Proc. Nati. Acad. Sci. USAVol. 77, No. 1, pp. 102-106, January 1980Biochemistry

Restoration of active transport of solutes and oxidativephosphorylatiorr by naphthoquinones in irradiatedmembrane vesicles from Mycobacterium phlei

(UV irradiation/specificity of naphthoquinones/protonmotive force/proline and calcium transport)

SOON-Ho LEE, THOMAS 0. SUTHERLAND, ROSA DEVE&, AND ARNOLD F. BRODWEDepartment of Biochemistry, University of Southern California School of Medicine, Los Angeles, California 90033

Communicated by David E. Green, September 14, 1979

ABSTRACT Irradiation of the inverted membrane vesiclesof Mycobacterium phlei with light at 360 nm inactivated thenatural menaquinone [MK9(II-H)J and resulted in a loss ofsubstrate oxidation, pH gradient, membrane potential, activetransport of proline .or calcium ions, and oxidative phos-phorylation. Restoration of the protonmotive force and activetransport occurred on addition of naphthoquinones such as vi-tamin KX, menadione, or lapachol to the irradiated membranevesicles. However, coupled phosphorylation was restored onlyby vitamin K1. Menadione and lapachol did not act as un-coupling agents. The magnitude of the pH gradient and mem-brane potential in the quinone-restored system was a reflectionof the rate of oxidation and was correlated with the rate of up-take of proline or Ca +. These results are consistent with thechemosmotic hypothesis proposed for the energy transducingmechanism for active transport and further demonstrate thatthe complete respiratory chain is not required to drive activetransport. In contrast, the data suggest that in addition to thedriving force (protonmotive force) necessary to establish oxi-dative phosphorylation, a specific spatial orientation of therespiratory components, such as the naphthaquinones, is es-sential for the utilization of the proton gradient or membranepotential or both. Bypass of electrons from the respiratory chainwith menadione may explain the inability of this quinone torestore oxidative phosphorylation; however, lapachol restoresoxidation by the same electron transport pathway as the naturalmenaquinone but fails to restore phosphorylation. Because allthree quinones restore the protonmotive force, other factors thatare discussed must be considered in understanding the mech-anism of oxidative phosphorylation.

The electron transport chain in Mycobacterium phlei has beenwell characterized (1-3). The natural quinone [MK9(II-H)]found in the electron transport particles (ETP) from M. phleican be selectively destroyed upon exposure to light at 360 nm.Light treatment has been shown to result in a loss of oxidation,phosphorylation, and active transport of solutes (4, 5). Resto-ration of oxidation has been shown to occur with a wide varietyof quinones; however, the, restoration of oxidative phos-phorylation occurs only with certain specific quinones (4, 6).

Studies of the structural requirements for restoration of ox-idation and phosphorylation by quinone after light treatmenthave revealed at least three different responses to quinone ad-dition (6, 7). The nature of the restoration was found to be de-pendent on the substitutions in the C2 and C3 position of thenaphthoquinone nucleus. The three types of restoration lighttreatment and quinone addition were as follows: (i) restorationof oxidation and phosphorylation, by quinones containing amethyl group in the C2 position of the naphthoquinone nucleusand a f3,y-unsaturated isoprenoid side chain of at least fivecarbons in the C3 position, such as that observed in the natural

quinone or vitamin K1; (ii) restoration of only oxidation, by thesame electron transport pathway as that observed with thenatural quinone, by quinones such as lapachol [2-hydroxyl-3-(3-methyl-2-butenyl-1,4-naphthoquinone)] or dihydrophytylvitamin K1; and (iii) restoration of oxidation by "bypassing"a segment of the electron transport pathway or by reactingdirectly with oxygen, by quinones such as menadione (2-methyl-1,4-naphthoquinone) or 2-methoxy-1,4-napthoqui-none.

In contrast to oxidative phosphorylation, active transport ofsolutes can be driven by menadione, lapachol, or artificialelectron acceptors (5, 8). The results obtained with the invertedirradiated membrane vesicles (360 nm) of M. phlei suggest thatthe bioenergetic mechanism for oxidative phosphorylationmight differ from that required to drive active transport ofsolutes.

According to Mitchell's chemosmotic hypothesis (9), ATPsynthesis is driven by the translocation of protons across themembrane as a result of the electrochemical gradient generatedby the passage of electrons through the respiratory chain. In anumber of bacterial systems it has been demonstrated that theprotonmotive force is also the driving force for the activetransport of solutes. Although the protonmotive force consistsof a membrane potential and a proton gradient, certain trans-port systems may be driven primarily or solely by the protongradient, the membrane potential, or both, depending upon thecharge of the substrate and characteristics of the specific carrier(10-12).

This communication describes the restoration of the pHgradient, membrane potential, and active transport of prolineor Ca2+ in light-inactivated ETP from M. phlei by the additionof vitamin K1, menadione, or lapachol. Coupled phosphoryl-ation was restored only with vitamin K1 even though all of thesecompounds restored the protonmotive force.

MATERIALS AND METHODSMaterials. All chemicals were obtained commercially and

were of reagent grade purity.Preparation of Inverted Vesicles. M. phlei (American Type

Culture Collection strain 354) was grown as described (13), and(ETP) were prepared by sonic disruption of cells as describedby Brodie (14) and suspended in 10 mM MgCl2 at a proteinconcentration of 10 mg/ml. Irradiated ETP were obtained bythe procedure previously described (1).

Preparation of Quinones. The quinones were suspended bysonication in 5.0 ml of 50 nmM Hepes/KOH buffer (pH 7.5)

Abbreviations: ETP, electron transport particles or inverted membranevesicles; 9-AA, 9-aminoacridine; TPD, N,N,N',N'-tetramethyl-p-phenylenediamine; CCCP, carbonyl cyanide m-chlorophenylhydra-zone; A4', membrane potential; LApH, transmembrane proton gradient;4LH+, protonmotive force.

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containing 37 mg of partially purified asolectin (15) or phos-pholipid from M. phlei. The preparation was sonicated in a10-kHz Raytheon sonic oscillator for 10 min.Measurement of Oxidation and Coupled Phosphorylation.

Oxygen consumption was measured polarographically andcoupled phosphorylation was measured manometrically at300C, as described (16).Measurement of pH Gradient (ApH). The pH gradient was

measured by the distribution of a weak base, [14C methylamine,using a flow dialysis apparatus (1.0 ml microsize, Bel-ArtProducts, Pequannock, NJ) described by Schuldiner et al. (17).The ApH was calculated by using the equation:

-ApH = log([methylaminelin/[methylamine]out).This is only valid when the external pH is in 1 or more pH unitsbelow the pK (10.65) of the weak base, because under theseconditions nearly all the base exists in the ionized form. Theconcentration gradient of methylamine taken up by themembrane vesicles was calculated by using an intravesicularvolume of 1.7 gtl per mg of membrane protein (18).Measurement of Membrane Potential (A{/). The membrane

potential was measured by the distribution of a negativelycharged permeant ion, [V5S]thiocyanate, using the flow dialysistechnique described above. The membrane potential was cal-culated from the Nernst equation, A; = 58.8 log([SCN-]in/[SCN-]OU). The total protonmotive force (A4H+) was calculatedaccording to the equation 4UH+ = - ZApH, in which Z =58.8 mV at 250C.Measurement of 9-Aminoacridine (9-AA) Fluorescence.

The formation of a proton gradient was also determined by themethod of Schuldiner et al. (19), using 9-AA as a fluorescenceprobe, as described by Lee and Brodie (20).

Assay of Proline Transport. The membrane vesicles (2.0 mgof protein) were suspended in 1.0 ml of a reaction mixturecontaining 50mM Hepes/KOH buffer (pH 7.5), 10mM MgC12,and 10 mM NaCl. After 10-min incubation at 30'C in thepresence and absence of naphthoquinones, the reaction wasstarted by the addition of 25 ,1 of proline (with [ 14C]proline ata final concentration of 1.0 ,uCi/ml; 1 Ci = 3.7 X 1010 bec-querels), with generated NADH as the substrate. The rate oftransport was measured by the procedure described earlier(21).Assay of Ca2+ Transport. The assay of Ca2+ uptake was

similar to that described for proline transport, except NaCI wasomitted from the reaction mixture and 0.5 mM CaC12 (con-taining 45Ca2+, 10 ,Ci/ml) was used.

Protein Estimation. Protein concentration was estimatedeither by the method of Lowry et al. (22) or by a modificationof the biuret method (23), with bovine serum albumin as astandard.

RESULTSRestoration of the Proton Gradient in Irradiated ETP.

Because the bulk of the ETP (approximately 90%) are invertedvesicles with respect to the normal orientation of the cellmembrane, they accumulate protons during substrate oxidation.Thus, in order to measure changes in internal pH during sub-strate oxidation, the distribution of '14C]methvlamine, a weakbase, was determined by using the flow dialysis technique.

As shown in Fig. 1A, the concentration of labeled methyl-amine in the dialysate decreased when an oxidizable substratewas added to untreated vesicles; the decrease from the levelsobserved in the absence of substrate indicated the energy-dependent level of uptake of the weak base. Furthermore, itwas observed that the addition of C'CCP, a proton-conducting

0n

Fraction1 0 20 30 40

FIG. 1. Flow dialysis determination of methylamine uptake bymembrane vesicles of M. phlei. The upper chamber contained 50 mMHepes/KOH buffer (pH 7.5), 10 mM MgCl2, 10 mM NaCf, substrate(1.0 mM NAD+, 20 mM hydrazine, 1 mg of alcohol dehydrogenase),and 2.0 mg of membrane protein in a total volume of 0.85 ml. Theexperiment was started by the addition of ['4C]methylamine at a finalconcentration of 27 MuM. The dialyzing buffer (Hepes/KOH, pH 7.5)was pumped through the lower chamber at a rate of 6.0 ml/min. Asindicated, ethanol or carbonyl cyanide m-chlorophenylhydrazone(CCCP) was added to the upper chamber to give final concentrationsof 50 MM and 10 MM, respectively. Fractions (2.0 ml) were collectedand assayed for radioactivity by liquid scintillation counter in Bray'ssolution. (A) ETP; (B) irradiated ETP; (C) irradiated ETP + vitaminK1 (0.5 mM); (D) irradiated ETP + menadione (0.4 mM); 0, withoutsubstrate; 0, with substrate.

uncoupler, caused the release of a great proportion of the me-thylamine taken up by the vesicles, as a consequence of thecollapse of the proton gradient. In contrast, the irradiated ETP(Fig. 1B) did not exhibit a CCCP-sensitive accumulation ofmethylamine, indicating the lack of formation of a protongradient; thus, it is assumed that a relatively small but constantportion of [14C]methylamine is absorbed into the membranevesicles; this portion is substrate-induced and CCCP-insensitive.This fraction was therefore subtracted in all cases, and thevalues given in Table 1 represent subtrate-induced CCCP-sensitive pH gradients.The substrate-induced (generated NADH) CCCP-sensitive

pH gradient observed with the ETP was 2.36 pH units; how-ever, after irradiation of the FTP, the pH gradient was abol-ished (Table 1). The addition of vitamin KI, menadione, orlapachol to the irradiated membrane vesicles resulted in therestoration of the pH gradient to a level similar to that observedwith the ETP (Fig. 1 C and D).

Restoration of Membrane Potential in Irradiated ETP.The membrane potential was measured by using a negativelycharged ion (SCN-) with the flow dialysis technique as wasshown for measurements of pH gradients. After the additionof substrate in ETP there was an accumulation of labeled SCN-,which indicated the formation of a potential across the invertedmembrane vesicles (Table 1). The uptake of SCN- was sensitiveto gramicidin D (data not shown). In irradiated ETP, in contrast

EtOH CCCPD

I II: I-- I I I

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Table 1. Restoration of ApH, A ,6, and 4LH+ by naphthoquinonesin irradiated membrane vesicles

Addition ApH A+, mV AWH+, mVETP 2.36 + 0.09 86 + 1.4 225 : 15.6Irradiated ETP 0 0 0Irradiated ETP+ vitamin K1 1.95 + 0.20 78 i 5.6 193 + 16.0

Irradiated ETP+ menadione 2.10 i 0.04 87 + 1.4 211 + 15.2

Irradiated ETP+ lapachol 2.05 ± 0.21 78 ± 7.1 199 1 13.5

The assay and reaction mixture were similar to those described inthe legend of Fig. 1. [14C]Methylamine (50.1 ptCi/mmol, 27 .M) andpotassium [35S~thiocyanate (66.4 mCi/mmol, 0.5 ,uM) were used tomeasure the ApH and A+t, respectively. Results are mean :1 SD.

to the ETP, there was a small amount of SCN- absorption oruptake, but it was found to be insensitive to gramicidin D.Therefore, all the data reported for the restored membranepotential were substrate-induced gramicidin-D sensitive. Asshown in Table 1, the membrane potential was restored afterthe addition of vitamin K1, menadione, or lapachol to the ir-radiated ETP. The total protonmotive force restored by theaddition of the different naphthoquinones was similar to thatobserved with the ETP.

Restoration of Quenching of Fluorescence. Proton uptakeinto the inverted vesicles was indicated by the quenching offluorescence of 9-AA during oxidation of NADH. Because thetransport of proline is Na+ dependent (24), the addition of bothNa+ and proline was required for the dissipation of the pHgradient (Fig. 2), suggesting that the proton gradient was uti-lized as the driving force for the proline transport. The addition

Time

FIG. 2. Quenching of 9-AA fluorescence. A 1-min interval isshown at the bottom of each panel. At the time indicated NADH (0.5mM) and proline (25 gM) plus NaCl (20 mM) were added. (A) Non-irradiated (-) and irradiated (--- ) ETP; (B) irradiated ETP + 0.5mM vitamin K1 (-) and irradiated ETP + dihydrophytyl vitaminK1 (- - -); (C) irradiated ETP + 0.4mM menadione (-) and irradi-ated ETP + 0.4 mM 2-methoxy-1,4-naphthoquinone (--- ); (D) ir-radiated ETP + 0.4mM lapachol (-) and irradiated ETP + naturalnaphthoquinone (---).

Time, min Time, min

FIG. 3. Restoration of proline (A) and Ca2+ (B) transport in ir-radiated membrane vesicles by naphthoquinones. Generated NADHwas used as the substrate. *0*, ETP; *----, irradiated ETP +0.4 mM menadione; 0-0, irradiated ETP + 0.4 mM lapachol;0---0, irradiated ETP + 0.5 mM vitamin K1; X-X, irradiatedETP.

of CCCP resulted in a complete collapse of the proton gradient,as evidenced by the increase in fluorescence to the level ob-served before the addition of substrate. Quenching of fluores-cence of 9-AA did not occur on addition of substrate to the ir-radiated vesicles; however, after the addition of naphthoqui-nones, quenching of fluorescence did occur (Fig. 2 B, C, andD). The further addition of CCCP resulted in the return to thefully fluorescent state of the 9-AA (data not shown).

Proline and Ca2+ Transport in Irradiated ETP. Activetransport of proline has been observed with ETP, couplingfactor/ATPase-depleted ETP (DETP), protoplast ghosts, andwhole cells of M. phlei (25). Active transport of proline wasinhibited in irradiated ETP (Fig. 3A), but was restored by theaddition of the naphthoquinones tested. The restoration ofproline transport was about 90% with menadione, 70% withlapachol, and 60% with vitamin K1. The extent of transportrestoration was consistent with the degree of restoration of theprotonmotive force and rate of oxidation with these quinones(Table 1 and Fig. 3A).The inverted membrane vesicles from M. phlei are also ca-

pable of transporting Ca2+ against a concentration gradient inthe presence of an oxidizable substrate or ATP (8). The uptakeof Ca2+ was noted to be much higher (approximately 100 times)than that observed with proline. The NADH-driven uptake ofCa2+ was inhibited in irradiated ETP, but the restoration ofCa2+ uptake occurred after addition of the naphthoquinones(Fig. 3B). The ATP-driven Ca2+ transport was not inhibited

Table 2. Effect of inhibitors on the restored activetransport of proline

Inhibition, %Irradiated ETP +

Vitamin Mena-Addition ETP K1 dione Lapachol

KCN (10 mM) 76 80 0 822-N-4-Hydroxy-

quinolineN-oxide (3 jig/ml) 71 77 18 69

Argon 71 84 77 82Dicumarol (1 mM) 80 70 65 79CCCP (20,uM) 76 53 77 82

The inhibitors were preincubated with vesicles in the reactionmixture at the indicated concentrations for 10 min at 300C. GeneratedNADH was used as substrate. The reactions proceded for 10 min at300C.

-AA B

Na'Proline

NAH -~ ~

NADH

9-AA D- I

Na'Proline

- I~~~~~~I

ND -4 -NADH

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Table 3. Effects of quinones on oxidation and phosphorylationQuinone, A02, APi,

Addition mM Amol (of 0) MmolExperiment 1

ETP 7.9 12.0ETP + vitamin K1 0.5 8.2 11.9ETP + menadione 0.4 8.7 11.4ETP + lapachol 0.4 12.3 10.6

Experiment IIETP 8.9 1.87Irradiated ETP 10.98 1.60Irradiated ETP + vitamin K1 0.5 12.95 1.47Irradiated ETP + menadione 0.4 10.36 1.47Irradiated ETP + lapachol 0.4 10.54 1.60

The measurements of oxidation and phosphorylation were similarto those previously described (16). The main compartment of thevessel contained 100 umol of Hepes/KOH buffer, pH 7.5,15 umol ofPi, 3 mg of yeast hexokinase, 50 Mmol of glucose, and 7.5 mg of proteinof ETP or irradiated ETP. The reaction was started by the additionof the substrate after a 10-min preincubation. The side arm containedeither 100 ,umol of ethanol, 1 1umol of NAD+, 50 ,mol of hydrazine,and 1 mg of alcohol dehydrogenase (generated NADH) (experimentI) or ascorbate/TPD (experiment II) as substrate. TPD was dissolvedin sodium ascorbate (pH 6.3) and added at final concentrations of 1.5mM and 17 mM. The reaction was terminated after 15 min by theaddition of 1.0 ml of 10% trichloroacetic acid.

by irradiation (360 nm), because the membrane-bound ATPasefrom M. phlei was found to be insensitive to near UV irradiation(26).

Effect of Inhibitors. As shown in Table 2, KCN, quino-line-N-oxide and anaerobiosis, established by bubbling withargon, inhibited the uptake of proline in ETP. However, res-toration of proline uptake in irradiated ETP by menadione wasinsensitive to KCN and quinoline-N-oxide, because the restoredelectron transport with menadione can bypass the cytochromeregion of the respiratory chain. Dicumarol (an analogue ofnaphthoquinones) and CCCP inhibited the uptake of prolinein reconstituted ETP, independent of the type of quinone usedto restore oxidation.

Possible Uncoupling Effect of Certain Quinones. Becausemenadione, lapachol, or the products of irradiation may act asuncoupling agents and thus restore only oxidation, it was im-portant to determine the effect of these quinones on phos-phorylation. The effects of vitamin K1, menadione, or lapacholon the phosphorylation associated with substrate oxidation wastested with two different substrates on the ETP and irradiatedET p.(a) generated NADH, utilizing the complete respiratorychain and (b) ascorbate/N,N,N',N'-tetramethyl-p-phenyl-enediamine (TPD), which enters the respiratory chain at thelevel of cytochrome c (above the quinone site). With the ETPthe naphthoquinones caused an increase in oxidation but had

little or no effect on the total amount of phosphate esterified(Table 3). Similar results were observed with the irradiated ETPwhen ascorbate and TPD were used as the electron donors. Itis pertinent to mention that the menaquinones did not affectthe membrane-bound coupling factor/latent ATPase activityin either nonirradiated or irradiated ETP (data not shown).

Relationship Between Oxidation, AH+, Proline Transport,Ca2+ Uptake and P/O Ratio. The treatment of membranevesicles with near UV light resulted in photoinactivation of thenatural quinone and in a loss of oxidation, pH gradient, mem-brane potential, active transport of solutes, and phosphorylation(Table 4). The proton-motive force and active transport ofproline and Ca2+ were restored by all three naphthoquinonestested, and there appeared to be a good correlation between thelevel of proline transport in the restored membrane vesicles andthe generation of a A#LH+, whereas Ca2+ transport appeared tobe dependent on the pH gradient. Vitamin K1, like the otheranalogues, was capable of restoring the protonmotive force, itwas the only one that in addition was capable of restoring oxi-dation and coupled phosphorylation.

DISCUSSIONStudies with numerous quinones (4, 6) have exhibited specificstructural and steric requirements for the quinones to restoreoxidative phosphorylation; the requirements differed fromthose for restoration of oxidation. To some degree, inability toreconstitute phosphorylation by certain quinones can be ex-plained by the fact that they bypass segments of the electrontransport pathway. However, it is not clear why quinones suchas lapachol or dihydrophytyl vitamin K1, which restore oxi-dation by the same electron transport pathway as the naturalmenaquinone, fail to restore phosphorylation.The inability of the quinones such as menadione or lapachol

to restore coupled phosphorylation in the irradiated membranevesicles is contrasted with the findings that these quinones wereable to restore the active transport of solutes, the pH gradient,and the membrane potential. Vitamin K1 was the only quinonetested capable of restouing 4UH+ active transport of solutes, andcoupled phosphorylation. These results indicated that it is notnecessary for oxidation to occur via the complete electrontransport chain in order to restore the 4LH+. There appears tobe a direct involvement of the pH gradient or membrane po-tential as the driving force for active transport of proline andCa +, as demonstrated previously in a model proteoliposomalsystem (20, 21, 27).

Because the chemosmotic theory proposes that both ATPsynthesis and accumulation of solutes are driven by the pro-tonmotive force, which is formed as a consequence of theelectron transport in intact membranes, the question still re-mains concerning the inability of certain quinones to restorecoupled phosphorylation in the irradiated ETP. The inability

Table 4. Restoration of oxidation, pH gradient, proline transport, Ca2+ uptake, and phosphorylation bynaphthoquinones in irradiated membrane vesicles

Active transportA02, Proline, Ca2+

nmol (of 0)/min pmol/min nmol/minAddition per mg protein 4UH+, mV per mg protein per mg protein P/O ratio

ETP 9.1 225 + 15.6 124 ± 5.7 17.1 ±0.85 0.86Irradiated ETP 0.1 0 23 ± 2.9 2.0 ± 0.12 0.00Irradiated ETP + vitamin K1 6.6 193 ± 16.0 72 ± 6.0 7.4 ± 0.76 0.61Irradiated ETP + menadione 14.5 211 + 15.2 113 ± 6.8 9.8 ± 0.70 0.02Irradiated ETP + lapachol 9.6 199 + 13.5 85 + 6.3 8.6 ±0.58 0.03Generated NADH was the substrate, and 0.5mM vitamin K1, 0.4mM menadione, or 0.4mM lapachol was used. The values given above represent

the mean of three to five determinations and their standard deviation.

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of menadione or lapachol to restore oxidative phosphorylationcould be explained by assuming that the photoinactivation hasdamaged the coupling factor/latent ATPase or that the in-tactness of the membrane has been affected during irradiation.However, both of these possibilities are ruled out because theirradiated preparations are capable of oxidative phosphoryl-ation in the absence of quinones with ascorbate/TPD or in thepresence of vitamin K1 with generated NADH. In addition, thequinone-restored membrane vesicles are capable of forminga 4LH+ and transporting solutes against a concentration gra-dient. Another possible explanation would be that menadioneand lapachol act as uncouplers by either inhibiting the synthesisof ATP or accelerating its breakdown. As shown in Table 3,neither of these compounds inhibit the level of phosphorylationassociated with oxidation. In addition, these analogues do notaffect the coupling factor latent ATPase.

Therefore the possibility exists that in addition to the AjiH+and the intactness of both the membrane and the couplingfactor, other factors might be involved in reestablishing oxi-dative phosphorylation. It is possible that restoration of coupledphosphorylation requires a conformational change in themembrane-bound coupling factor or other closely associatedmembrane proteins (28); such a change may be dependent oncertain essential features of the quinones such as the longlipophilic side chain on C3 and the methyl group on C2 that arenecessary for restoration of phosphorylation.

Williams (29) has proposed that local charges in the mem-brane generated by the separation of protons from electrons bydislocated reactions can be utilized as the driving force for ATPformation. Thus, the ability of certain quinones to restore oxi-dative phosphorylation may be a reflection of their specificlocalization in the membrane, where they would be able toproduce a local proton gradient.

Green and his collaborators (30, 31) have proposed that thetransducing assembly of the mitochondrial inner membraneis a polymeric continuum of repeating structures, each of whichcontains three transducing complexes: an electron transfercomplex (ETC), an ion transfer complex in close associationwith the ETC (ETC-ITC), and another type of ion transfercomplex that is in direct interaction not only with the ETC butalso with the site of ATP synthesis and hydrolysis (TRU-ITC).The results in this paper can be explained if we assume thatvitamin K1 can induce the required interaction between theETC and both classes of ITC, whereas menadione and lapacholcan interact only with ETC-ITC and thus they fail to restorephosphorylation.

This work was supported by grants from the National Institute ofHealth (AI-05637-17) and the Hastings Foundation of the Universityof Southern California. R.D. was supported in part by U.S. PublicHealth Service International Research Fellowship (F05 TW02822).

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