleniT izrdeba fermentis substratTan Tvisoba. fer-

mentis konformaciuli cvlileba inducirdeba fosfolipidis an

substratis ganawilebiT arawylovan fazaSi. wamlebis metabolizmis

kvlevisas naCvenebi iqna lipidebis monawileoba substratis ganawi-

lebaze mikrosomebsa da wyalxsnarebs Soris. dadgenil iqna agreTve,

rom buferisa da heptanis xsnarSi N,N-dimeTilaminis umuxto war-

moebulebi umTavresad hidrofobul, muxtianebi ki wylovan fazaSi

nawildebian [49].

substratis ganawilebis koeficientis gansazRvrisas aRmoCnda,

rom igi damokidebulia lipidis koncentraciaze; garda amisa, cito-

qrom P450-is aRdgenis xarisxic korelaciaSia lipidis koncentra-

ciasTan, rac imaze miuTiTebs, rom es ukanaskneli gavlenas axdens

citoqrom P450-substratis kompleqsis warmoqmnaze. dasaSvebadaa

miCneuli lecitinis araspecifikuri rolic, romelic xsnadi mo-

nooqsigenazuri sistemis komponentTa efeqturi urTierTqmedebisaT-

vis aucilebeli hidrofobuli zonis SeqmnaSi mdgomareobs. es va-

raudi, upirveles yovlisa, gamomdinareobs im ufro hidrofobul

dioleil- da dilauroil-fosfatidilqolinebis Semcvel sistemaTa

umaRlesi aqtivobebidan, romlebic RviZlis lecitinebis Semadgen-

lobaSi ar Sedian. dadgenilia, rom zogierT detergents, romlebic

wylovan fazaSi maRalhidrofobuli Sida zedapiris mqone micelebs

warmoqmnian, unari aqvT rekonstruirebul sistemaSi Secvalon fos-

folipidebi. amasTan dakavSirebiT gasaTvaliswinebelia, rom xsnad

rekonstruirebul sistemaSi, sadac lecitinis Tanamyofobisas mem-

branismagvari struqturebi ar warmoiqmnebian, monooqsigenazisa da

fosfolipidebisagan Semdgari, advilad disocirebadi kompleqsebis

arseboba mainc savsebiT mosalodnelia.

sayuradReboa, rom rekonstruirebuli monooqsigenazuri sistemis amoqmedeba mxolod detergentebis saSualebiT gamoyofil reduqtazas SeuZlia. izolirebuli fermentis trifsiniT damuSaveba reduqtazas am Tvisebas ukargavs, magram citoqrom c-s aRdgenis unars unarCunebs. naC-venebia, rom proteolizi ferments polipeptiduri jaWvis nawils

acilebs. Tvlian, rom reduqtazis molekulis es nawili fosfoli-

pidur matriqsSi Taviseburi „Ruzis” rols asrulebs da NADPH-

277

reduqtazas membranaSi lateraluri gadamoZravebis saSualebas aZ-

levs. gamovlenilia erTi, metad sayuradRebo faqtic: mikrosomul

membranaSi SesaZlebelia detergentuli reduqtazis damatebiTi CaSe-

neba. amis Sedegad monooqsigenazis katalizuri aqtivoba mniSvnelov-

nad izrdeba. amasTan dakavSirebiT Camoyalibebulia reduqtazisa da

citoqrom P450-is kompleqsis e.w. „xisti” organizaciisa da ur-

TierTqmedebis alternatiuli sqema, romelSic flavoproteinis erT

molekulaze hemoproteinis 12_20 molekula modis [48].

mocemuli fermentebi membranaSi Tu marTlac iZlevian steqio-

metrul kompleqss, maSin maTi Tanafardoba yvela SemTxvevaSi mudmi-

vi unda iyos. miuxedavad amisa, monooqsigenazuri sistemis komponen-

tebze amis Tqma ar SeiZleba. mag., axaldabadebul virTagvebSi

NADPH-citoqrom P450-reduqtaza ufro adre Cndeba, vidre cito-qrom P450. cxovelSi fenobarbitalis Seyvanis Semdeg reduqtazis

Semcveloba RviZlSi dauyovnebliv matulobs, maSin rodesac cito-

qrom P450-is raodenobis zrda mxolod ineqciidan 5 sT-is Semdeg iwyeba. es faqtebi ar gamoricxaven kompleqsebis arsebobis SesaZ-

leblobas. kompleqsis calkeul komponentebs SeuZliaT erTmaneTisa-

gan damoukideblad sinTezireba, xolo kompleqsebad maTi „Seg-

roveba” SeiZleba mogvianebiT moxdes.

Catarebul eqsperimentul monacemebze dayrdnobiT stieri, sakme-

ni da estabruki [50] mividnen daskvnamde, romlis Tanaxmadac cito-

qrom P450 reduqtazianad lokalizebulia mikrosomuli fosfoli-

pidebis „specializebul” zonaSi, romelic fosfolipiduri mat-

riqsis ZiriTadi masisagan gansxvavdeba Tavisi dabali aqtivobiT da

Txevadkristaluridan Txevad mdgomareobaSi fazur gadasvlas ganic-

dis. navaraudebia, rom am zonaSi imyofeba mikrosomuli fosfolipi-

dis araumetes 20%-isa, romlebic monooqsigenazis komponentebs So-

ris kontaqtis damyarebis garda, citoqrom P450-isaken lipofiluri

substratebis lateralur difuziasac uzrunvelyofen.

igive avtorTa mier miRebuli monacemebis safuZvelze warmodge-

nil iqna mikrosomul membranaSi monooqsigenazuri kompleqsis orga-

nizaciis modeli [50]. citoqrom P450-isa da NADPH-citoqrom P450-reduqtazis cnobili molekuluri masebidan gamomdinare, komp-

leqsSi am komponentTa molekuluri Tanafardoba 20 : 1-is tolia.

gamoTvlilia, rom swraf fazaSi citoqrom P450-is 50%-iT aRsadge-

nad dabal temperaturaze aucilebelia reduqtazis yoveli mole-

kulis 8_12 molekula citoqromiT garemocva. fosfolipidur ma-

278

triqsSi monooqsigenazuri sistemis komponentTa am kompleqss mog-

vianebiT, rogorc aRniSnuli iyo, „klasteri” ewoda. varaudoben,

rom temperaturis cvlilebisas „klasteris” zoma varirebs komp-

leqsis SigniT da gareT citoqrom P450-is molekulaTa cvlis

gansxvavebuli siCqaris gamo.

reduqtazuli reaqciis neli fazis Sefasebisas im garemoebas iT-

valiswineben, rom igi axasiaTebs reduqtazisagan mocilebuli (e.i.

„klasteris” gareT myofi) citoqrom P450-is aRdgenas da Tvlian,

rom hemoproteinis mocemuli molekulebisaTvis SesaZlebelia mi-

krosomul fosfolipidebSi lateraluri difuzia. neli fazis ma-

Rali aqtivaciis energia (fazuri gadasvlis zeviT temperaturaze),

rogorc Cans, unda aixsnas „klasteris” SigniT da gareT citoqrom

P450-is molekulebis cvliT [51].

saWirod migvaCnia mokled SevexoT fosfolipidebis monawileo-

bas estabrukisa da hildenbrandtis sqemis Semdgom reaqciebSi. mono-

oqsigenazur reaqciebSi NADH-isa da NADPH-is calkeulad an

erTdroulad gamoyenebisas citoqrom b5-is Jangva-aRdgenis kinetikis

analizma SesaZlebeli gaxada gakeTebuliyo daskvna, rom am cito-

qromis aRdgenili forma aris sammagi kompleqsisaTvis meore eleq-

tronis aucilebeli donori. amasTan, rekonstruirebul sistemaze

da mikrosomul membranebSi redoqs-jaWvebis ganlagebis sqemebze

pirdapiri monacemebis Tanaxmad miRebulia, rom fosfolipiduri kom-

ponenti saWiroa b5-sa da P450-s Soris urTierTqmedebisaTvis. rekonstruirebul monooqsigenazur sistemaSi naCvenebia, rom ma-

hidroqsilirebel agents superoqsiduli anion-radikali ( •−2O ) war-

moadgens. misi monawileoba aseT sistemaSi mowmdeba dismutazis

(eriTrokupreinis) da taironis (superoqsiduli radikalebis damWe-

ris) gamoyenebiT. NADPH-iTa da citoqrom P450-reduqtaziT, agreTve qsantin/qsantioqsidazuri sistemiT an arafermentuli fo-

toqimiuri sistemis (riboflavinisa da meTioninis Semcveli) supe-

roqsidis generacia erTnairad moiTxovs fosfolipidis aucilebel

Tanamyofobas. garda amisa, arsebobs monacemebi, romlebic uCveneben

fosfolipidebis mniSvnelovan rols glukuroniltransferazuli

fermentiT monooqsigenazuri reaqciis produqtTa gamoyofaSi.

ganxiluli faqtebis dajamebiT SeiZleba sqematurad warmovadgi-

noT membranuli fosfolipidebis monawileoba lipofiluri qsenobi-

otikebis JangviT hidroqsilirebaSi (ix. qveTavi 1.5, nax. 1.8).

membranis monooqsigenazuri sistemisaTvis fosfolipidebis auci-

279

leblobas ramdenime zogadi principi arsebobs [48]:

1. fermentuli aqtivobis daqveiTeba fosfolipidis mocilebisas;

2. fermentis reaqtivacia fosfolipidis damatebisas;

3. fermentis aqtiuri asociacia membranis fosfolipidebTan;

4. citoqrom P450-is funqciurad aqtiuri konformaciis SenarCuneba; 5. eleqtronebis transportis modulaciis uzrunvelyofa.

monooqsigenazuri sistemisaTvis CamoTvlili kanonzomierebebi

biologiurad ganmtkicebul niSan-Tvisebas warmoadgens, radgan baq-

teriebisa da Tirkmelzeda jirkvlis mitoqondriebis mahidroqsili-

rebeli sistemac Tavisi aqtivobebisaTvis aseve amJRavneben fosfoli-

pidebisadmi mkacr damokidebulebas.

amgvarad, dabejiTebiT SeiZleba iTqvas, rom mikrosomuli membranis fosfolipiduri komponenti absoluturad aucilebelia mTeli rigi

siCqaremalimitirebeli reaqciebis msvlelobisa da monooqsigenazuri

sistemis funqcionirebis regulirebisaTvis. organul qsenobiotikTa

biotransformaciaSi substratis membranaSi SeRwevis, citoqrom

P450-is molekulis hidrofobul ubnebTan misi dakavSirebis, mikro-

somuli gadamtanebis cila-lipidur kompleqsebTan mimagrebisa da eleq-

tronTa gadatanis jaWvSi reduqtazuli reaqciebis yvela stadia mem-

branuli lipidebisadmi uSualo damokidebulebaSi imyofeba. cxadia,

uaRresad mniSvnelovania fosfolipiduri komponentis im yvelaze „pers-peqtiuli” funqciis gamovlena, romelic gansazRvravs mis rolsa da

adgils „Tavisufali Jangvis” mTlian sistemaSi.

4.4.3 NADH-citoqrom b5-reduqtazisa da citoqrom b5-is

damokidebuleba membranul fosfolipidebze

NADH-specifikuri flavoproteinis aqtivoba Cveulebriv fasde-

ba fericianidis an citoqrom c-s aRdgenis siCqariT [48]. es reaqcie-bi gansxvavebul mgrZnobiarobas amJRavneben im agentebis mimarT,

romlebic mikrosomebSi hidrofobuli urTierTqmedebis dazianebas

iwveven. mag., mikrosomuli fraqciis acetoniT damuSaveba NADH-

citoqrom c-reduqtazul aqtivobas 80_85%-iT Trgunavs. aseT

fraqciaze ultrabgeriT damuSavebuli fosfolipiduri micelebis

damateba, romelic 70%-mde fosfatidilqolins Seicavs, reduqtazis

aramarto srul aRdgenas, aramed sawyisTan SedarebiT gaoTxmagebul

stimulacias iwvevs.

280

fericianidTan mimarTebaSi fermentis aqtivoba 10_15%-iT qveiT-

deba trifsiniT damuSavebisas da praqtikulad ar icvleba C da D tipis fosfolipazebis Tanamyofobisas [49]. aqtivobis ∼30%-iani

vardna SeiniSneba natriumis dezoqsiqolatiT, buTanoliTa da tri-

ton X-100-iT zemoqmedebisas. mxolod fosfolipaza A iwvevs feri-

cianidis NADH-damokidebuli aRdgenis reaqciis siCqaris orjer

daqveiTebas. aRniSnuli fermentuli aqtivobis gaZliereba ar xdeba

sainkubacio xsnarSi fosfolipidebis damatebiT. es faqtebi membrana-

Si NADH-damJangveli redoqs-jaWvis ganiv ganlagebaze (anu fosfo-

lipiduri matriqsis gadakveTaze) miuTiTeben.

dezoqsiqolatis Semcvel gelze qromatografiis saSualebiT mi-

Rebul fosfolipidebisagan Tavisufal mikrosomul fraqciaSi ci-

toqrom b5-is aRdgenis siCqare 80%-iTaa inhibirebuli. mikrosomebis

saerTo fraqciidan miRebuli fosfolipidebisagan momzadebuli li-

posomebis damateba ki fermentis srul reaqtivacias iwvevs.

reduqtazis qimotrifsiniT an katefsiniT damuSavebisas ferments

scildeba polipeptiduri fragmentebi, romlebic 65%-ze met hi-

drofobuli aminomJavebis naSTebs Seicaven. sayuradReboa erTi gare-

moeba: e.w. P-reduqtaza (molekuluri masa 28 kD), romelic membra-

nidan solubilizebulia kobras SxamSi arsebuli fosfolipazebis

nareviT, mikrosomaSi CaSenebis unars ar amJRavnebs da citoqrom b5-

is mimarT sust reduqtazul aqtivobas flobs. meore mxriv, nebis-

mieri sxva saSualebiT solubilizebuli fermenti NADH-isa da

citoqrom b5-is mimarT erTnairi TvisobiT xasiaTdeba. miRebuli Se-

degebi imaze metyveleben, rom NADH-citoqrom b5-reduqtazis amfi-

paturi molekulis katalizurad aqtiuri centri membranis hidro-

filur nawilSi imyofeba.

moyvanili faqtebi da agreTve D-reduqtazis (triton X-1000-isa da dezoqsiqolatis nareviT solubilizebuli fermenti, molekulu-

ri masiT 45 kD) unari, erTnairad efeqturad imoqmedos gamoyofil

da membranasTan bmul citoqrom b5-Tan [48], saSualebas iZleva ga-

keTdes daskvna, rom fermentis es forma natiuria. misi arapolaru-

li nawili molekulis 25%-s Seadgens da unari aqvs mikrosomul

membranaSi im raodenobiT CaSendes, rac ori rigiT aRemateba mis en-

dogenur Semcvelobas. naCvenebia, rom mikrosomuli membranis fos-

folipidebi warmoadgenen adgils, romelsac ukavSirdebian rogorc

endogenuri, aseve damatebiT CaSenebuli reduqtazebi. ufro metic,

fosfolipidebi gansazRvraven fermentis maqsimalurad dakavSirebis

281

dones, mis funqciur identurobas endogenur reduqtazasTan da ci-

toqrom b5-is dakavSirebis saerTo wertilebisaTvis konkurencias.

mikrosomuli membranidan katefsiniT reduqtazis mocilebisas

supernatantSi citoqrom b5-is 6%-ze meti ar gadmodis [49]. 50%-

ze naklebi reduqtazis mocilebisas NADH-iT citoqrom b5-is aRd-

geniT sawyisi (swrafi) siCqare ar icvleba, magram mniSvnelovnad

qveiTdeba xsnarSi reduqtazis gadasvlis zrdasTan erTad. maSasadame,

NADH-citoqrom b5-reduqtaza da citoqrom b5 membranaSi unda

qmnidnen „mtkice kompleqsebs”, romelTagan TiToeuli 5 molekula

reduqtazas da 50 molekula citoqroms Seicavs. N-eTilmaleimidiT

reduqtazis 90%-iani inaqtivaciis drosac ki citoqrom b5 dabali

siCqariT, magram mainc srul aRdgenas ganicdis. ufro metic, endo-

genur reduqtazas SeuZlia eleqtronebis maRali siCqariT gadacema

mikrosomul membranaSi maqsimalurad CaSenebuli egzogenuri cito-

qrom b5-is araumetes 60%-ze. es monacemebi ki TavisTavad uaryofen

raime „kompleqsis” arsebobas da pirdapir miuTiTeben, rom reduqta-

zas „tivtivas” msgavsad membranis gaswvriv lateraluri moZraoba

SeuZlia da citoqrom b5-Tan misi urTierTqmedeba SemTxveviT dajax-

ebebzea damokidebuli.

literaturaSi gvxvdeba monacemebi membranaSi citoqrom b5-is

lateraluri difuziis SesaZleblobis Sesaxeb [49]. es varaudi

mtkicdeba hemoproteinis aRdgenis siCqaris gazrdiT mikrosomaSi

misi damatebiTi CaSenebis Semdeg. am dros mrudze, romelic gviCve-

nebs aRdgenis siCqaris konstantis damokidebulebas temperaturis

Sebrunebul mniSvnelobasTan, texili Cndeba. amasTan, reduqtazuli

reaqciis aqtivaciis energiis uaRresad dabali mniSvneloba (28 kj/moli), citoqrom b5-is aRdgenis difuziur bunebaze miuTiTebs.

arsebobs imis mcdelobac, rom citoqrom b5-isaTvis ganisazRvros

lateraluri difuziis koeficienti. amasTan dakavSirebiT pirobiTad

miRebul iqna mikrosomuli buStis saSualo farTobi _ 12.6.10-10sm2-

is, buStis moculoba _ 1.26.10-15 sm3-is, citoqrom b5-isa da misi

reduqtazis jamuri radiusi 3.10-7 sm-is tolad da am monacemebze

dayrdnobiT gamoangariSebulia aRniSnuli sidide, romelic 10°C-ze 0.92.10-12 sm2

/wm-s udris. citoqrom b5-isa da reduqtazis moleku-

laTa efeqturi dajaxebebis wilis gaTvaliswinebiT lateraluri

difuziis koeficientis WeSmariti mniSvneloba 3.10-9 sm2/wm-is toli

aRmoCnda. saWiroa xazi gaesvas im garemoebas, rom citoqrom b5-is

lateraluri difuzia fosfolipiduri matriqsis denadobis kidev

282

erTi gamovlenaa, ramdenadac am hemoproteinisaTvis lateraluri

difuziis koeficienti mikrosomul fosfolipidebTan SedarebiT

100-jer naklebia. Ggarkveuli azriT SeiZleba iTqvas, rom hemopro-

teinis molekulebis lateraluri difuziis siCqare sruladaa

uzrunvelyofili „gamxsnelis”, anu fosfolipidis molekulebis

lateraluri difuziis siCqariT.

sadReisod SeuZlebelia amomwuravad vupasuxoT kiTxvas, Tu ra

funqciuri datvirTva aqvs endoplazmur membranaSi citoqrom b5-s

da saerTod, NADH-specifikur redoqs-jaWvs. arsebobs monacemebi

imis Sesaxeb, rom citoqrom b5 monawileobs cximovan mJavaTa desa-

turaciis reaqciebSi da rom am citoqroms sxva xsnadi gadamtanebis

dauxmareblad SeuZlia ganaxorcielos eleqtronTa membranaTaSorisi

gadatana. monooqsigenazuri sistemis funqcionirebis sqemaSi (ix.

qveTavi 1.3.4.1, sqema 1.2) citoqrom b5-s daTmobili aqvs sammag kom-

pleqsSi _ citoqrom P450–substrati_Jangbadi meore eleqtronis

„Semomtanis” roli. arCakovs da mis TanamSromlebs [1, 4] miRebuli

aqvT sarwmuno monacemebi citoqrom b5-is monawileobis Sesaxeb

NADPH-is JangvasTan qsenobiotikTa hidroqsilirebis SeuRlebul

reaqciebSi.

cnobilia, rom citoqrom b5 gacilebiT advilad solubilizdeba

proteazuli damuSavebiT, vidre organuli gamxsnelebiT, triton X-

1000-iT an A tipis fosfolipaziT. fosfolipaza C saerTod ar

moqmedebs mikrosomebSi citoqrom b5-is Semcvelobaze.

P-reduqtazasTan dakavSirebuli citoqrom b5 (P_b5) 11 kD mo-

lekuluri masis mqone monomeri aRmoCnda, romelsac mikrosomul

membranaSi CaSenebis unari ar gaaCnia. naCvenebi iqna, rom igi cito-

qrom b5-is molekulis nawils warmoadgens, Seicavs hems da pasuxis-

mgebelia donorul-aqceptorul urTierTqmedebaze. natiuri saxiT D-b5 gamoyofilia detergentebis gamoyenebiT [49]. igi 141 aminomJavur naSTs Seicavs da 16.7 kD molekuluri masa gaaCnia; wyalxsnarebSi

agregacias ganicdis da oqtameris formas iRebs, romlis moleku-

luri masa ∼120 kD-ia. specialurad Catarebuli gamokvlevebiT Se-

saZlebeli gaxda dadgeniliyo, rom triton X-100-Tan citoqrom b5-

is „delipidizebuli” xsnadi kompleqsebis warmoqmnisas detergenti

citoqromis hidrofobul da ara hidrofilur globulur nawils

ukavSirdeba. trifsiniT damuSavebisas citoqrom b5-s scildeba ka-

talizurad aqtiuri polipeptiduri komponenti, romelic 97 ami-nomJavur naSTs Seicavs. damatebiTi komponenti 44 aminomJavuri naS-

283

Tis Semcvelia, romelTagan 60% arapolarulia.

citoqrom b5-isa da fosfolipidebis urTierTqmedebaze gansaku-

TrebiT saintereso monacemebi iqna mopovebuli hemoproteinis hi-

drofobuli komponentis aRmoCenis Semdeg [48]. swored es komponen-

ti, romelsac ∼4 kD molekuluri masa aqvs, warmoadgens hidrofo-

bul „kuds”, romliTac D-b5 membranas emagreba. amasTan, mikrosomu-

li suspenziis ultrabgeriT da homogenizebiT Semdgomi damuSavebi-

sas es komponenti supernatantSi ar gadadis.

citoqrom b5-isaTvis damaxasiaTebelia unari, CaSendes mikrosoma-

Si hemoproteinis endogenur SemcvelobasTan SedarebiT 5–20-jeradi siWarbiT [49]. damatebiT CaSenebuli citoqromis dakavSirebis ubnebi

fosfolipidebi aRmoCndnen. amfipaturi cilis damatebiTi dakavSire-

bis fenomeni SeiZleba biomembranebis Senebis „mozaikuri” modeliT

aixsnas [48]. misi Teoria iTvaliswinebs fosfolipiduri matriqsis

gansxvavebuli ubnebis (zonebis) SesaZleblobas, sxvadasxva tempera-

turaze fazuri gadasvlebi ganaxorcielon. rogorc irkveva, kon-

kretul pirobebSi biomembranebSi fosfolipidebi SeiZleba imyofe-

bodnen „lateraluri gamyofi fazis” mdgomareobaSi, rodesac Txe-

vadkristaluri da Txevadi zonebis Tanafardoba matriqsis garkveu-

li ubnebis lateralur „kumSvas” gansazRvravs. Tvlian, rom late-

raluri gamyofi faza da lateraluri kumSva ganapirobeben cilis

molekulis aramarto konformaciul cvlilebebs, aramed biomembra-

nebSi maT damatebiT CaSenebas.

hidrofobul polipeptidur fragmentSi arsebuli triftofanis

fluorescenciis unaris gamoyenebiT dadgenil iqna, rom erTi mole-

kula citoqrom b5-is dakavSirebisaTvis fosfatidilqolinis araum-

cires 35 molekulaa saWiro. es cila/lipidis woniTi Tanafardobis

0.5 koeficients Seesabameba. garemos temperaturis cvlilebisas ci-

toqrom b5–lecitinis kompleqsis fluorescenciis cvlilebam Se-

saZlebeli gaxada gamoTqmuliyo mosazreba, rom hemoproteinis hi-

drofobuli nawili aqtiurad asocirdeba mikrosomuli lecitinebis

cximovani mJavebis naSTebTan.

4.4.4 monooqsigenazebis da lipidebis erTiani sistema

endoplazmur membranebSi arsebuli qsenobiotikTa mJangveli fer-

mentebi Tavis aqtivobas ar kargaven marilTa maRali koncent-

raciebiT an xelatorebiT damuSavebisas [48]. reagentebi, romlebic

284

cilis molekulis konfiguracias arRveven (Sardovana, guanidini), an

maT hidrolizs iwveven (trifsini), fermentTa solubilizacias ver

axdenen. mxolod iseTi detergentebi, romlebsac membranis lipidur

matriqsTan uaRresad mtkiced dakavSirebuli cilebi xsnad mdgoma-

reobaSi gadahyavT (Na-is qolati da dezoqsiqolati, triton X-100 da tvin-80), axdenen am fermentul sistemaTa calkeuli komponente-

bis solubilizacias. aqedan cxadi xdeba, rom endoplazmuri membra-

nebis sunTqviTi jaWvis komponentebi garegan, periferiul cilebs ar

miekuTvneba. isini ZiriTadi, integraluri cila-fermentebia, romleb-

ic membranis siRrmeSi iWrebian da mtkice kontaqts amyareben mis

fosfolipidur matriqsTan.

mikrosomul redoqs-sistemaTa cxovelmyofelobaSi fosfolipi-

debis gadamwyvet mniSvnelobaze miuTiTebs maTi gavleniT sxvadasxva

qsenobiotikebis metabolizmis reaqtivacia [49]. reagentebi, romleb-

ic uaryofiTad moqmedeben membranaTa hidrofobul Tvisebebze, ro-

gorc wesi, aqveiTeben masSi myof mJangvel fermentTa aqtivobebsac.

am viTarebaSi egzogenurad damatebul fosfatidilqolins unari

aqvs sakontrolo donemde aRadginos zogierTi procesis siCqare,

mag., Na-is dezoqsiqolatiT daqveiTebuli aminopirinis N-deme-

Tilirebis reaqcia, an buTanoliTa da acetoniT daTrgunuli 3,4-benzpirenis daqveiTebuli hidroqsilireba. amasve adasturebs Cvens

laboratoriaSi [19] naCvenebi mcenareSi triton X-100-is damTrgun-veli (65-85%-iT) moqmedeba aminopirinis, dimeTilanilinis N-

demeTilirebaze da anilinis p-hidroqsilirebaze, rac detalurad

iyo ganxiluli qveTavSi 1.5.

mTel rig laboratoriebSi [48, 49] naCvenebia, rom rekonstrui-

rebul proteoliposomebSi (sinTezur mikrosomebSi) samkurnalo

preparatebis, hormonebis, cximovani mJavebis, najeri naxSirwyalbade-

bisa da kancerogenebis metabolizeba mxolod lipiduri bunebis

mqone Termostabiluri faqtoris Tanamyofobisas xorcieldeba da

igi identificirebulia rogorc fosfatidilqolini. gamosaxul

reaqtivaciul unars flobs rogorc „nedli”, aseve sinTezuri le-

citini.

mikrosomebis damuSavebisas ultrabgeriT, maRali koncentraciis

marilebis xsnarebiT an gayinva-gaTbobiT NADPH-citoqrom P450-reduqtazis membranidan solubilizeba ar xdeba. mxolod proteo-

lizuri fermentebiT, lipaziT an detergentebiT zemoqmedebisas xde-

ba membranaTa sxva komponentebisagan misi izolireba. iTvleba, rom

285

intaqtursa da fenobarbitaliT an qolantreniT inducirebul da

Semdeg izolirebul NADPH-specifikur flavoproteins Soris ar-

sebiTi gansxvaveba araa.

proteoliziT da detergentiT gamoyofili flavoproteinebi

erTmaneTisagan molekuluri masebiT gansxvavdeba. detergentiT miRe-

buli fermentis molekuluri masa ∼8 kD-iT metia. amasTan dakavSi-

rebiT gamoTqmulia mosazreba, rom flavoproteini mikrosomul mem-

branas ukavSirdeba Tavisi hidrofobuli nawiliT, romelic 50_100 aminomJavur (upiratesad arapolarul) naSTs Seicavs da mikrosome-

bis proteolizurad damuSavebisas ferments mxolod hidrofiluri

nawili wydeba, hidrofobuli ki membranis siRrmeSi rCeba. proteo-

lizisagan gansxvavebiT detergenti saSualebas iZleva fermenti ga-

moyofil iqnas Tavis „hidrofobul kudTan” erTad.

Semdgomma gamokvlevebma [49] aCvenes, rom flavoproteinis hidrofo-buli „kudi” arsebiT rols ar asrulebs flavinebis Jangvisas aRZrul speqtrul cvlilebebSi, eleqtronis aqceptorTa aRdgenasa da ferment-inhibitoris urTierTqmedebaSi. yvela es Tviseba fermentis hidrofi-luri nawilis funqcia aRmoCnda. dadginda isic, rom fermenti, ro-

melic proteolizuradaa miRebuli, rekonstruirebul membranaSi Semd-gomi „CaSenebisas” qsenobiotikTa gardaqmnis unars veRar amJRavnebs.

samagierod, rekonstruirebul sistemaSi „CaSenebuli” detergentuli

flavoproteini aqtiurad monawileobs qsenobiotikTa JangviT kata-

bolizmSi da am Tvisebas kargavs trifsiniT damuSavebisas, Tumca

lipidebis Jangvis unari SenarCunebuli aqvs.

rac Seexeba mikrosomuli sunTqviTi jaWvis terminalur kompo-

nents _ citoqrom P450-s, igi gamoyofis manipulaciebis mimarT sak-

maod mdgradia. eleqtroforezulad homogenur mdgomareobaSi ga-

moyofili fermentis monomeri, romlis molekuluri masa 49 kD-is

tolia, 409 aminomJavur naSTs Seicavs da maTgan 50% arapolaru-

lia. maSasadame, fermenti erT-erT yvelaze hidrofobul membranul

cilas warmoadgens. solubilizebuli fermentis mikrosomul membra-

naSi „CamontaJebisas” misi mJangveli unari mkveTrad izrdeba. amJamad

dadgenilia, rom fermenti Tavisi substratebisagan lipiduri „ba-

rieriTaa” ganmxoloebuli. amitom Jangvas win uZRvis lipidur matriqs-Si substratTa SeRwevis aqti. mTeli rigi cikluri da alifaturi nax-Sirwyalbadebis unari, warmoqmnan citoqrom P450-Tan katalizurad

aqtiuri kompleqsi, damokidebulia im siCqareze, romliTac es qseno-

biotikebi toveben membranis polarul fazas da gadaadgildebian ara-

286

polarulisaken, romelic fosfolipidebis naxSirwyalbadovani jaW-

vebiTaa warmodgenili da romelSic citoqrom P450 imyofeba. maSasadame, rodesac organul naerTTa biotransformaciaze vmsje-

lobT, am procesSi vgulisxmobT substratis membranaSi SeRwevis,

citoqrom P450-is molekulis hidrofobul ubnebTan misi dakavSire-

bis, membranis lipid-cilovan kompleqsTan mikrosomuli gadamtanebis

mimagrebis da eleqtronTa transportis reduqtazuli reaqciebis

mTlian Tanmimdevrobas.

monooqsigenazur reaqciaTa substratebi gacilebiT intensiurad

ukavSirdebian ujer fosfolipidebs (mag., nedl lecitins), vidre

najers (mag., dipalmitoillecitins). citoqrom P450 da misi re-duqtaza lokalizebulia fosfolipidebis „specializebul” zonaSi,

romelic SedarebiT araaqtiur mdgomareobaSi imyofeba, vidre fos-

folipiduri matriqsis ZiriTadi masa. am zonisaTvis damaxasiaTebe-

lia fazuri gadasvla Txevad-kristaluridan Txevad mdgomareobaSi.

varaudoben, rom am zonaSi 20%-mde mikrosomuli fosfolipidi im-

yofeba. isini aramarto monooqsigenazuri sistemis calkeul kompo-

nentTa urTierTkontaqts amyareben, aramed citoqrom P450-isaken lipiduri substratebis lateralur difuziasac uzrunvelyofen.

molekuluri organizaciis ra upiratesobas iZleva wyalSi xsnad

formebTan SedarebiT monooqsigenazuri sistemis membranaSi evolu-

ciuri CamontaJeba? principSi SesaZlebelia am fermentul sistemaTa

organizaciis ori tipis arseboba. pirvelia garkveuli steqio-

metriis mqone, mtkice, mravalkomponentiani fermentuli sistemebis

membranaSi funqcionireba. eleqtronTa gadatana SesaZlebelia mxo-

lod aseTi „klasteris” SigniT. modeli, romelsac „klasterul

organizacias” uwodeben, reduqtazisa da citoqrom P450-is mole-

kulebs Soris xist kavSirs iTvaliswinebs [51]. mikrosomul membra-

nasTan maqsimalurad miaxloebul sistemebze Catarebulma cdebma

saWiro gaxada monooqsigenazuri sistemis funqciuri integraciis

arsebobis aRiareba. naCvenebi iqna, rom citoqrom P450 mikrosomul

membranaSi warmoqmnis ara umcires ori molekulisagan Semdgar kom-

pleqss [51-53]. nanaxia kompleqsis warmoqmnis SesaZlebloba hemo-

proteinsa da Sesabamis reduqtazas Soris TanafardobiT 2 : 1. aseTi binaruli kompleqsis arseboba marTlac gamovlenilia integrirebul

mikrosomebSi. aRmoCnda, rom misi warmoqmna 0°C-zec ki xdeba, rode-sac molekulaTa moZraobis siCqare mniSvnelovnadaa daqveiTebuli.

aqedan SeiZleba gakeTdes daskvna, rom hemoproteini da misi reduq-

287

taza membranaSi uSualo siaxloveSi imyofebian. iTvleba, rom qseno-

biotikTa degradaciaSi oqsigenazaTa klasteruli organizacia am

fermentul sistemas umaRles katalizur aqtivobas aniWebs. arse-

bobs monacemebi, rom fosfatidilqolini klasteruli kompleqsis

aucilebeli Semadgeneli komponentia.

monooqsigenazebis molekuluri organizaciis meore tipi fer-

ment-substratis urTierTqmedebaSi gulisxmobs endoplazmur membra-

nebSi gadamtanebis SemTxveviT Sejaxebebs (lateralur difuzias) [7,

54]. rekonstruirebul aramembranul sistemebze da agreTve gadam-

tanebiT „aRWurvil” liposomebze Catarebuli cdebiT SesaZlebeli

gaxda am tipis realurad arsebobis dadgena. cxadi gaxda, rom ga-

damtanebi metad Zvradia, advilad difundireben membranis sibrtyeSi

da SemTxveviTi dajaxebisas adgili aqvs eleqtronis gadatanas. amas-

Tan, bunebrivia, warmoiqmneba muxtis gadamtani Sesabamisi binaruli

kompleqsebi. imis gamo, rom endoplazmuri membranebi Txevadkrista-

luri struqturisaa, romelSic cilis molekulebs lateraluri

moZraoba SeuZlia, swored amitom gadamtanebis erTmaneTTan Tvisobis

maxasiaTebeli konstanta unda iyos is sidide, romelic membranaSi

fermentul sistemaTa molekulur organizacias gansazRvravs. sa-

dReisod, ramdenadac CvenTvis cnobilia, endoplazmur membranebSi eleq-

tronTa gadamtanebisaTvis am sididis raodenobrivi Sefaseba ar momxda-ra. amitom monooqsigenazuri sistemis molekuluri organizaciis problema isev gadauWrelia. amasTan dakavSirebiT intensiuri kvleva Ca-tarda lipiduri biSris mikrosiblanteze eleqtronTa gadatanis siCqa-ris damokidebulebis gamosavlenad. sawyis kriteriumad iqca is varaudi, rom membranis fosfolipiduri komponentis mikrosiblantis daqveiTebi-sas (rac mikrosomidan qolesterolis mocilebiT miiRweva), SesaZlebe-

li gaxdeboda dadgeniliyo difuziaze damokidebuli eleqtronTa gada-tanis reaqciebi da am gziT gamovleniliyo eleqtronis gadamtani Tavi-sufali difuziis unaris mqone cila.

aRniSnuli midgomiT cnobili gaxda, rom NAD(P)H-citoqrom b5-

reduqtazuli reaqcia damokidebulia difuziis siCqareze, NAD(P)H-

citoqrom P450-reduqtazuli ki ara [49]. es Sedegebi SeiZleba gan-

xilul iqnas, rogorc NADH-damokidebuli JangviTi jaWvis ori

sawyisi komponentis _ flavoproteinis da citoqrom b5-is latera-

luri Zvradobis pirdapiri eqsperimentuli dadastureba. isini sarw-

munod gviCvenebs membranaSi am gadamtanTa uwesrigo ganawilebas.

maSasadame, citoqrom P450-isa da NADPH-specifikuri flavo-

288

proteinis SemTxvevaSi adgili unda hqondes SedarebiT mtkice kom-

pleqsis warmoqmnas, vidre am flavoproteinisa da citoqrom b5-is

SemTxvevaSia. ganxiluli Sedegebi gvafiqrebinebs, rom membranuli da

wyalSi xsnadi monooqsigenazuri sistemebis molekulur organiza-

ciaSi principuli gansxvaveba ar arsebobs.

4.4.5 lipidebis fermentuli peroqsidacia da qsenobiotikebis

Jangva endoplazmur membranebSi erTi da igive eleqtronis

satransporto sistemiT katalizdeba

fosfolipidTa ujeri rigis cximovani mJavebis fermentuli ze-

Janguri Jangva reaqciaTa im erT gansakuTrebul klass warmoadgens,

romelic endoplazmur membranebSi lokalizebuli fermentuli kom-

pleqsebiT xorcieldeba. gansakuTrebul mniSvnelobas iZens am pro-

cesSi mikrosomuli NADPH-specifikuri flavoproteinisa da cito-

qrom P450-is monawileobis sakiTxi.

pirveli monacemebi imis Sesaxeb, rom lipidTa peroqsidacia da

qsenobiotikTa Jangva erTidaimave fermentebiT katalizdeba,

ernstersa da mis TanamSromlebs [55, 56] ekuTvnis. am daskvnamde av-

torebi Semdeg faqtebze dayrdnobiT mividnen:

1. qsenobiotikTa Jangvis produqtebi ainhibireben lipidTa zeJangur

Jangvas;

2. mikrosomuli preparatebis „daberebisas”, rodesac qsenobiotikTa

Jangvis mkveTri daqveiTeba xdeba, ixsneba am produqtTa mier

NADPH-damokidebuli zeJanguri Jangvis inhibireba.

aRniSnuli faqtebis asaxsnelad avtorTa mier wamoyenebul iqna

hipoTeza, romlis Tanaxmadac orive procesSi monawileobs NADPH-is damJangavi eleqtronis transportis saerTo fermentuli sistema.

axlad gamoyofil mikrosomebSi qsenobiotikebi am sistemaze „baton-

debian”, ris gamoc NADPH-is ZiriTadi fondi maT degradaciaSia

CarTuli, xolo lipidTa zeJanguri Jangva daTrgunulia. „monopo-

lirebis” efeqti ar SeiniSneba „asakovan” mikrosomebSi da NADPH-is mJangavi sistema aqtiurad monawileobs lipidur peroqsidaciaSi.

sadReisod mopovebuli eqsperimentuli monacemebi [48] adasture-

ben NADPH-specifikuri flavoproteinis gadamwyvet rols membra-

nuli lipidebis zeJangur JangvaSi. igi warmatebiT akatalizebs am

process ADP–Fe2+-isa da EDTA–Fe2+-is kompleqsebTan Tanamyofobi-

289

sas. orive SemTxvevaSi adgili aqvs reaqciis saboloo produqtis _

malonis dialdehidis erTnairi raodenobiT dagrovebas. rogorc

irkveva, mikrosomebSi lokalizebulia lipidTa peroqsidaciis, sul

mcire, sami fermentuli sistema, romlebic daaxloebiT erTnairi

siCqariT, magram gansxvavebuli steqiometriiT axorcieleben zeJan-

gur Jangvas, radgan TiToeul maTgans fardobis [O2] : [malonis di-

aldehidi] sxvadasxva mniSvneloba gaaCnia. samidan ori sistema dakav-

Sirebulia NADPH-specifikur, xolo erTi – NADH-specifikur flavoproteinis funqcionirebasTan. pirvelis Taviseburebas is war-

moadgens, rom mas gaaCnia Zlier stabiluri aRdgenili mdgomareoba,

ris gamoc funqcionirebs rogorc erTeleqtroniani gadamtani da

advilad gadadis mTlianad aRdgenil mdgomareobaSi.

unda aRiniSnos, rom flavoproteinis katalizuri aqtivoba erT-

nairia xsnarsa da fosfolipidur biSreSi [49]. es amfifiluri ci-

lebisaTvis damaxasiaTebeli Tvisebaa, rac imaSi mdgomareobas, rom

isini konformaciuli cvlilebebis mimarT mdgradobas amJRavneben

wylidan lipidur fazaSi gadasvlisas. igive xdeba citoqromebis b5-isa da P450-is SemTxvevaSi. am ukanasknels membranaSi funqcioni-

rebisas konformaciuli cvlilebebi ar sWirdeba, radgan mis aqtiur

centrs organizebuli mdgradi sistema aqvs, rac agreTve misi moqme-

debis safuZvelsac warmoadgens.

detergentiT solubilizebuli NADPH-specifikuri flavopro-

teini rekonstruirebul sistemebSi CaSenebisas funqciurad aqtiuria

rogorc qsenobiotikebis metabolizmSi, aseve lipidTa peroqsidaca-

Si. am faqts wina planze gamoyavs aRniSnul reaqciebSi fermentis

hidrofobuli fragmentis roli. eWvgareSea, rom am komponents ar-

sebiTi mniSvneloba aqvs flavoproteinis funqciis realizaciaSi.

ucnobi rCeba mxolod is, monawileobs Tu ara igi fermentis membra-

nasTan dakavSirebisa da misi gansazRvruli sivrcobrivi orientaciis

CamoyalibebaSi, Tu katalizuri Tvisebebic gaaCnia.

RviZlis mikrosomebidan gamoyofili flavoproteinis xelovnur

liposomaSi CaSenebis Semdeg lipidTa zeJanguri Jangvis igive done

registrirdeba. es Sedegi TavisTavad imaze metyvelebs, rom es fer-

menti araa zeJanguri Jangvis malimitirebeli faqtori.

detergenti, romelic endoplazmuri membranebSi lipid-cilur da

lipid-lipidur urTierTqmedebaTa cvlilebebs iwvevs, rkinis ionebi-

saTvis dakavSirebis ubanTa ricxvsac cvlis [48]. rogorc aRniSnuli

iyo, membranasTan dakavSirebuli rkinis ionebi radikalwarmoqmnis

290

centrebs qmnian da lipidur fazaSi jaWvuri reaqciebis ganviTarebas

iwyeben. safiqrebelia, rom anionuri detergentebi zrdian rkinis

ionTa dakavSirebis centrTa ricxvs da am gziT lipidTa peroqsida-

cias aZliereben. kationuri detergenti _ acetiltrimeTilamonium-

bromidi sapirispiro efeqts avlens, xolo araionuri detergenti _

triton X-100 – peroqsidaciis siCqareze praqtikulad ar moqmedebs.

es Sedegebi membranul struqturebSi lipidTa zeJangur Jangvaze

hidrofobuli urTierTqmedebis cvlilebebis maregulirebeli gavle-

nis saukeTeso magaliTs warmoadgens.

arsebobs monacemebi [48], romlebic lipidTa fermentul zeJan-

gur JangvaSi da qsenobiotikTa hidroqsilirebaSi NADPH-speci-fikuri flavoproteinis udavo monawileobaze miuTiTeben. maTi ana-

lizi saSualebas iZleva gakeTdes Semdegi daskvnebi:

1. orive procesi maRalspecifikuria NADPH-is mimarT; 2. NADPH-citoqrom P450-reduqtazis konkurentuli inhibitori _

daJanguli NADP efeqturad Trgunavs lipidTa fermentul zeJan-

gur Jangvas da qsenobiotikTa hidroqsilirebas;

3. arsebobs sruli Sesabamisoba sxvadasxva tipis membranebSi

NADPH-citoqrom P450 reduqtazul aqtivobasa da lipidTa fer-

mentul zeJangur Jangvas Soris;

4. fermentuli reaqciis samive tipi _ NADPH-citoqrom P450-is Jangva-aRdgena, NADPH-damokidebuli hidroqsilireba da NADPH-damokidebuli lipidTa fermentuli zeJanguri Jangva substratis

mimarT erTnairad stereospecifikuria;

5. NADPH-specifikuri flavoproteinisadmi Zlier maRali Tvisobis

mqone xelovnuri aqceptori menadioni (vitamini K3) erTnairad

efeqturad Trgunavs NADPH-damokidebul procesebs – dimeTila-

nilinis Jangvasa da lipidTa peroqsidacias;

6. NADPH-is oqsidoreduqcia, qsenobiotikTa Jangva da lipidTa

fermentuli zeJanguri Jangva uaRresad mgrZnobiarea proteazebis

moqmedebis mimarT. flavoproteinis solubilizacia mikrosomuli

membranebis trifsiniTa da pronaziT xanmokle damuSavebisas hi-

droqsilirebisa da lipidTa peroqsidaciis mkveTr inhibirebas

axdens;

7. eleqtronTa gadatanis NADPH-specifikuri sistema da lipidTa

fermentuli zeJanguri Jangva mikrosomuli membranis im saerTo

fragmentebSia lokalizebuli, romlebic fraqciis ultrabgeriT

an izoamilis spirtiT damuSavebisas miiReba.

291

CamoTvlili argumentebis miuxedavad, arsebobs faqtebi, romelTa

Tanaxmadac orive procesis gansaxorcieleblad flavoproteinis

garda, saWiroa eleqtronis gadamtani sxva komponentebis arsebobac.

sakmarisia aRiniSnos, rom es fermenti membranis zedapirzea ganlage-

buli da masTan dasakavSireblad gamoiyeneba ara lipidebi, aramed

membranuli cilebis aminomJavuri naSTebi (amis mizezia proteazebis

maRali masolubilizebeli aqtivoba). membranul cilebTan susti

kontaqti gvafiqrebinebs, rom zeJanguri Jangvis iniciaciaSi flavo-

proteini uSualod ar monawileobs. amisaTvis saWiroa radikalebis

warmomqmnel centrebsa da fermentul zeJangur Jangvas Soris sivr-

cobrivi siaxlove. amitom Tvlian [49], rom lipidTa fermentul

peroqsidacias warmatebiT ganxorcielebisaTvis NADPH-specifikuri flavoproteini aucilebelia, magram procesis iniciacia mainc ara am

pirvel komponentTan, aramed ufro moSorebiT, sadRac Sualed an

saboloo ubanze xdeba.

endoplazmur membranebSi NADPH-specifikuri eleqtronis sa-

transporto jaWvis momdevno komponenti sabolood cnobili araa.

varaudoben, rom flavoproteinsa da citoqrom P450-s Soris aqcep-toris yvelaze Seucvlel komponents arahemuri, feredoqsinis msgav-

si rkinis Semcveli cila unda warmoadgendes, romelic zogierT

rekonstruirebul sistemaSi marTlac efeqturad funqcionirebs. am

cilis arseboba dadgenilia Tirkmelzeda jirkvlis mitoqondriul

membranaSi (ix. qveTavi 2). RviZlis mikrosomul fraqciaSi aseTi

cilis identifikaciis yvela mcdeloba uSedego aRmoCnda. amitom,

sadReisod iTvleba, rom mJangveli sistemis am ubanze citoqrom b5 funqcionirebs [48].

endoplazmuri membranebis NADPH-specifikuri redoqs-sistemis

calkeuli komponentis TvisebaTa ganxilva ar iZleva romelime maT-

ganze lipidTa peroqsidaciis zusti lokalizaciis dadgenis saSua-

lebas, radgan Tavisufali radikalebi, romlebic zeJangur Jangvas

inicireben, SeiZleba warmoiqmnan rogorc sawyis (TviTJangvadi fla-

vinis), aseve terminalur (citoqrom P450-is) ubnebze. miTumetes,

Znelia am procesSi eleqtronis satransporto jaWvis Sualeduri

ubnis rolze msjeloba.

axla ganvixiloT NADPH-specifikuri redoqs-sistemis saboloo

eleqtronuli aqceptoris, citoqrom P450-is SesaZlo monawileoba

lipidTa fermentul zeJangur JangvaSi. amTaviTve unda iTqvas, rom

mTeli rigi mkvlevarebisa uaryofs am faqts da amis safuZvlad

292

asaxeleben sakmaod damajerebel arguments: aRniSnuli hemoproteinis

klasikuri inhibitori _ naxSirbadis monooqsidi fermentul zeJan-

gur Jangvaze araviTar gavlenas ar axdens. ra Tqma unda, es argu-

menti samarTliani iqneboda im SemTxvevaSi, Tu CavTvliT, rom zeJan-

guri JangvisaTvis aucilebelia eleqtronis gadatana hemoproteinis

hemur ubanze. amasTan, gasaTvaliswinebelia citoqrom P450-is sami metad sayuradRebo Tavisebureba [48]:

1. apofermentis SH-jgufebi, rogorc ligandebi, monawileoben hemu-

ri rkinis dakavSirebaSi;

2. fermenti lokalizebulia membranis hidrofobul zonaSi, anu ze-

Janguri Jangvis substratTa uSualo siaxloveSi;

3. citoqrom P450 warmoadgens redoqs-sistemis im komponents, ro-melzec yvelaze metadaa mosalodneli Tavisufali radikalebis

warmoqmna. hidroqsilis radikalebTan erTad gamoricxuli araa

organuli radikalebis, kerZod, aminomJavaTa radikalebis generi-

rebac. yovelive zemoTqmulis gaTvaliswinebiT arCakovma warmoad-

gina lipidTa fermentul zeJangur JangvaSi citoqrom P450-is monawileobis ramdenadme uCveulo meqanizmis sqema (nax. 4.7). am mkvlevaris [4] Tanaxmad, hemoproteinis aqtiur centrSi myof

apofermentis SH-jgufTan rkinis pirofosfatis dakavSirebisas xde-ba Tavisufali radikalebis generirebadi axali centris Camoyalibe-

ba (sqemaze FePP), romlis aqtivobac naxSirbadis monooqsidiT ar

inhibirdeba. am SemTxvevaSi citoqrom P450 funqcionirebs ara ro-gorc hemoproteini, aramed rogorc arahemuri gogirdSemcveli rki-

naproteini. mikrosomebis eleqtronebis satrnsporto sistemaSi ci-

toqrom P450 warmoadgens im erTaderT gadamtans, romelsac aqtiur

centrSi SH-jgufi gaaCnia da eleqtronis gadatanaSi monawileobis

miReba SeuZlia. zeJanguri Jangvis fermentuli sistema ki mgrZno-

biarea SH-jgufebis iseTi Sxamebis mimarT, rogoric p-qlor-

merkuribenzoati, HgCl2 da mersalilia. rogorc Cans, SH-jgufi lipidur peroqsidaciaSi mxolod endogenur rkinasTan kompleq-

sirebisas erTveba. arafermentul, askorbatdamokidebul zeJangur

JangvaSi SH-jgufebis raime roli gamoricxulia, radgan es procesi

sulfhidriluri jgufebis Sxamebis mimarT aramgrZnobiarea.

soiis nazardebidan miRebul mikrosomul fraqciaze Cvens labo-

ratoriaSi Catarebuli gamokvlevebiT [19] dadgenilia, rom lipope-

roqsidaciis rogorc fermentuli, aseve arafermentuli procesi

qsenobiotikis (dimeTilanilinis) Jangvis gaZlierebas iwvevs. Fe2+-

293

isa da NADPH-is Tanamyofobisas lipidTa zeJanguri Jangvis orive

reaqcia TiTqmis sinergulad jamdeba. paralelurad adgili aqvs

fraqciis N-demeTilazuri aqtivobis zrdas. fosfatidilqolini,

romelsac antioqsidanturi Tvisebebi gaaCnia, lipidTa zeJanguri

Jangvis inhibirebasTan erTad aRniSnuli qsenobiotikis N-demeTilirebas astimulirebs. fosfatidileTanolamins aseTi gamok-

veTili moqmedeba ar gaaCnia. rogorc Cans, misi naxSirwyalbadovani

naSTis ujeri bmebi TviTon warmoadgens Tavisufali radikalebisaT-

vis moxerxebul samiznes, razec miuTiTebs lipoperoqsidaciis erT-

erTi saboloo produqtis – malonis dialdehidis dagroveba sareaq-

cio areSi. miRebuli monacemebis safuZvelze dasaSvebad miviCnieT

mosazreba, rom qsenobiotikis Jangvis procesi radikalur stadias

Seicavs, Tumca igi lipiduri peroqsidaciisadmi damoukideblobiT

xasiaTdeba.

qsenobiotikis

hidroqsileba

eleqtronis

gadatanis jaWvi

hemis

Fe

SH

FePP

O2

lipidTa

zeJanguri Jangva

citoqrom P450-is apofermenti

a.

b. eleqtronis

gadatanis jaWvi

hemis

Fe

SH

citoqrom P450-is apofermenti

O2

nax. 4.7. citoqrom P450-is monawileoba qsenobiotikTa hidroqsili-

rebisa (a) da lipidTa fermentuli zeJanguri Jangvis (b) reaqciebSi

(ganmarteba ix. teqstSi) [4].

294

arsebiTia membranis hidrofobul zonaSi citoqrom P450-is lo-

kalizaciis faqtoric: danarCeni gadamtanebisagan gansxvavebiT, misi

solubilizacia mxolod lipazebiTa da detergentebiT xerxdeba

[49]. hemoproteini membranasTan mtkiced bmuli fermentia da misTvis

daculia Tavisufali radikalebis iniciaciis centrebisa da zeJan-

guri Jangvis substratTa sivrcobrivi siaxlovis piroba. fermentul

zeJangur JangvaSi citoqrom P450-is monawileobaze miuTiTebs am

procesis daTrgunva hemoproteinis iseTi specifikuri inhibitore-

biT, rogorebic SKF-S25A (β,β-dieTilaminoeTildifenilpropil-

acetati) da nikotinamidia.

amgvarad, arsebul monacemebze dayrdnobiT SegviZlia miviRoT,

rom:

1. endoplazmur membranebSi arsebobs lipidTa zeJanguri Jangvis

ori sistema, romelic erTmaneTisagan principulad gansxvavdeba

iniciaciis stadiiT. NADPH-specifikuri sistemis SemTxvevaSi

radikalwarmomqmneli centrebi _ rkinis (II) ionebi eleqtronis

gadamtani jaWviT fermentulad aRdgeba, xolo arafermentul

sistemaSi rkinis aRdgena askorbatiT an Sesaferisi redoqs-

potencialis mqone sxva aRmdgeneliT xdeba;

2. orive procesis kinetika da steqiometria imis sasargeblod me-

tyvelebs, rom am reaqciaTa ganviTarebas erTiani, radikalur-

jaWvuri meqanizmi udevs safuZvlad;

3. Fe2+ an Fe3+

ukavSirdeba cilis garkveul funqciur jgufs, ris

Sedegadac es ukanaskneli radikalebis warmoqmnis (reaqciis in-

iciaciis) centri xdeba. lipidTaAfermentuli zeJanguri Jangvis

SemTxvevaSi aseTi jgufi SeiZleba iyos citoqrom P450-is aq-tiur centrSi arsebuli sulfhidriluri jgufi, xolo arafer-

mentul zeJangur JangvaSi es roli SeiZleba Seasrulon im mem-

branuli cilebis funqciurma jgufebma, romlebic hidrofobuli

fazidan ar solubilizdeba.

4.5 regulacia citoqrom P450-is qimiuri modifikaciis doneze

fermentebze arsebuli tradiciuli Sexeduleba imis Sesaxeb, rom

katalizuri aqtis dasrulebis Sedegad isini cvlilebebs ar ganic-

dian, ukanaskneli ori aTeuli wlis manZilze kritikulad iqna ga-

dasinjuli. aRmoCnda, rom mravali fermenti reaqciis msvlelobisas

295

qimiur modifikacias ganicdis da es gansakuTrebiT im fermentebs

exebaT, romelTa funqcionirebac Tavisufali radikalebis, Jangbadis

aqtiuri formebis da reaqciisunariani intermediatebis generirebas

an realizacias ukavSirdebian [57-61]. modifikacia, upirveles yov-

lisa, inaqtivaciaSi vlindeba (cxrili 4.1). es movlena “TviTinaqti-

vaciis” saxelwodebiTaa cnobili.

cxrili 4.1

katalizis procesSi Jangbadis aqtiuri formebiT

fermentTa qimiuri modifikacia [58]

Tavisufali radikalebis dagroveba ujredisaTvis gansakuTrebiT

saxifaToa, radgan isini mravali paTologiuri procesis iniciacias

iwveven. rodesac maTi koncentracia saSiS zRvars aWarbebs da

ujredidan mocileba SeuZlebeli xdeba, ujredi apoptozs, anu

winaswar daprogramebul “TviTmkvlelobas” mimarTavs. termini

“apoptozi” (berZn. “foTolcvena”) SesaniSnavad gamoxatavs movlenis

arss. mTeli rigi procesebis CarTvis Sedegad dazianebuli ujredi

ki ar nekrozdeba, aramed ganlevas ganicdis. organelebi iSleba,

makromolekulebi hidrolizdeba da isini sxva ujredebis mier sakveb

da saamSeneblo masalad gamoiyeneba.

fermentebi inaqtivaciis

gamomwvevi agentiqimiuri modifikaciis mizezi

citoqromi P450 2B4 H2O2 cisteinis da meTioninis Jangva,

hemis dakargva

CuZn-superoqsiddismutaza H2O2 histidinis Jangva

Fe-superoqsiddismutaza H2O2 triftofanis, histidinisa da

cisteinis Jangva

D-glukozooqsidaza H2O2 triftofanis, histidinisa da

cisteinis Jangva

qsantinoqsidaza H2O2 triftofanis, histidinisa da

cisteinis Jangva

qlorperoqsidaza H2O2 triftofanis, histidinisa da

cisteinis Jangva

laqtoperoqsidaza •HO triftofanis, histidinisa da

cisteinis Jangva

glutaTionperoqsidaza −•

2O triftofanis, histidinisa da

cisteinis Jangva

mieloperoqsidaza H2O2, ClO_ meTioninis, Tirozinis Jangva

NADH-oqsidaza H2O2, ClO_ hemis dakargva

296

fermentTa TviTinaqtivaciis movlena SeiZleba ganxilul iqnas,

rogorc “apoptozi fermentul doneze”, ramdenadac am dros Tavisu-

fali radikalebis generatori fermenti TviTon gamodis mwyobridan

da ujredisaTvis arasasurveli intermediatebis warmoqmna safuZ-

velSive ispoba. aqve unda aRvniSnoT amJamad arsebuli, sakmaod dama-

jerebeli mosazreba imis Sesaxeb, rom modifikaciis gziT makromo-

lekulebis inaqtivacia CarTuli unda iyos Sidaujreduli cilebis

brunvis meqanizmSi, romlis saSualebiTac ujredidan maTi moleku-

lebis mocileba regulirdeba. es garemoeba uaRresad mniSvnelovania

yvela im situaciisaTvis, romelic qsenobiotikebisa da sxvadasxva

wamlebis Warbi raodenobis organizmidan gamodevnasTanaa dakavSi-

rebuli [57]. fermentTa inaqtivaciis molekuluri meqanizmi da

ujredSi misi WeSmariti roli jer kidev safuZvlian Seswavlas

moiTxovs. am mimarTebiT mogvepoveba Semdegi faqtebi: Jangbadis

aqtiuri formebiT mravali fermenti JangviT modifikacias ganicdis;

inaqtivacia SeiZleba ganxorcieldes fermentulad, qimiurad an

radiolizurad generirebuli reaqciisunariani Jangbadis formebiT;

Jangbadovani radikalebi cilis aqtiur centrTan axlos myofi

aminomJavuri naSTebis modifikaciis unars amJRavneben da es reaqcia

metad specifikuria; aqtiuri centris axlos aRZrulma struq-

turulma cvlilebebma konformaciuli Zvrebi da proteolizisadmi

mgrZnobiarobis gazrda SeiZleba gamoiwvion.

ZuZumwovrebis ujredebSi Jangbadis aqtiuri formebis mTavari

producentebia membranuli NADPH-oqsidazebi da mitoqondriebis

eleqtron-satransporto jaWvebis komponentebi [62]. Jangbadis aqti-

uri formebi garkveul fiziologiuri rols asruleben citoqrom

P450-iT mdidar qsovilebsa da ujredebSi: hepatocitebSi, epiTe-

liur ujredebSi da endoTeliocitebSi [63]. naCvenebia, rom anTebiTi

procesebisas da iSemiiT gamowveuli JangviTi stresis dros

Jangbadis aqtiuri formebis generacia mniSvnelovania sisxlZarRvebis

tonusis regulaciisaTvis, sxvadasxva biologiuri procesebis

mediatorebisa da regulatorebis biosinTezis warmarTvisaTvis [64-

66]. magram umetes SemTxvevebSi Jangbadis aqtiuri formebis warmo-

qmna saziano procesebis inicirebas iwvevs, magaliTad, alkoholis

qronikuli moxmarebisas es agresiuli nawilakebi citoqrom Р450 2Е-is katalizuri aqtiurobis Sedegad RviZlis cirozis ganviTa-

rebas iwveven [67]. citoqrom P450-is mkvlevarTa erT-erTi avtori-

tetuli jgufi (v. liaxoviCi da misi TanamSromlebi _ ruseTis

297

mecnierebaTa akademiis cimbiris filialidan) Tvlis, rom mikroso-

muli monooqsigenazebis mier Jangbadis aqtiuri formebis warmoqmna TviT qsenobiotikebis metabolizmSi monawile fermentuli sistemebis funqcionirebisTavisaa mniSvnelovani, ramdenadac am dros xorcieldeba citoqrom P450-is sapirispiro, anu negatiuri regulacia, inducirdeba qsenobiotikebis meore metabolizmis fazis (koniugaciis) fermentebi, izrdeba ujredebis antioqsidanturi dacva [62].

citoqrom P450-is sapirispiro, anu negatiuri regulacia (e.w.

downregulation) Jangbadis aqtiuri formebis monawilebiT SeiZleba

mimdinarebdes transkripciul doneze, birTvuli NF1-faqtoris inaqtivaciiT, an proteasomuri degradaciis gaZlierebiT. cnobilia,

rom glukokortikoidebi, anTebis sawinaaRmdego citokinebi (simsiv-

nis nekrozis α-faqtori, γ-interferoni, interleikinebi), zrdis

faqtorebi da baqteriuli lipopolisaqaridebi ainhibireben citoq-

rom P450-is mravali izoformis (CYP1A, CYP3A, CYP2B, CYP2Е) genebis eqspresias, rac Jangbadis aqtiuri formebis generaciis

gaZlierebisas NF1-faqtoris inhibirebiT realizdeba [68]. NF1-faqtori sinergiulad moqmedebs policikluri aromatuli naxSir-

wyalbadebis AhR-receptorTan. igi Seicavs cisteinis naSTs

(Cys427), romlis daJangvis SemTxvevaSic NF1-faqtori veRar ukav-Sirdeba dnm-s Sesabamis regulatorul ubans [69]. amas adasturebs

Semdegi faqtebi: wyalbadis zeJangis an katalazas inhibitoris _ 3-

amino-1,2,4-triazolis damateba virTagvebis hepatocitebze, rom-

lebic inkubirdebian induqciisaTvis xelisSemwyob pirobebSi (feno-

barbitalis Semcvel areSi), iwvevs citoqrom P450-is izoformis _ CYP2B1-is translaciaSi monawile matriculi rnm-is raodenobis

mkveTr Semcirebas, xolo 5 mM N-acetilcisteinis damateba piri-

qiT, 5-10-jer zrdis induqciis xarisxs [97]. Jangbadis aqtiuri formebis produqcia mikrosomuli mono-

oqsigenazebis mier mravali cilis, maT Soris citoqrom P450-is JangviT modifikacias iwvevs [71, 72]. Jangbadis aqtiuri formebis

mimarT gansakuTrebiT maRalmgrZnobiare izoformaa CYP2E1, romelic

sxva izoformebTan (CYP1A1, CYP1A2, CYP2B1, CYP2B2 da CYP3A) SedarebiT gacilebiT xanmokle sicocxlisunarianobiT xasiaTdeba.

virTagvis RviZlis mikrosomebSi JangviTi substratis ararsebobisas

CYP2E1 izoformis Semcveloba 6-7 sT-Si naxevrdeba, xolo eTano-

lis damatebis SemTxvevaSi naxevardaSlis periodi 37 sT-mde

xangrZlivdeba [73]. CYP2E1-is degradacias aCqarebs NADPH (1 mM),

298

xolo antioqsidantebi troloqsi (50 µM) da α-tokoferoli (20 µM), iseve rogorc rkinis ionebis xelatorebi deferoqsamini (40 µM) da (100 µM) xels uwyoben mikrosomebSi hemoproteinis natiuri

formiT SenarCunebas.

katalizuri ciklis procesSi citoqrom P450-is inaqtivacia

sxvadasxva meqanizmiT SeiZleba ganxorcieldes (nax. 4.8).

nax. 4.8. oqsidazur reaqciebSi aqtiuri intermediatebis warmoqmna.

RH – substrati; Fe – citoqrom P450-is hemuri rkina [58].

pirveli meqanizmi am hemoproteinis peroqsi-kompleqsis daSlis

Sedegad aqtiuri droebiTi substratebis warmoqmnasTanaa dakavSi-

rebuli. aseT reaqciisunarian intermediatebs miekuTvneba Tavisu-

fali organuli radikalebi, epoqsidebi, N-oqsidebi, S-oqsidebi, aldehidebi, ketonebi da sxv. isini Tavis mxriv kovalenturad

ukavSirdebian apocitoqrom P450-s da mis modifikacias iwveven.

inaqtivaciis es meqanizmi SeiniSneba e.w. “gamanadgurebeli” subst-

ratebis Jangvisas, romlebic Seuqcevadad an TiTqmis Seuqcevadad

ROH

RH

O2

OH2

C OR

RCR

O

H

R N R SRO

O

RHFe

RHFe

RH(FeO2)RH(FeO2)

NADP

NADPH

(peroqsi kompleqsi)

(oqsi kompleqsi)

,

O O

, ,R, ROH, ,

Fe3+

3+

2+

2+1+

+

e_

e_

.., H2O2, HO, HO2O2._

299

ainhibireben ferments. substratis reaqciisunariani jgufebis aqti-

vacias Tan sdevs prosTetul jgufTan an apofermentTan kova-

lenturad dakavSirebuli reaqciisunariani metabolitebis for-

mireba. substratebs, romlebic citoqrom P450-is aseTi gziT inaq-tivacias iwveven, “gamanadgurebeli” substratebi ewodebaT (mag.,

qloramfenikoli ukavSirdeba apofermentis lizinis naSTs; para-

Tioni, qloroformi ukavSirdebian cisteins da a.S.). isini fer-

mentisadmi maRal specifikurobas avlenen.

citoqrom P450-is inaqtivaciis meore meqanizmi dakavSirebulia

katalizur ciklSi fermentis aqtiur centrze Jangbadis aqtiuri

formebis (−•

2O -is, •HO -isa da H2O2-is) generirebasTan. es SeiZleba

araSeuRlebuli monooqsigenazuri reaqciebis Sedegic iyos, rodesac

NADPH-is aRmdgeneli eqvivalentebi arasrulad xmardeba endoge-

nuri Tu egzogenuri naerTebis hidroqsilirebas. superoqsiduli

anionis generireba ZiriTadad citoqrom P450-is prerogativaa,

radgan am procesSi sxva mikrosomuli gadamtanebis wvlili Zalian

mcirea. citoqrom P450 upiratesad mis aqtiur centrze

formirebuli H2O2-iT inaqtivirdeba, maSin rodesac −•

2O -is dismuta-

ciiT miRebuli H2O2 inaqtivacis umniSvnelo efeqts iZleva [74-82].

saerTod, substratis qimiuri bunebidan gamomdinare, citoqrom

P450-is inaqtivacia sami gziT SeiZleba ganxorcieldes [77]. esenia: -

1) apofermentTan metabolitebis Seuqcevadi dakavSireba; 2) meta-

bolitebis hemis rkinasTan TiTqmis Seuqcevadi dakavSireba; 3) hemis

alkilireba an destruqcia. pirveli da mesame SeiZleba kombini-

rebuli iyos. hemis modifikacia misi Semdgomi dazianebiT, Seqcevad

inaqtivacias iwvevs, maSin rodesac apofermentis modifikacia

Seuqcevadia [83, 84].

amgvarad, “gamanadgurebeli” substratebis Jangvisas citoqrom

P450-is inaqtivacias warmoqmnili reaqciisunariani intermediatebi

(Jangbadis nawilobriv aRdgenili formebi) axdenen, romlebic hemis

an apofermentis qimiur modifikacias axorcieleben. inaqtivacia

ufro xSirad hemis degradaciis Sedegia, vidre apoproteinis

destruqcia. aRniSnul gamokvlevebSi citoqrom P450-is P420-ad konversia ar daregistrirda. amasTan dakavSirebiT, ar SeiZleba

gverdi avuaroT im gansxvavebuli monacemebis ganxilvas, romlebic

arCakovis [85] laboratoriaSia mopovebuli: diTionitiT reduci-

rebuli izolirebuli citoqrom P450-is izoforma 2B4 swraf

inaqtivacias ganicdis autooqsidaciisas warmoqmnili Jangbadis

300

aqtiuri tipebiT da es inaqtivacia araaqtiur citoqrom P420-ad konversiaSi gadasvlis fonze mimdinareobs. mikrosomul an

liposomaSi CaSenebuli citoqrom P450 metad stabiluria feri- da

fero-mdgomareobebSi [86]. aqedan gamomdinare, gamoiTqva mosazreba,

rom citoqrom P450-is inaqtivaciisaTvis aucilebelia Tavisufal-

radikaluri safexuri, xolo citoqrom P420-ad konversia misi

degradaciis Sualed safexurad SeiZleba CaiTvalos, radgan igi

sakmaod arastabiluria da O2-is Tanamyofobisas advilad kargavs

hems [87].

oqsidazur da oqsigenazur reaqciebSi formirebuli H2O2-iT

hemis daJangva an dazianeba “gasaRebis” rols asrulebs citoqrom

P450-is inaqtivaciaSi. mravali fermenti oqsidazuri sistemebiT

ganicdis inaqtivacias. maT Sorisaa TviT citoqrom P450, NAD(P)H-

oqsidazebi, qsantinoqsidaza da arafermentuli sistemebi, romlebic

Seicaven askorbats, O2-s da Fe(III)-s an Fe(II)-s [88-90]. sakmaod

detaluradaa Seswavlili baqteriuli glutaminsinTetazas JangviTi

inaqtivaciis meqanizmi [91]. naCvenebia, rom inaqtivacia damokidebulia

O2-isa da NAD(P)H-is Tanamyofobaze: stimulirebas ganicdis Fe(III)-iT da inhibirdeba katalaziT, Mn(II)-iT an EDTA-iT. sisteme-

bisaTvis, romlebic arahemuri rkinis cilas (feredoqsins, puti-

daredoqsins) Seicaven, inaqtivacia inhibirdeba dimeTilsulfo-

qsidisa da manitolis tipis radikalebiT. dadgenilia, rom am

fermentis JangviTi modifikacia asocirebulia erT suberTeulze

histidinis erTi naSTis dakargvasTan da karbonilis jgufis

gaCenasTan. fermentis Semdgomi Jangva meore histidinis dazianebas

iwvevs, magram inaqtivacia ar exeba meTioninis an SH-jgufebis

daJangvas.

reaqciisunarianobis mxriv Jangbadis aqtivaciis produqtebi

erTmaneTisagan gansxvavdeba. TavisTavad H2O2 da −•

2O ar arian Zlie-

ri damJangvelebi. −•

2O -s dabali redoqs-potenciali gaaCnia, xolo

H2O2 SedarebiT stabiluria. amdenad safiqrebelia, rom makromole-

kulebs isini efeqturad pirdapir ver uteven. amave dros cnobilia −•

2O -is inaqtivaciuri unari iseT fermentebTan urTierTqmedebisas,

rogoricaa katalaza, peroqsidaza, transferaza, laqtatdehidroge-

naza da sxv. aSkaraa agreTve makromolekulebze −•

2O -is pirdapiri

destruqciuli moqmedeba. amitom safiqrebelia, rom inaqtivaciis

procesSi ZiriTad damJangvels •HO -radikali warmoadgens, romelic

H2O2-dan haber-veisisa da fentonis reaqciis Sedegad miiReba:

301

H2O2 + O2 O2 + HO + OH _.._

jamurad:

Fe + H2O22+ .Fe + HO + OH3+ _

Fe + O23+ Fe + O2

2+

hidroqsil-radikalebma, romelTac metad xanmokle sicocxlisu-

narianoba aqvT, SeiZleba imoqmedon, rogorc Zlierma damJangvelebma

uSualod maTi generirebis adgilze, maSin rodesac H2O2 da −•

2O

formirebis adgilidan difuziis gziT did manZilze gadaadgilde-

bian. H2O2-ma SeiZleba gaiaros ujredul da Sidaujredul membra-

nul barierSi, xolo −•

2O difuzias mxolod specifikuri aniongam-

tari arxebis gavliT axerxebs. superoqsid-anioni metad reaqciisu-

nariani xdeba hidrofobul an metalTan koordinirebul ubnebSi.

misi Tanamyofobisas hidroqsilis radikalebi cilebis fragmentacias

iwveven. O2-s SeuZlia •HO -damokidebuli Jangvis pirvelad produq-

tebTan urTierTqmedeba, rac makromolekulebis destruqcias iwvevs

[92].

citoqrom P450-is inaqtivacias sagrZnoblad amcireben fosfoli-

pidebi da maTi es efeqti damokidebuli araa membranebSi fosfolipi-

debis Semadgenlobaze. inaqtivacia sustdeba anaerobul pirobebSi da

aseve naxSirbadis monooqsidis an citoqrom b5-is Tanamyofobisas

[49].

hemis alkilirebiT gamowveuli inaqtivaciis erT-erTi

TvalsaCino magaliTia bocvris RviZlis mikrosomebSi dodecinmJavas

citoqrom P450-damokidebuli Jangva. am dros warmoqmnili aqtiuri

intermediati _ keteni hemis acilirebas iwvevs, rac citoqrom

P450-is inaqtivaciis mizezi xdeba [93]. amasTan dakavSirebiT

uaRresad sayuradRebo Sedegebi miiRes helvigma da Tanaavt. [94,

95], rodesac maT analogiuri gamokvlevebi Caatares cercvidan

miRebul citoqrom P450-ze da daadgines, rom cxovelurisagan

gansxvavebiT, mcenareSi inaqtivaciis mTavari mizezi fermentis

aqtiur centrSi hemTan axlos myofi erT-erTi aminomJavas

(savaraudod, cisteinis an histidinis) nukleofiluri naSTis

acilirebaa (nax. 4.9).

amgvarad, cxoveluri da mcenareuli citoqrom P450-is dodecinmJavaTi TviTinaqtivaciis procesSi erTi arsebiTi gansxvaveba

vlindeba: cxovelur hemoproteinSi qimiur modifikacias hemi

302

ganicdis, xolo mcenareulSi apofermenti. cxovelur citoqrom

P450-Si hemi amdros srulad iSleba, fermenti mwyobridan gamodis

da amis Sedegad kargavs Tavis katalizur aqtivobas. sxva viTarebaa

gamovlenili mcenareul citoqrom P450-is SemTxvevaSi. Cvens

laboratoriaSi [96] Catarebulma kvlevebma damajereblad gviCvenes,

rom hemoproteini inaqtivacias ganicdis rogorc monooqsigenaza,

magram imavdroulad maRal peroqsidazul aqtivobas amJRavnebs.

sxvagvarad rom iTqvas, garkveul pirobebSi qimiuri modifikaciis

Sedegad monooqsigenazas aqtiuri centri peroqsidazas aqtiuri

centris analogad transformirdeba. gamovlenil efeqts adgili

aqvs rogorc mikrosomul fraqciaSi (in vitro pirobebSi), aseve

mTlian mcenareul qsovilSi (in vivo pirobebSi).

COOH

COOH

O

C

COOH

O

COOH

COOHHis

N

N N

NFe

CO R

CO R

citoqrom P450-is aqtiuri centri

hemi

dodecinmJava

keteni

dodekanmJavahistidinis

naSTi

nax. 4.9. citoqrom P450-is inaqtivaciis savaraudo sqema helvigis

da Tanaavt. [95] mixedviT. vertikaluri StrixiT naCvenebia mcena-

reuli citoqrom P450-is qimiuri modifikaciis Sedegad aminomJavas naSTTan warmoqmnili acilis jgufi, xolo horizontaluri Strix-

iT _ cxoveluri citoqrom P450-is hemTan warmoqmnili acilis

jgufi.

303

katalizur reaqciebSi mniSvnelovani roli peroqsidazas xuT

Jangva-aRdgeniT mdgomareobas ekuTvnis. esenia aRdgenili (Fe+2 anu

martivad, mdgomareoba 2), daJanguli (Fe+3 anu 3), I kompleqsi (Fe+5

anu 5), II kompleqsi (Fe+4 anu 4) dabolos, mdgomareoba Fe+6

(oqsi-

peroqsidazaSi), romelic aRdgenili fermentis molekulur

JangbadTan urTierTqmedebiT miiReba [97]. CvenTvis sayuradReboa I da

II kompleqsebi.

I kompleqsi (mwvane feris) xasiaTdeba STanTqmis maqsimumebiT

407- da 658 nm-ebze. jorjis hipoTezis [98] Tanaxmad wyalbadis

zeJangTan fermentis urTierTqmedebisas hemis rkina +3-dan

daJangulobis Zlier maRal (+5) mdgomareobaSi gadadis da amis

Sedegad oqso-ionebi FeO3+ (oqsenoidis analogi) warmoiqmneba.

I kompleqsis warmoqmnis kinetika safuZvlianadaa Seswavlili

Cansis [99] klasikuri gamokvlevebiT. mis mier peroqsidazisa da

H2O2-is urTierTqmedeba Semdegi martivi reaqciiT SeiZleba

gamoisaxos:

FeOH2 FeO 3++ H2O2

K3.5 3++ 2H2O (1)

am SemTxvevaSi fermentis porfirini da mexuTe aqsialuri ligandi sibrtyis qveviT imyofebian, xolo reaqciis siCqaris konstanta (K3) bimolekulur pirdapir reaqcias da peroqsidazas Jangvis mdgomareobis +3-dan +5-mde cvlilebas axasiaTebs. es reaqcia sinamdvileSi arc

ise martivia, rogorc zeviTaa gamosaxuli: procesis malimitirebel

stadias win uswrebs wonasworobis damyareba, xolo TviT procesi

SeiZleba mravali stadiisagan Sedgebodes.

H2O2-Tan an sxva damJangvelebTan peroqsidazas urTierTqmedebis

siCqare pH-ze praqtikulad araa damokidebuli. katalazasTan

ZmarmJavas zeJangis urTierTqmedebis Seswavlam aCvena, rom mJavas

aradisocirebuli forma fermentTan gacilebiT swrafad reagirebs,

vidre misi anioni. peroqsidazasTan H2O2-is reaqciisas ar SeiZleba

gamoiricxos −2HO -is upiratesi monawileoba, ramdenadac es anioni

ufro Zlieri ligandia.

I kompleqsis eleqtronuli struqtura didi xnis ganmavlobaSi

diskusiis sagans warmoadgenda, vidre mesbaueris speqtroskopulma

gamokvlevebma I da II kompleqsebSi rkinis ionis mdgomareobis ana-

logia ar daadastures. am monacemebis Tanaxmad, Fe+5-is formaluri

mdgomareobidan gansxvavebiT, I kompleqsSi Fe+4 mdgomareoba realiz-

deba, radgan rkinis ioni erT eleqtrons hemis porfirinis birTvi-

304

dan iRebs, romelic I kompleqsSi kation-radikalis (RO+) saxiT

imyofeba. dolfinma [100] aCvena, rom peroqsidazisa da katalazas I kompleqsebis STanTqmis speqtrebi mTeli rigi metalporfirinuli

kompleqsebis π-kation-radikalebis speqtrebis analogiuria, anu

Jangvis maRali xarisxis mdgomareobaSi myofi rkinis arseboba

SeiZleba aixsnas porfirinis birTvis da rkinis atomze lokali-

zebul eleqtronebis spinuri urTierTqmedebiT. amgvarad, peroqsi-

dazas I kompleqsSi erTi mJangveli eqvivalenti rkinis ionzea

lokalizebuli, xolo meore _ hemoproteinis porfirinis birTvze.

sayuradReboa, rom citoqrom c-peroqsidazaSi erTi mJangveli

eqvivalenti fermentis aqtiuri centris erT-erT aminomJavur

naSTzea lokalizebuli da ara porfirinul birTvze, rogorc

peroqsidazaSia.

II kompleqsi, anu mdgomareoba 4 (wiTeli feris) xasiaTdeba

STanTqmis maqsimumebiT 417, 530 da 561 nm-ebze. igi miiReba H2O2-Tan peroqsidazas urTierTqmedebisas sxvadasxva aRmdgenlebis (aroma-

tuli aminebis, fenolebis, askorbatis, ferocitoqrom c-s, feroci-anidis, iodidis, nitritisa da sxv.) Tanamyofobisas. or ukanasknel

aRmdgenelTan reaqcias Tan axlavs protonis moxmareba. varaudoben,

rom II kompleqss feril-ionis (FeO2+) struqtura gaaCnia da +4

Jangvis xarisxis rkinis (II) ions Seicavs. amas adasturebs

mesbaueris speqtrebic. II kompleqsi radikaluri meqanizmiT reagirebs

aRmdgenelTa molekulebTan da sawyis ferments Fe+3 Jangvis

xarisxiT ubrundeba. II kompleqsis warmoqmnisa da moxmarebis zo-

gierT kanonzomierebas gamosaxavs me-2 da me-3 gardaqmnebi:

3+Fe + H2O + AH.

FeO 3+ + AH2

FeO 2+ + AH2

FeO (2)

I kompleqsi II kompleqsi

(3)

2++ H2O + AH.

zogierTi mkvlevari peroqsidazebisa da sxva hemoproteinebis he-

muri rkinis maRal daJangul mdgomareobis arsebobas undoblobiT

ekideba, Tumca rkinis ionisaTvis aseTi mdgomareobebi araorganul

qimiaSi sakmaod kargadaa cnobili [101]. FeO2+ da FeO3+

ionebi arse-

boben mJava xsnarebSi da Sesabamis kationebs, xolo tute xsnarebSi

Sesabamis hidroqsidebs warmoqmnian. perferitioni )FeO( 23

− da fera-

tioni )FeO( 24

− rkinis +4 da +6 mdgomareobebiT xasiaTdebian da

305

arseboben tute areSi, maSin rodesac mJava areSi isini swrafad iS-

lebian Jangbadis gamoyofiT. peroqsidazebSi rkinis maRali valentu-

ri mdgomareobis stabilizaciaSi did rols asruleben misi ligan-

duri garemocva da aqtiuri centris aminomJavuri naSTebi. ase maga-

liTad, pirSuSxas peroqsidazas II kompleqsSi Fe+4 mdgomareoba Sem-

degnairadaa stabilizebuli: erTi mJangveli eqvivalenti rkinis

atomze, xolo meore porfirinis birTvzea lokalizebuli, romelic

π-kation-radikalis saxiT arsebobs. naCvenebia aseTi radikalis war-

moqmna pirSuSxas cink-peroqsidazaSi [102]. misi daJangvisas K2IrCl6-

iT, erTi eqvivalentiT Raribi peroqsidaza miiReba, romlis STanT-

qmis speqtri peroqsidazebis I kompleqsis msgavsia, xolo epr-signali 2g-faqtoriT xasiaTdeba. daJanguli cink-peroqsidaza SeiZ-

leba xelaxla aRdges sawyis mdgomareobamde K3IrCl6-iT. rkinis ma-

Ralvalenturi mdgomareobis stabilizaciaSi arsebiTi roli SeiZle-

ba Seasrulos fermentis aqtiurma centrma. mag., citoqrom c-peroqsidazas I kompleqsi stabilizebulia fermentis aqtiuri cent-

ris aminomJavebis naSTebis Tavisufali radikalebiT. I kompleqsis

warmoqmnasa da stabilizaciaSi, sul cota, citoqrom c-peroqsidazas aqtiuri centris oTxi aminomJavuri naSTi mainc mona-

wileobs: distaluri histidini-52, arginini-48, proqsimaluri hi-

stidini-174 da distaluri triftofani-51.

arsebobs monacemebi imis Sesaxeb, rom fermentis Sualedi forme-

bis (I da II kompleqsebis) substratebTan urTierTqmedebis dros

adgili aqvs peroqsidazas, wyalbadis zeJangsa da substrats Soris

sammagi kompleqsis warmoqmnas [103], citoqrom P450-is katalizuri

ciklis msgavsad.

mkvlevarTa ramdenime jgufma [104-111] miiRo oqsenoidis anu I da

II kompleqsebis analogebi, romlebsac aramarto fermentTa Suale-

duri kompleqsebis struqturis imitacia, aramed maTTvis damaxasia-

Tebel reaqciebSi monawileobac SeuZliaT.

miRebulia citoqrom P450-is, peroqsidazisa da katalazas II

kompleqsis struqturuli da funqciuri analogebi [108-111]. zo-

gierTi rkinis (III) porfirinebis orbirTvuli zeJanguri kompleqsebi

N-meTilimidazolis, piridinis an piperidinis moqmedebisas

[PFeIV=O]2+-tipis naerTebad gardaiqmneba [109-111]. am SemTxvevaSi ori

SesaZlo reaqcia mimdinareobs:

2PFeIII_OO_FeIIIP → 2PFeIII_O_FeIIIP + O2 (4)

306

2PFeIII_OO_FeIIIP + 2B → 2[BPFeIV=O] (5)

sadac, B – piridini, piperidini an N-meTilimidazolia.

[BPFeIV=O]-Si rkinis ionis mdgomareoba I da II kompleqsebSi

misi mdgomareobis analogiuria (cxrili 4.2).

cxrili 4.2

citoqrom P450-isa da misi kompleqsebis struqturuli modelebi

sadac OEP _ 2,3,7,8,12,13,17,18-oqtaeTilporfirinis dianionia.

1972 wels o’braienis [112] laboratoriaSi pirvelad iqna naCve-

nebi, rom citoqrom P450-s peroqsidazas Tvisebebi gaaCnia. fermenti sterolebis hidrozeJangebis spirtebad aRdgenas akatalizebs wyal-

badis iseTi donoris Tanamyofobisas, rogoric N,N,N,N’-tetrameTil-p-fenilen-diaminia (TMPD). am SemTxvevaSi peroqsidazul

aqtivobaze gavlenas ar axdenen CO, azoti, EDTA da 2-fenil-2-

propanoli. mxolod mcire mainhibirebel (20%-iT) moqmedebas av-

lenda natriumis azidi. mikrosomebis 80°C-mde gacxelebiT peroqsi-

dazuli aqtivoba 95%-iT qveiTdeboda.

RviZlis mikrosomebis peroqsidazul reaqciebze damTrgunvel

moqmedebas amJRavneben mikrosomuli redoqs-jaWvis substratebi,

kerZod, I tipis substratebi: androstendioni, testosteroni, 17β-estradioli, aminopirini, heqsobarbitali da linolis mJava. maT

mier gamowveuli inhibireba 65-34%-s aRwevs, maSin rodesac II tipis substratebi _ anilini, imidazoli, piridini, kortikosteroli da

n-oqtilamini peroqsidazul aqtivobas 83-50%-iT aqveiTeben.

aRmoCnda agreTve, rom sxva hemSemcveli naerTebisagan (hematini,

methemoglobini, citoqromi c, citoqrom P420) gansxvavebiT yvelaze

maRali peroqsidazuli aqtivoba citoqrom P450-s gaaCnia.

citoqrom P450-is peroqsidazul reaqciebSi wyalbadis dono-

rebad SeiZleba gamoyenebul iqnas NADH an NADPH [113]. eqspe-

rimentul monacemTa erToblioba [112, 113] imaze miuTiTebs, rom

modeluri sistema modelis daxasiaTeba

OEP_FeIIICl + 2-iodozo-

meta-qsiloli

oqsenoidis, anu citoqrom P450-is kompleqs I-is, katalazisa da peroqsidazas modeli: [FeV=O]3+

PFe_OO_FeP + N-meTil-

imidazoli [PFeIV=O] citoqrom P450-is kompleqs II-is, katalazisa da

peroqsidazas modeli: [FeIV=O]2+

307

peroqsidazul aqtivobas citoqrom P450-is daJanguli forma

flobs. am aqtivobas CO imitom ar ainhibirebs, rom igi kompleq-

sirebas mxolod hemoproteinis aRdgenil formasTan ganicdis.

gamoTqmulia mosazreba, rom hidrozeJangebTan citoqrom P450-is urTierTqmedebisas warmoiqmneba peroqsidazas I kompleqsis analo-

giuri forma [114], romelSic hemuri rkina SeiZleba Jangvis maRal

xarisxSi imyofebodes:

P450_Fe3+

ROOH ROH

TMPD NAD(P)HP450_Fe5+ (6)

cnobilia, rom pirveladi alifaturi spirtebi peroqsidazas

substratebs ar warmoadgenen [115]. miuxedavad amisa, virTxis

RviZlis mikrosomebi da maRali sisufTavis citoqrom P450 akata-lizebs spirtebis aldehidebamde Jangvis reaqciebs [116]. kumilis

hidrozeJangiT da citoqrom P450-is an katalazas monawileobiT

spirtebis daJangvis unari alifaturi radikalis zomis zrdis

mixedviT mcirdeba. citoqrom P450 gansakuTrebiT efeqtur

katalizators warmoadgens kumilis hidrozeJangiT eTanolis daJan-

gvaSi, maSin rodesac citoqrom c, hemoglobini da peroqsidaza spir-

tis Jangvas praqtikulad ar axorcieleben. fenobarbitaliT mikro-

somebis induqcia 5-jer zrdis kumilis hidrozeJangiT eTanolis

Jangvas [117].

arasasurveli damatebiTi procesi, romelic Tan axlavs mravali

substratis hidrozeJangur Jangvas, TviT am naerTebis mxridan cit-

oqrom P450-is destruqciaa [118, 119]. kumilisa da mesameuli

buTilis hidrozeJangebi aramarto citoqrom P450-s, aramed

mikrosomul jaWvSi eleqtronTa transportis mniSvnelovan

komponents _ citoqrom b5-sac Slian [119].

arCakovisa da misi TanamS. mier [120, 121] Catarebul iqna

gamokvleva, romelic miznad isaxavda mikrosomuli Jangvis tipiuri

substratis _ N,N-dimeTilanilinis (DMA-s) monooqsigenazuri

meqanizmiT Jangvis Seswavlas, anu DMA-sTan kompleqsSi citoqrom

P450-iT stimulirebuli aRdgenis gavlenis dadgenas am substratis

N-demeTilirebis, p-hidroqsilirebisa da N-Jangvis reaqciaTa

siCqareebze. NADPH-citoqrom P450-reduqtazuli reaqciis mastimu-

lirebel faqtorad gamoiyeneboda Mg2+-is ionebi [122, 123]. aseTi midgoma sakmaod originaluri iyo hidroqsilirebis meqanizmSi komp-

308

leqsis aRdgenis reaqciis rolis srulad gamosavlenad: hidro-

qsilirebis jaWvSi reduqtazuli reaqcia Tu marTlac malimi-

tirebelia, maSin Mg2+-is daxmarebiT misi siCqaris gazrdas erTi

substratis Jangvis samive tipis reaqciis stimulireba unda

moexdina. am SemTxvevaSi substratad DMA-s gamoyeneba amis srul

saSualebas iZleva. misi gardaqmnis SesaZlo gzebi mocemulia sqemaze

(nax. 4.10).

Mg2+-is sxvadasxva koncentraciebis moqmedebis Seswavlam DMA-s

N-demeTilirebasa da p-hidroqsilirebaze gansxvavebuli mastimuli-

rebeli efeqtebi gamoavlina. aqtivaciis koeficientebi Sesabamisad

1.5 da 2.2-s Seadgenda. N-demeTilirebis maqsimaluri stimulacia

miiRweoda 15 mmoli MgCl2-is Tanamyofobisas. p-hidroqsilirebi-

saTvis aucilebeli aRmoCnda Mg2+-is ufro maRali koncentraciebi.

rac Seexeba DMA-s N-Jangvas, masze am metalis gamaaqtiurebeli

moqmedeba ar gamovlinda da am Sedegs avtorebma orgvari axsna

misces: demeTilireba da hidroqsilireba an NADPH-specifikuri

flavoproteinis meSveobiT ar xorcieldeba, an N-JangvaSi sxva,

Mg2+-isadmi aramgrZnobiare flavoproteini monawileobs [120].

DMA-s peroqsidazuli gziT Jangvis procesi neiloris [124]

mieraa Seswavlili. am avtors detalurad aqvT ganxiluli cdis

msvleloba da Cvenc aq ucvlelad mogvaqvs misi aRwera (nax. 4.11).

309

NCH3 CH3

NCH3 CH3

NCH3 CH3

OH

NH CH3

O

NADPH + O2

N,N-dimeTilanilini

(DMA)N-oqsid-N,N-dimeTilanilini

N-monomeTilanilini

N,N-dimeTil-p-aminofenoli

+ CH2ON-demeTilireba

N-Jangva

p-hidroqsilireba

+ H2O

+ H2O

nax. 4.10. dimeTilanilinis Jangvis gzebi RviZlis mikrosomebSi.

nax. 4.11. dimeTilanilinis peroqsidazuli meqanizmiT Jangva [124].

N(CH3)2

C6H5N(CH3)2, CH3COOH-Si+

C6H5N(CH3)2, CH3COOH-Si+

C6H5N(CH3)2, CH3COOH-Si+

(CH3)2N N(CH3)2

ufero

II

(CH3)2N N(CH3)2+

mwvane

III

N(CH3)2

N(CH3)2

(CH3)2N N(CH3)2

I adimeTilanilini

lurji

Seferiloba

narinjisferi

IV++

+

yviTeli

. I

310

ZmarmJavaSi (pH 4.5) ganzavebul DMA-s xsnars emateboda

fermenti da H2O2. sareaqcio are jer yviTldeboda (I stadia),

magram male Seferiloba muq mwvaneSi gadadioda (II stadia).

warmoiqmneboda cisferi naleqi, xolo xsnari mwvanedan mewamul-

lurj Seferilobas iRebda. mwvane xsnars Tu Warbad daemateboda

wyalbadis zeJangi da peroqsidaza, Cndeboda Jolosferi, romelic

swrafad brundeboda mwvane ferSi (III stadia). Jangvis damTavrebis Semdeg naleqi ifiltreboda da calkeuli

komponentebi qromatografiiT calkevdeboda. kvalis saxiT bevri

Seferili nivTiereba gamovlinda, xolo ZiriTad produqts TMPD

warmoadgenda (gavixsenoT, rom es nivTiereba, sterolebis

peroqsidazuli meqanizmiT JangvaSi wyalbadis donoris rols

asrulebs [119]).

sqemaSi moyvanili II naerTis warmosaqmnelad, romelic DMA-s p-mdgomareobidan wyalbadis mocilebis Sedegad miiReba, aRniSnulma

mkvlevarebma ori alternatuli meqanizmi warmoadgines: maTgan

umartivesis Tanaxmad, Tavdapirvelad generirdeba I naerTis

Tavisufali radikalebi, romlebic tetrameTilbenzidins (II-s) warmoqmnian. meore meqanizmi gulisxmobs, rom reaqciis pirvel

stadias warmoadgens dimeTilanilinidan eleqtronis mogleja da

ion-radikalis (I-is) warmoqmna. aseT nawilakebs protonTa

eliminirebiT SeuZliaT gaormageba da H-is mocileba. rogorc ar

unda iyos, swrafad qrobadi yviTeli Seferiloba (I stadia)

SesaZloa labiluri radikaluri nawilakebis (I-isa da II-is) TanamyofobiT iyos ganpirobebuli.

warmoqmnili TMPD-s nawili naleqad gamoyofas aswrebs, maSin,

rodesac darCenili nawili Semdgom Jangvas ganicdis. marTlac,

ganzavebul ZmarmJavaSi am naerTis peroqsidazuli Jangvis

substratad gamoyenebisas, pirveldawyebiTi momwvano-yviTeli

Seferiloba muq mwvane SeferilobaSi gadadioda. Semdgomi JangviT

xsnari narinjisfer Seferilobas iRebda. amgvarad, SeiniSneboda

igive Seferilobebi, rogorsac adgili hqonda DMA-s Jangvisas II da III stadiebze.

xsnaris mwvane feri ganpirobebulia III kation-radikalis

TanamyofobiT, romelic TMPD-dan eleqtronis mowyvetisas

warmoiqmneba. Semdgomi Jangvisas kidev erTi eleqtroni ixarjeba,

ris Sedegadac IV-is qinoiduri struqtura miiReba. masze gadasvla

311

dakavSirebulia rezonansis SesaZleblobaTa SemcirebasTan da amitom

Tan unda sdevdes feris intensiur matebas. varaudobeb, rom IV

stadiaze narinjisferi Seferiloba ganpirobebulia IV-is

dikationiT, romelic Sualedi naerTis rols TamaSobs mcire

raodenobis Seferil nivTierebaTa warmoqmnaSi da romlebic

TavisTavad muxtis gadamtan kompleqsebs warmoadgenen. sayuradReboa

erTi faqti: DMA-s arc monooqsigenazur da arc peroqsidazul

JangviT garaqmnebSi oqsidazuri sistemebis Mg2+-iT gaaqtivebis

SemTxvevaSic ki ar figurirebs am substratis N-Jangvis Semdeg N-oqsidis warmoqmna. rogorc zeviT aRiniSna, N-JangvaSi monawile

flavoproteini aramgrZnobiarea magniumis ionebis mimarT [120].

Cvens mier [96, 125, 126] dadgenilia, rom praqtikulad yvela is

JangviTi procesi, romelic mikrosomebSi mimdinareobs, met-naklebi

xarisxiT citoqromi P450-is araaqtiur citoqrom P420-ad Seuq-

cevad konversias iwvevs. simindis 7-dRiani aRmonacenebis fesvebSi

mikrosomebis citoqromuli komponentebis Semadgenlobis Seswavlam

aCvena, rom masSi ZiriTadad citoqromi P450 dominirebs. mikro-

somebis “asakSi SesvlasTan” (7-9 dRiani) dakavSirebiT SeiniSneba

citoqrom P450-is Semcireba da citoqrom P420-is Semcvelobis

zrda, rasac imavdroulad fraqciaSi peroqsidazuli aqtivobis

ganuxreli zrdac Tan axlavs (nax. 4.12). analogiuri suraTi

SeiniSnaeba “axalgazrda” (7-9 dRiani) nazardebidan gamoyofili

mikrosomuli fraqciis 120 wT-iani intensiuri aeraciisas: citoq-

romebis P420/P450-is Tanafardobis 5-jer zrdas peroqsidazuli

aqtivobis 1.5-jer mateba Seesabameba (nax. 4.13).

“xandazmuli” (14-dRiani) mcenareebidan gamoyofili mikroso-

mebisa da pirSuSxas peroqsidazuli preparatis speqtrulma ana-

lizma maTi absoluturi identuroba dagvidastura, kerZod, orive

STanTqmis maqsimums 420 nm-ze amJRavnebs, xolo diTionitiT

aRdgenisas maqsimumis 432 nm-ze wanacvleba xdeba. wyalbadis zeJangs

hemi kvlav daJangul mdgomareobaSi gadahyavs. analogiuri speqtrebi

gaaCniaT naxSirbadis monoqsidiT aRdgenil maT kompleqsebsac. aqedan

gamomdinare, naTeli xdeba, rom mikrosomaSi arsebobs hemoproteini,

romlis hemis mdgomareoba peroqsidazas hemis identuria.

gel-filtraciuli qromatografiis analiziT dadginda, rom

mikrosomuli fraqcia or hemoproteins Seicavs, romelTagan pero-

qsidazuli aqtivoba mxolod ∼100 kD molekuluri masis mqones

gaaCnia (nax. 4.14). cilis es masa ar Seesabameba peroqsidazis

312

fermentuli cilis preparatis molekulur masas (40 kD). meore,

SedarebiT dabali molekuluri masis (10-15 kD) mqone hemoproteini

citoqrom b5-s warmoadgens. fraqciaSi identificirebulia arahemuri

cilac (∼30-40 kD), savaraudod _NADPH-citoqrom P450-reduqtaza.

nax.4.12. mikrosomebSi citoqrom P450-isa da P420-is Semcvelobis da peroqsidazuli aqtivobis cv-lilebebi mcenaris asakisagan dam-okidebulebiT [96]. 1 _ citoqromi P450; 2 _ citoqromi P420; 3 _ peroqsidazuli aqtivoba.

nax.4.13. citoqromebis P420/P450-is Tanafardobisagan damokidebulebiT. peroqsidazu-li aqtivobis cvlilebebi mikro-somebis 120 wT-iani aeraciis pirobebSi [96]. 1 _ peroqsidazuli aqtivoba; 2 _ P420/P450.

monooqsigenazuri meqanizmis peroqsidazuliT Senacvlebis suraTi savsebiT aSkarad gamoikveTa flavonoidebis mikrosomuli Jangvis

SeswavliT [125]. am mizniT Cvens mier gamocdil iqna meToqsilirebis

gansxvavebuli xarisxis mqone oTxi flavonoidi. maTi SerCeva im mosazrebiT moxda, rom ori maTgani (nobileteni da 5-OH-nobiletini) unda daJanguliyo monooqsigenirebiT (O-demeTilirebiT), xolo ori (limocitrini da kvercetini), romlebic ukve hidroqsilirebul produqtebs warmoadgenen, peroqsidazuli Jangvis meqanizmiT unda gardaqmniliyvnen. garda amisa, cxovelur obieqtebSi es nivTierebebi monooqsigenazebis mZlavr induqtorebad iTvlebian [127].

2.0

6 8 10 12 140

10

20

30

40

citoqrom P420-isa da P450-is

Semcveloba, pmoli mg cilaze

asaki, dRe-Rame

..

....

..

....

1.0

.. .....

∆A450/wT mg cilaze

peroqsidazuli aqtivoba,

1

2

3

0

10

20

30

40

citoqromebis P420/P450

Tanafardoba

dro, wT

.40 80 120

0.2

0.3

0.4

0.5

0.6

...

....

∆A450/wT mg cilaze

peroqsidazuli aqtivoba,

1

2

313

nax. 4.14. mikrosomuli

fraqciis gelfiltraciis

qromatograma [96].

1 _ A280; 2 _ A432; 3 _ peroqsidazuli aqtivo-

ba.

sveti _ 50×1.6 sm; geli _ ULTROGEL AcA 44; eluenti _ 0.05% Na-SDS 1/30 M fosfatis buferSi (pH 7.4).

O

OOCH

OCH

OCH

OCH

OC

CO

3

3

3

3

H3

H3

O

OOH

OCH

OH

OCH

OH

33

nobiletini

(5,6,7,8,3',4'-heqsameToqsiflavoni)

limocitrini

(3,5,7,4'-tetraoqsi-8,3'-dimeToqsiflavoni)

O

OOH

OH

OHOHO

OOH

OCH

OCH

OCH

OC

CO

3

3

3

H3

H3

5-OH-nobiletini

(5-oqsi-6,7,8,3',4'-hentameToqsiflavoni)

kvercetini

(3,5,7,3',4'-pentaoqsiflavoni)

nobiletini da 5-OH-nobiletini mikrosomebSi mxolod NADPH-is Tanamyofobisas iJangebian (nax. 4.15). amis Sedegad sareaqcio areSi adgili aqvs formaldehidis dagrovebas. kosubstratad H2O2-is gamoye-neba mocemuli reaqciis msvlelobaze gavlenas ar axdens, e.i. aRniSnuli flavonoidebidan meTilis jgufis mocileba monooqsigenirebis gziT xorcieldeba. kinetikuri mrudis platoze gasvlis Semdeg NADPH-is ganmeorebiTi damateba registrirebad cvlilebebs aRar iZleva. es imaze miuTiTebs, rom am momentSi O-demeTilirebis substrati mTlianadaa daxarjuli da reaqcia damTavrebulia. warmoqmnili produqtebis

10 30 50 70

0.2

0.4

1.0

2.0

ABS ∆A450/wT mg cilaze

peroqsidazuli aqtivoba,

eluentis moculoba,ml

. ..........

.............

..

........ .. ...............

1

2

3

314

Semdgomi Jangva (misi koncentraciis Semcireba) am SemTxvevaSi ukve H2O2-damokidebuli xdeba, anu monooqsigenirebuli flavonoidi amjerad ukve peroqsidazas substratadaa gadaqceuli. limocitrinisa da kvercetinis NADPH-damokidebuli Jangva mikrosomul fraqciaSi saerTod ar xorcieldeba da reaqcia mxolod H2O2-is TanamyofobiT mimdinareobs (kvercetinis Jangvis mrudi mocemulia nax. 4.16-ze).

amgvarad, mcenareul mikrosomebSi meToqsilirebuli flavonoide-

bi monooqsigenazuri, xolo hidroqsilirebuli _ peroqsidazuli

meqanizmebiT gardaqmnebian. am ori reaqciis Tanmimdevruli Senacvle-

ba citoqrom P450-is qimiuri modifikaciis anu araaqtiur citoqrom P420-ad misi konversiis Sedegia.

nax. 4.15. nobiletinis Jangva eTiolirebuli si-mindis 7-dRiani nazardebis fesvebis mikrosomebSi. sainkubacio xsnaris (3 ml) Semadgenloba: 1/15M fos-fatis buferi pH 7.4; 0.33 mg/ml mikrosomuli cila; 100 µM NADPH; 15 µM H2O2; 25 µM nobiletini [125].

nax. 4.16. kvercetinis Jang-va eTiolirebul simindis 14-dRiani nazardebis fes-vebis mikrosomebSi. sainku-bacio xsnaris (3 ml) Se-madgenloba: 1/15 M fosfa-tis buferi pH 7.4; 0.33 mg/ml mikrosomuli cila: 100 µM NADPH; 15 µM H2O2, 25 µM kvercetini [125].

citoqrom P450-is iseT qimiur modifikacias, romlis drosac

NADPH

A365 NADPH

dro, wT10 20

1.0

2.0

H2O2

30

NADPHA365

dro, wT10 20

0.5

1.0

H2O2

315

adgili aqvs fermentuli moqmedebis meqanizmis Senacvlebas, ufro

esadageba termini “transformacia”, radgan igi ukeTesad asaxavs

movlenis arss.

cotoqrom P450-is qimiur modifikaciaSi mniSvnelovan adgils

ikavebs NADPH-is Tavisufali (hidroqsilirebasTan araSeuRle-

buli) Jangva. am procesis msvlelobas citoqromebis P420/P450-is Tanafardobis 10-jer zrda da peroqsidazuli aqtivobis 1.8-jer

zrda Seesabameba. aseve, mikrosomul membranaSi intensiuri

lipoperoqsidaciisas peroqsidazuli aqtivoba ormagdeba. citoqrom

P450-is peroqsidazad maqsimaluri transformacia miiReba

mikrosomuli Jangvis substratis _ 3,4-benzpirenis gardaqmnisas,

rodesac P420/P450 Tanafardobis 25-jer zrdisas peroqsidazuli

aqtivoba sammagdeba. SedarebiT dabali xarisxis transformacia

axlavs mikrosomuli redoqs-jaWvis meore tipiuri substratis _

dimeTilanilinis Jangvas (monacemebi diagramebis saxiT

warmodgenilia nax. 4.17-ze).

nax. 4.17. simindis 7-dRiani eTiolirebuli nazardebis mikrosomebSi

peroqsidazuli aqtivobebis cvlilebebi aeraciisa da

sxvadasxva substratebis Jangvisas [126].

miRebuli Sedegebis asaxsnelad Cvens mier gamoTqmul iqna

0

1

2

3

4

5

6

kontroli

aeracia

NADPH-is

Tavisufali

Jangva

lipoperoqsidacia

3,4-benzpirenis

Jangva

N,N-

dimeTilanilinis

Jangva

P420

/P45

0

0

50

100

150

200

250

300

350

peroqsidazuli aqtivoba, %

citoqromebis P420/P450 Tanafardoba peroqsidazuli aqtivoba

NA

DPH

N,N

- P P

316

mosazreba, rom citoqrom P450-is amgvar transformacias sain-kubacio areSi warmoqmnili Tavisufali radikalebi unda iwvevdnen.

cnobilia, mag., rom NADPH-is Tavisufali Jangvis procesi oTxi

gansxvavebuli gziT SeiZleba warimarTos (ix. qveTavi 4.1.2.2) [18].

pirveli sami reaqcia oqsidazuria, xolo meoTxe hipoTezuri

endogenuri substratis (XH) monooqsigenazuri meqanizmiT Jangvas

asaxavs. pirveli reaqciis Sedegad superoqsiduli anion-radikali

)O( 2−•

warmoiqmneba. mikrosomuli fraqciis aeraciisas Jangbadis

mudmivi miwodeba aCqarebs sareaqcio areSi endogenuri NADPH-isa

da substratebis Jangvas. imis gamo, rom am ukanasknelTa raodenoba

uaRresad limitirebulia, Sesabamisad efeqtic dabalia. am mosaz-rebis Sesamowmeblad SevecadeT gamogvevlina is aqtiuri inter-mediati, romelic citoqrom P450-is citoqrom P420-Si gadasvlas

iwvevs da monooqsigenazuri aqtivobis peroqsidazuliT Secvlas

ganapirobebs; kerZod, SevecadeT dagvedgina, aris Tu ara es nawilaki

superoqsiduli anion-radikali, romelic, rogorc cnobilia,

zogierTi substratis Jangvisas uSualod citoqrom P450-is aqtiur centrze generirdeba [57-59]. am mizniT Cvens mier Catarebul iqna

eqsperimenti [126]: mikrosomul fraqciaSi TanmimdevrobiT Segvqonda

NADPH, 3,4-benzpireni, taironi (4,5-dihidroqsi-1,3-disulfomJava),

katalaza (EC 1.11.1.6) da am pirobebSi polarografiulad vakvir-

debodiT Jangbadis cvlilebebis dinamikas (nax. 4.18).

polarografiuli mrudidan Cans, rom pirveli ori naerTis dama-

tebisas registrirdeba Jangbadis STanTqma, xolo taironi, romelic

superoqsiddismutazas (EC 1.15.1.1) dabalmolekuluri, membranaSi

ganvladi analogia da superoqsiduli anion-radikalebis damWerad

iTvleba (2H++2 −•

2O →H2O2+O2), Jangbadis moxmarebis mrudis

mimarTulebis mkveTr cvlilebas iwvevs, rac Jangbadis gamoyofis

maCvenebelia. analogiuri efeqti gaaCnia katalazasac, romlis

moqmedebiTac warmoqmnili wyalbadis zeJangi aseve iSleba Jangbadis

gamoyofiT (2H2O2 → H2O + O2). am eqsperimentiT (Tumca arapirdapiri gziT) dasturdeba, rom

3,4-benzpirenis NADPH-damokidebuli Jangvisas marTlac adgili

aqvs superoqsiduli radikalebis warmoqmnas, romlebic moqmedeben

citoqrom P450-is hemze da mis Tvisobriv da raodenobriv

cvlilebebs iwveven.

317

0

5

10

15

20

25

250 500 750 1000 1250Jangbadis koncentracia, nM

dro,wT

katalaza

taironi

3,4-benzpireni

NADPH

0

nax. 4.18. simindis eTiolirebuli nazardebis mikrosomuli fraqciis

mier 3,4-benzpirenis NADPH-damokidebuli Jangvis polarograma.

0.067 M KH2PO4-Na2HPO4 buferi, pH 7.4;K3,4-benzpireni – 0.01 mM; NADPH – 0.24 mM, mikrosomuli fraqcia 1.0 mg/ml [126].

taironi da katalaza gamocdil iqna N,N-dimeTilanilinis NADPH-

damokidebuli Jangvisa da Fe2+-iT inicirebuli mikrosomuli arafer-mentuli lipoperoqsidaciis procesis mimarTac: arc erT SemTxvevaSi adgili ar aqvs Jangbadis gamoyofas; e.i. aRniSnul procesSi super-oqsiduli radikalebi ar generirdebian. rogoc Cans, lipidTa zeJanguri Jangvisas Jangbadis sxva tipis aqtiuri formebi warmoiqmnebian.

citoqrom P450-is peroqsidazad transformiaciis procesi aSkarad SeiniSneba mcenaris asakSi SesvlasTan dakavSirebiTac. simindis 7-dRiani eTiolirebuli nazardebis fesvebidan miRebul mikrosomul fraqciaSi citoqrom P450-is raodenoba da, Sesabamisad, monooqsigenazuri aqtivoba maqsimaluria. momdevno dReebSi citoqromi P450 citoqrom P420-ad Seuqcevad konversias ganicdis, rasac paralelurad Tan axlavs peroqsidazuli aqtivobis zrda. 7-dRianTan SedarebiT 14-dRiani nazardebis mikrosomebSi P420/P450 Tanafardoba 30-32-jer, xolo

peroqsidazuli aqtivoba 5-6-jeraa gazrdili [128].

bunebrivia, asakze damokidebul citoqrom P450-is trans-

318

formacias Tan unda axldes citoqrom P450-damokidebuli biosin-Tezuri procesebis intensivobis cvlilebebic. am sakiTxis kvle-

visas, Sidaujreduli procesis modelad SerCeul iqna ligninis

biosinTezis fenilpropanoiduri gza, romelSic ramdenime citoqrom

P450-Semcveli monooqsigenaza monawileobs [129-131]. maTgan umTav-

resia dariCinmJava-4-hidroqsilaza, romelic fenilalanin-amiak-lia-

zasTan erTad biosinTezis umniSvnelovanes ferments warmoadgens.

kvlevis mizans Seadgenda imis dadgena, Tu rogor icvleba aRniS-

nuli procesis intensivoba citoqrom P450-is transformaciis

pirobebSi. amisaTvis Seiswavleboda biosinTezSi monawile zemoT-aRniSnuli fermentebis aqtivoba 7- da 14-dRiani nazardebis mikro-somebSi. aRmoCnda, rom 7-dRianTan SedarebiT 14-dRiani nazardebis

mikrosomebSi orive fermentis aqtivoba mkveTrad mcirdeba [128], e.i.

citoqrom P450-is transformaciis pirobebSi mikrosomebSi wydeba

dariCinmJavas hidroqsilirebiT mimdinare metaboluri gza, rasac

Tan axlavs wina, amiak-liazuri reaqciis blokirebac.

eTiolirebul nazardebSi, sadac fotosinTezi gamoricxulia, ligninis biosinTezi mniSvnelovnad unda iyos damokidebuli citoqrom P450-is aqtivobaze, ramdenadac am procesSi erT-erTi siCqare-

malimitirebeli stadia _ dariCinmJavas p-kumarmJavad gardaqmna citoqrom P450-Semcveli monooqsigenaziT _ dariCinmJava-4-

hidroqsilaziT (CYP73) katalizdeba [29]. rodesac mcenaris zrdas Tan sdevs citoqrom P450-is transformacia, cxadia, lignifikaciis procesic unda Seicvalos. amis dasadgenad vakvirdebodiT ligninis fraqciaSi radioaqtiuri (4-14C)dariCinmJavas niSandebuli naxSirbadis CarTvas [25, 128]. aRmoCnda, rom ligninSi 14C-is CarTva maqsimaluria 7-9 dRis intervalSi, Semdeg ki mkveTrad mcirdeba (nax. 4.19).

ligninSi radioaqtiuri niSnis CarTvis Semcirebis periodi mce-

naris asakTan dakavSirebul citoqrom P450-is transformacias emTx-veva. am eqsperimentis Sedegebi gviCveneben, rom citoqrom P450-is transformacia bunebrivi procesia da im bioqimiuri reaqciebis

intensivobis daqveiTebas iwvevs, romlebic am hemoproteiniT kata-

lizdebian.

319

nax. 4.19. ligninis fraqciaSi dariCinmJavas 14C-niSnis CarTvis dina-

mika simindis eTiolirebuli nazardebis asakTan damokidebulebiT

[26].

4.5.1 NADPH-citoqrom P450-reduqtazas SesaZlo funqciebi

citoqrom P450-is peroqsidazad transformaciis pirobebSi

citoqrom P450-is qimiuri modifikaciis Sedegad cxadia, monooq-

sigenazuri sistemis danarCeni komponentebi axal funqciur dat-

virTvas unda iZendnen. Cvens mier [97] dadgenil iqna, rom am

hemoproteinis peroqsidazad transformirebis fonze NADPH-

citoqrom P450-reduqtaza aqtivobis maRal dones mainc inarCunebs

(nax. 4.20), miuxedavad imisa, rom peroqsidazuli aqtivobis

gamovlenaSi masze generirebuli superoqsiduli radikalebi ar

monawileoben. aRniSnulidan gamomdinare, bunebrivad ibadeba kiTxva:

Seqmnil viTarebaSi ra funqciur rols asrulebs NADPH-citoqrom

P450-reduqtaza?

100

50

7 9 11 13 15

ligninis fraqciis

radioaqtiuroba, %

..

.. .

asaki, dRe-Rame

320

nax. 4.20. mikrosomebSi citoqrom P450-is Semcvelobisa da NADPH-

citoqrom P450-reduqtazuli aqtivobis cvlilebebi mcena-ris asakisagan damokidebulebiT.

1 _ citoqromi P450; 2 _ NADPH-citoqrom P450-reduqtazuli aqtivoba [97].

aRmoCnda, rom reduqtazaze generirebuli −•

2O upiratesad lipi-

dur peroqsidaciaSia CarTuli (nax. 4.21). rogorc gamoirkva,

lipidTa zeJangur Jangvas citoqromi P450 superoqsiduli

radikalebis generatorad sWirdeba da hemoproteinis modifikaciis

SemTxvevaSi am funqcias mTlianad reduqtaza iTavsebs. garda amisa,

Cven SevZeliT gveCvenebina, rom lipidur peroqsidacias reduq-

tazuli aqtivobis mxolod naxevari (50%-mde) xmardeba da es

savsebiT sakmarisia aRniSnuli procesis normaluri msvlelo-

bisaTvis (nax.4.21) [97]. reduqtazas darCenili enzimuri simZlavre

mikrosomuli redoqs-jaWvis sawyisi komponentebis saSualebiT

ujredis energetikul procesebs emsaxureba [128]. aseTi kavSiris

ganxorcieleba mikrosomidan mitoqondriaze eleqtronTa transmemb-

ranuli migraciis gziTaa SesaZlebeli [17, 132].

µΜ/wT mg cilaze

NADPH-citoqrom P450-reduqtazuli aqtivoba,

6 8 10 12 140

10

20

citoqrom P450-is

Semcveloba,

pmoli mg cilaze

asaki, dRe-Rame

5.0

.1*

. . . ..*

** * *

2

321

nax. 4.21. NADPH-citoqrom P450-reduqtazas SesaZlo funqciebi

citoqrom P450-is transformirebis pirobebSi. eleqtronis trans-

portis is gza, romelic am dros aRar funqcionirebs, gadaxazulia

[128].

mcenaris ujredSi mikrosomebidan mitoqondriaze eleqtronebis

gadatanis SesaZlo gzebis arsebobisa da meqanizmebis dasadgenad Cven

farTo eqsperimentuli kvleva CavatareT [128, 132]. cdebis erT

seriaSi mikrosomul fraqcias vainkubirebdiT NADPH-Tan, raTa

redoqs-jaWvis komponentebi winaswar aRdgeniliyvnen (nax. 4.22).

nikotinamiduri kofermentis srul xarjvas vafasebdiT 340 nm-ze

misi STanTqmis maqsimumis SemcirebiT. citoqrom b5-is aRdgenis

maqsimumis miRwevis Semdeg mikrosomebs vamatebdiT mitoqondriebis

fraqcias. citoqrom b5-is Jangvis paralelurad am SemTxvevaSi

SeiniSneboda citoqromebis c1c-isa da aa3-s aRdgena. maSasadame,

ujredis am organelaTa uSualo kontaqtisas mikrosomidan

mitoqondriaze redoqs-eqvivalentebis transmembranuli migracia

xorcieldeba da am procesSi pirdapir arian CarTuli mikrosomuli

NADPH-citoqrom P450-reduqtaza da citoqromi b5. aqve unda

SevniSnoT, rom cxovelur obieqtebSi mikrosomebsa da

mitoqondriebs Soris reducirebadi eqvivalentebis ormxrivi

transmembranuli migracia da masSi am organelaTa citoqrom b5-ebis

SesaZlo monawileoba gamovlenilia gasuli saukunis 70-80-ian

wlebSi [9, 133-134].

e

ee

H2O2

NADPH

NADPH-citoqrom P450-reduqtaza

energetikuli

procesebi 50%

lipidebis zeJanguri

Jangva 50%

transformacia

peroqsidaza

._O2

citoqrom P450

_

_

_

322

nax. 4.22. mikrosomuli da mitoqondriuli citoqromebisa da

NADPH-is aRdgenis kinetika kombinirebul sistemaSi mitoqondria+

mikrosoma [132].

ganxiluli monacemebi yovelgvar safuZvels gvaZleven misaRebad

CavTvaloT mosazreba imis Sesaxeb, rom citoqrom P450 yovelTvis

ar warmoadgens mikrosomuli redoqs-jaWvis mudmiv terminalur

komponents da saerTod, monooqsigenazuri sistema membranaSi,

Cveulebriv, disocirebuli, da ara erT kompleqsad Sekruli

agregatis saxiT unda arsebobdes. rogorc Cans, NADPH-citoqrom

P450-reduqtazasTan da citoqrom b5-Tan dakavSireba da

monooqsigenazur sistemaSi gaerTianeba mas Semdeg xdeba, rodesac

citoqrom P450 substrats ikavSirebs da Tanmimdevrulad ferment-

substratis pirveli an sammagi kompleqsis meore eleqtroniT

aRdgena xdeba saWiro. am hemoproteinTan endogenuri an egzogenuri

substratis dakavSireba im regulatorul signals warmoadgens,

romlis Sedegadac disocirebul mdgomareobaSi myofi redoqs-jaWvis

komponentebisagan monooqsigenazuri kompleqsi yalibdeba. sxva

SemTxvevaSi, rodesac amis saWiroeba araa (mag., NADPH Tavisuflad

iJangeba, citoqrom P450 substrats ar Jangavs, an peroqsidazadaa

Zn2+

....................................................

aRdgenili

citoqromebi, %

100

50

100

50

aRdgenili

NADPH, %

b5

NADPH aa3

c1c

mitoqondriamikrosoma+NADPH

dro wT

0 10 20

323

transformirebuli), NADPH-citoqrom P450-reduqtazasaTvis eleq-tronis aqceptoris funqcia SeiZleba citoqromoqsidazurma sistemam

Seasrulos. amisaTvis aucilebelia ujredSi mikrosomebisa da

mitoqondriebis uSualo kontaqti da am SemTxvevaSi eleqtronTa

gadatana am ori struqturuli warmonaqmnis b-tipis citoqromebis gziT ganxorcieldeba. imis gamo, rom mikrosomuli citoqrom b5-is

redoqs-potenciali (+24 mV) yvelaze axlosaa mitoqondriuli

citoqrom b-s redoqs-potencialTan, aRmdgeneli eqvivalentebis am

gadatanas ar unda axldes energiis raime SesamCnevi kargva.

cnobilia, rom Zn2+ (10-6 M) sunTqviTi jaWvis III kompleqsSi b da c1 citoqromebs Soris arsebuli FeSIII-klasteris blokirebas

iwvevs [15, 135]. amitom mcenaridan miRebul mikrosomebisa da

mitoqondriebis kombinirebul sistemaze dayenebul Cvens cdebSi

movelodiT, rom citoqrom b-ze eleqtronebis migraciisas Zn2+

daaqveiTebda citoqromebis c1c-sa da aa3-is aRdgenis siCqares.

sinamdvileSi miviReT sapirispiro Sedegebi: Zn2+-is moqmedebis

Sedegad adgili hqonda mitoqondriul citoqromebze eleqtronTa

gadatanis siCqaris SesamCnev zrdas (nax.4.22). amasTan dakavSirebiT

gamovTqviT varaudi, rom sunTqviTi jaWvis FeSIII-klasteris

blokirebisas SeiZleba eleqtronis satransporto gzis Suntireba

moxdes da eleqtronebi mitoqondriuli citoqrom b-s gverdis

avliT, mikrosomuli citoqrom b5-idan uSualod c1c- da aa3-

citoqromebze moxvdnen (nax. 4.23). am SemTxvevaSic Sualed

gadamtanad perimitoqondriuli citoqrom b5 unda funqcionirebdes

[128].

amrigad, citoqrom P450-is peroqsidazad qimiuri modifikaciisas NADPH-citoqrom P450-reduqtaza da citoqrom b5 axal, energe-

tikul funqcias iZenen, rac mitoqondriuli sunTqvis jaWvis

aRmdgeneli eqvivalentebiT damatebiT uzrunvelyofaSi mdgomareobs.

qsenobiotikis ujredSi moxvedrisas NADPH-is mikrosomuli Jangvis

SeuRlebuli sistema energetikulidan kvlav detoqsikaciur reJims

ubrundeba.

324

nax. 4.23. mikrosomuli redoqs-jaWvidan mitoqondriaze eleqtronebis

migraciis SesaZlo gzebi. daStrixuli isriT aRniSnulia

Zn2+-iT blokirebuli ubani [132].

qimiuri modifikaciis gziT citoqrom P450-is peroqsidazad

transformacia ujredis saerTo detoqsikaciur potencials ki ar

amcirebs, aramed garkveulwilad aZlierebs kidevac. am dros

monooqsigenazas peroqsidaza enacvleba, romelic qsenobiotikebis

JangviT degradaciaSi aranakleb rols asrulebs. saqme imaSia, rom

zogierTi mkvlevaris TvalsazrisiT mcenareebSi organuli toqsi-

kuri naerTebis ZiriTadi nawili peroqsidazebiT iJangeba [136]. es

hipoTeza eqsperimentulad dasabuTebul Semdeg mosazrebebs eyrdnoba:

_ mcenareebSi peroqsidazas farTo gavrcelebas;

_ fermentis maRal swrafvas sxvadasxva qimiuri struqturis mqone

organuli qsenobiotikebis mimarT;

_ universalur substratul specifikurobas.

NADPH

citoqrom b5modificirebuli

citoqrom P450

mikrosoma

mitoqondria

citoqrom

bFeSIII

klasteri

citoqrom

c1

citoqrom

ccitoqrom

acitoqrom

a3

I kompleqsi

NADPH-CoQ-reduqtaza

NADPH-citoqromP450-reduqtaza

perimitoqondriuli citoqrom b5

325

es Taviseburebebi gansazRvravs peroqsidazas aqtiur monawi-

leobas mTel rig detoqsikaciur procesebSi. qsenobiotikebis

hidroqsilirebis reaqciebSi mcenareuli peroqsidazebis monawile-

obaze mravali faqti miuTiTebs, mag., sxvadasxva mcenareebis peroq-

sidazebs SeuZliaT daJangon N,N-dimeTilanilini [137], 3,4-benz-pireni, 4-nitro-o-fenilendiamini [138], 4-qloranilini [139], fenoli,

aminofluoreni, paraoqsiacetanilidi, dieTilstilbestroli, hidro-

qsibuTiltoluoli, hidroqsianizolebi da sxv. [140]. triTiumiT

niSandebuli meTilis jgufis Semcveli [C3H3] TNT-s gamoyenebiT naCvenebia, rom pirSuSxas peroqsidazas sufTa preparats SeuZlia

iseTi Znelad dasaJangi jgufic ki daJangos, rogorsac TNT-s meTilis jgufi warmoadgens [141].

mravali ucxo naerTis Jangvis dasawyisSi citoqrom P450-s SeuZ-lia funqcionireba, rogorc monooqsigenazas, SemdgomSi ki Jangva

gaagrZelos peroqsidazad transformirebulma hemoproteinma. am

SemTxvevaSi qsenobiotikis degradacia fermentTa urTierTSenacv-

lebis principiT xorcieldeba. Cvens laboratoriaSi naCvenebi iqna,

rom qsenobiotikTa Jangvis procesSi erTmaneTs SeiZleba

Seenacvlon aramxolod monooqsigenaza da peroqsidaza, aramed

fenoloqsidazac [142-144]. am fermentebis koordinirebuli moqme-

deba mravali ucxo naerTis JangviTi degradaciis procesis mTavar

regulatorul meqanizms unda warmoadgendes.

ganxiluli faqtebis Sejerebas erT mniSvnelovan daskvnamde

mivyavarT:

cxoveluri ujredebisagan gansxvavebiT, romelSic Tavisufali

radikalebi hemis modifikaciis da misi Semdgomi destruqciis gziT

citoqrom P450-is srul inaqtivacias iwveven, mcenareul ujredSi

am hemoproteinis qimiuri modifikacia ise xorcieldeba, rom

citoqrom P450 Tavis katalizur aqtivobas peroqsidazas saxiT

inarCunebs. e.i. mcenareuli fermenti mxolod Tvisobriv gardaqmnas

_ transformacias ganicdis. am dros citoqrom P450-damokidebuli

Sidaujreduli metaboluri procesebis intensivoba qveiTdeba. cito-

qrom P450-is transformaciis Sedegad monooqsigenazuri sistemis

Semadgenel komponentebs Soris adre arsebuli funqciuri kavSiri

wydeba da isini axal funqciebs iZenen. aseTi transformaciiT

mcenareuli ujredi, erTi mxriv, cxoveluris msgavsad Tavidan

icilebs agresiuli intermediatebis generators _ citoqrom P450-s, meore mxriv, cxovelurisagan gansxvavebiT, amave ferments

326

peroqsidazas funqcias aniWebs. am gziT ujredi iZens ferments,

romelsac erTdroulad qsenobiotikebis Jangva da agresiuli H2O2-is likvidacia SeuZlia. swored amaSi unda mdgomareobdes qimiuri

modifikaciis gziT citoqrom P450-is transformaciis biologiuri

azri.

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