Ž .Biochimica et Biophysica Acta 1343 1997 16–22

Selective inhibition of the sperm-specific lactate dehydrogenaseisozyme-C4 by N-isopropyl oxamate

Carlos Wong ), Lorena Rodrıguez-Paez, Benjamın Nogueda, Alfredo Perez, Isabel Baeza´ ´ ´ ´Departamento de Bioquımica, Escuela Nacional de Ciencias Biologicas, Instituto Politecnico Nacional. Apdo. Postal 4-897, Admon. 4,´ ´ ´

Mexico 06401 D.F., Mexico´

Received 5 February 1997; accepted 12 May 1997

Abstract

In the present study, we demonstrated that the attachment of the nonpolar isopropylic carbon chain in the nitrogen ofoxamate, converted this competitive inhibitor of LDH isozymes into a powerful selective inhibitor of mouse LDH-C4. Thecomparative study of the inhibitory effect of oxamate and N-isopropyl oxamate on mouse LDH isozymes pointed out thatthe isopropylic carbon chain conferred upon N-isopropyl oxamate a high affinity for LDH-C4 and a marked decrease in the

Ž .affinity for the other isozymes since oxamate showed more inhibitory effect on LDH-1 K s0.06 mM and LDH-5iŽ . Ž .K s0.08 mM , and less inhibitory effect on LDH-C4 K s0.25 mM . On the other hand, N-isopropyl oxamate showedi i

Ž . Ž .the highest inhibitory effect on LDH-C4 K s0.014 mM and poor inhibitory effect on LDH-1 K s0.4 mM and LDH-5i iŽ .K s0.8 mM . Apparently, the enzymatic inactivation proceeded through a reversible binding of N-isopropyl oxamate,i

facilitated by nonpolar interactions with a hydrophobic region present only in the active site of mouse LDH-C4, resulting ina selective inhibition of this isozyme in comparison with the other LDH isozymes. N-isopropyl oxamate was also a

Ž . Ž .powerful competitive inhibitor of LDH-C4 K s0.015 mM compared with oxamate K s0.35 mM , using a-ketoiso-i i

caproate as a substrate. q 1997 Elsevier Science B.V.

Keywords: LDH-C4 inhibition; N-Isopropyl oxamate; Hydrophobic interaction; LDH isozyme; LDH x

1. Introduction

ŽLactate dehydrogenase LDH: L-lactate-NAD ox-.idoreductase EC 1.1.1.27 in animals occurs in five

common isozyme forms, each being one of the possi-ble random tetrameric combinations of two different

w xpolypeptide chains, designated A and B, 1 or M andw xH polypeptides 2 . The relative distribution of en-

zyme activity among the five isozymes is specific foreach tissue. A special form of lactate dehydrogenasewas demonstrated in mature testes and spermatozoa

) Corresponding author. Fax: q52 5 396 3503.

of many species and designated isozyme X by Blancow xand Zinkham 3 . However, most authors prefer now

the designation LDH-C4, which appears more conve-nient for the molecular form unique to testes andspermatozoa.

The isozyme LDH-C4 accounts for 80–100% ofthe LDH activity in human and rabbit spermatozoaw x4 . The existence of this unique molecular form oflactate dehydrogenase in cells of spermatogenic linesuggests that it must be adapted to fulfil very special-ized functions.

The oxidation of fructose, glucose and lactate byw xthe spermatozoa from different species 5 generates

0167-4838r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved.Ž .PII S0167-4838 97 00090-3

( )C. Wong et al.rBiochimica et Biophysica Acta 1343 1997 16–22 17

NADH in the cytoplasm and requires the existence ofmechanisms to transfer reducing equivalents to themitochondria. It appears that the malateraspartate

w xshuttle is functional in spermatozoa 6 . LDH-C4 hasbeen demonstrated in the cytosol of mice spermato-

w xzoa and in the matrix of sperm-type mitochondria 7 .This observation was recently confirmed by immuno-

w xcytochemistry 8 . This dual intracellular distributionof LDH-C4 led Blanco and his group to propose an

w xadditional shuttle system 9 that utilizes branchedchain a-keto acids as hydrogen acceptors in mouse

w xspermatozoa 10 . The a-keto acid produced bytransamination of branched-chain amino acids couldbe reduced by cytoplasmic NADH in a reactioncatalyzed by the cytoplasmatic LDH-C4. The a-hy-droxy acids thus formed would penetrate into themitochondria and be oxidized by the mitochondrialLDH-C4, transferring the hydrogens to NAD and

w xthen to the respiratory chain 11 . Therefore, it ap-pears that LDH-C4 is related to metabolic processesthat provide energy for motility and survival of sper-matozoa.

Due to the unique presence of LDH-C4 in sperma-tozoa and spermatogenic cells, LDH-C4 has been

w xused as a target enzyme in antifertility studies 12since LDH-C4 has been also localized on the plasma

w xmembrane of rabbit and mouse sperm 13–15 , and,therefore, is accessible to antibodies. Fertility inhibi-tion by active and passive immunization against thesperm-specific LDH-C4 has been obtained in micew x w x w x16 , rabbits 13 and in both man and woman 17–19 .The purpose of these immunological studies and oth-

w xers on epitopes and antigenic sites of LDH-C4 20 isw xto develop a contraceptive vaccine 21 .

Gossypol, a polyhydroxylated binaphthalene ex-tracted from cotton seed oil, has been studied exten-

w xsively as a human antifertility agent in China 22 .Gossypol inhibits sperm motility and glycolysisw x w x23,24 . Studies by Lee and Malling 25 and Stephens

w xet al. 24 suggest that gossypol acts by inhibitingselectively the intracellular LDH-C4, thus affectingenergy metabolism. On the other hand, there is someevidence that inhibition of LDH-C4 may not be themajor mode of action of gossypol, since gossypol

w xalso inhibits other dehydrogenases 26 .According to the above-mentioned metabolic and

fertility implications of LDH-C4, it is evident that thedevelopment of selective inhibitors for LDH-C4 can

be completely justified because they could be usefulas biochemical tools for further characterization ofthe metabolic and fertility implications of LDH-C4.Therefore, the objective of this study was to developa new selective inhibitor for mouse LDH-C4.

Based on substrate specificity differences betweenmouse LDH isozymes and LDH-C4 isozymes from

w xdifferent species 9 , as well as in the susceptibility ofthe LDH isozymes to oxamate and other N-sub-

w xstituted oxamates inhibition 27 , we assumed thatN-isopropyl oxamate would fulfil the structural andsteric requirements to be a selective inhibitor ofmouse LDH-C4. Therefore, in the present study westart with the synthesis of N-isopropyl oxamate andthen we try to determine if this substance actuallyinhibits mouse LDH-C4 and if the inhibition is selec-tive for this isozyme.

2. Materials and methods

2.1. Materials

LDH isozyme-C4 from adult Swiss albino mousetestes was purified by the method reported by one of

w xus 28 . LDH isozyme-1 from mouse heart and LDHisozyme-5 from mouse skeletal muscle were purifiedby a procedure described by Gerez de Burgos et al.w x w x29 , slightly modified by Blanco et al. 9 . All theisozyme preparations showed a single band on poly-acrylamide gel electrophoresis. Succinic dehydroge-nase was obtained and partially purified from rat

w xheart 30 . Pyruvate, oxamate, a-ketoisocaproate, DL-isocitrate, a-ketoglutarate, DL-malate, succinate,NAD, NADH, NADP, malic dehydrogenase from

Ž .bovine heart, L-glutamic dehydrogenase type I fromŽ .bovine liver, isocitric dehydrogenase NADP from

porcine heart. were obtained from Sigma ChemicalŽ .Co. St. Louis, MO, USA . All other chemicals were

of reagent grade.

2.2. Methods

N-Isopropyl oxamate was synthetized by a pub-w xlished procedure 31 , and the structure of the product

was confirmed by spectroscopy.Malate dehydrogenase was assayed by the method

w xdescribed by Yoshida 32 , glutamate dehydrogenase

( )C. Wong et al.rBiochimica et Biophysica Acta 1343 1997 16–2218

w xby the method described by Strecker 33 , succinatedehydrogenase by the method described by Veerger

w xet al. 34 and isocitrate dehydrogenase by the methodw xdescribed by Plaut 35 . Lactate dehydrogenase activ-

ity was determined by recording the absorbancechange at 340 nm produced by the oxidation ofNADH. Assays were performed at 378C. The reagentmixture contained 0.115 mM NADH, 50 mM sodiumphosphate buffer, pH 7.4, sodium pyruvate 0.2 mMfor LDH-C4, 0.5 mM for LDH , 1.5 mM for LDH ,1 5

and the enzyme preparation, diluted with phosphatebuffer, pH 7.4, to provide a D E of 0.06–0.07 per340

minute when the activity was assayed in a 1 cm lightpath. For determination of K values, the isozymes,i

Ž .the inhibitor oxamate or N-isopropyl oxamate andthe coenzyme were incubated with the buffer used inthe assay for 10 min at 378C before starting thereaction by adding the substrate. The K values werei

determined from those of K and V obtained withm

and without oxamate or N-isopropyl oxamate addedto the assay mixture using various concentrations ofsubstrate at a constant inhibitor concentration, and

Ž .plotting the slope K rV against inhibitor concen-mw xtrations 36 .

3. Results

Fig. 1 shows the inhibitory effect of low concen-trations of N-isopropyl oxamate on the activity of

Fig. 1. Effect of N-isopropyl oxamate on mouse LDH isozymes.Velocities were calculated taking the maximum activity without

Ž . Ž .the inhibitor N-isopropyl oxamate as 100%. ` LDH-C4, '

Ž .LDH-1, I LDH-5.

Fig. 2. Effect of pyruvate on the inhibitory activity of N-isopro-pyl oxamate on mouse LDH-C4. Reciprocal values of V werecalculated taking the maximum activity without inhibitor as100%. The concentrations of pyruvate used were 0.025, 0.035,0.05, 0.1 and 0.2 mM. NADH concentration was kept at 0.115

Ž . Ž .mM. ` Assays without inhibitor, I assays with 0.02 mM,Ž . Ž .' 0.04 mM and v 0.06 mM of the inhibitor N-isopropyloxamate. The K for pyruvate was 0.045 mM. Upper left:m

determination of K from replot of slope values against inhibitori

concentrations.

LDH isozymes. Under the experimental conditionsemployed, only LDH-C4 was inhibited at 0.1 mMconcentration of N-isopropyl oxamate, LDH-C4 wasinhibited up to 70% without affecting the enzymaticactivity of the other two isozymes.

Fig. 2 shows double reciprocal plots of initialvelocities at different substrate concentrations and theinhibitory effect of N-isopropyl oxamate on mouseLDH-C4. The inhibition was competitive; the Km

value with respect to pyruvate was 0.045 mM.The K values for LDH-C4, LDH-1, and LDH-5m

determined from Lineweaver-Burk double-reciprocalplots are listed in Table 1. The K values werem

w xsimilar to those reported by others 9 . The K valuesi

were determined as indicated in Section 2.2, and theresults are presented in Table 1. In all cases, theinhibition was of the competitive type. Maximummouse LDH-C4 activity was attained at 0.2 mM ofthe substrate pyruvate and at 0.1 mM of a-ketoiso-caproate; both a-keto acids presented strong in-

w xhibitory action at higher concentrations 9 . Recipro-cals of initial velocity with substrate concentrationsbelow the above-mentioned concentrations gave lin-

( )C. Wong et al.rBiochimica et Biophysica Acta 1343 1997 16–22 19

Table 1Kinetic parameters for the inactivation of LDH-isozymes by oxamate and N-isopropyl oxamate

Ž . Ž .Substrate Isozymes K mM K mMm i

Oxamate N-Isopropyl oxamate

Pyruvate LDH-C4 0.045 0.250 0.014Pyruvate LDH-1 0.100 0.060 0.400Pyruvate LDH-5 0.170 0.080 0.800a-Ketoisocaproate LDH-C4 0.040 0.350 0.015

The K values were calculated by Lineweaver-Burk plots. The K values were determined by using various concentrations of substratesm iw xat a constant inhibitor concentration 36 .

w xear plots with these substrates. 9 . In addition, wefound that N-isopropyl oxamate, at very high concen-trations, can also inhibit LDH-1 and LDH-5. The

Ž .inhibition was also competitive not shown , but theconcentrations of N-isopropyl oxamate employed inthese kinetic studies were 100 times higher than those

Ž .used for LDH-C4 Table 2 .The effect of N-isopropyl oxamate on the activity

of LDH isozymes and other dehydrogenases was alsocompared. Initial velocities were determined in thepresence and absence of N-isopropyl oxamate. Con-centrations of the substrates as well as those of thecoenzymes and inhibitor are indicated in Table 2.Maximum enzymatic activity was attained at theconcentrations of substrates and coenzymes indicatedfor each dehydrogenase. A strong inhibition of LDH-C4 was observed with a concentration of 0.1 mM ofthe inhibitor. At higher concentrations of N-isopropyl

Ž .oxamate 100 times higher , LDH-1 and LDH-5 werealso inhibited; whereas, glutamate, malate and succi-nate dehydrogenases were only slightly inhibited byN-isopropyl oxamate. Isocitrate dehydrogenase was

Ž .not affected by this substance Table 2 . These results

also indicated that N-isopropyl oxamate was a pow-erful and selective inhibitor of mouse LDH-C4.

4. Discussion

Comparative studies of catalytic properties haverevealed differences between LDH-C4 and the other

w xlactate dehydrogenase isozymes 43 . All LDHisozymes catalyzed the reaction from pyruvate tolactate and from lactate to pyruvate. However, theLDH-C4 from different species also showed activityagainst a-keto and a-hydroxy acids of longer carbon

w xchain than those of pyruvate and lactate 28,37–46 .w xBlanco et al. 9 re-examined this problem of sub-

strate specificity of LDH-C4 from different speciesand they found that mouse LDH-C4 presented thebroadest spectrum of substrate specificity. It exhib-ited very similar K values for a variety of a-ketom

and a-hydroxy acids from 4 to 6 nonpolar linear orŽ . w xbranched 5 to 7 carbon chain 9 . Whereas, a-keto

and a-hydroxy dicarboxylic acids with a polar groupat the end of the carbon chain such as oxalacetate,

Table 2Effect of N-isopropyl oxamate on the activity of LDH isozymes and other dehydrogenases

Ž . Ž . Ž .Enzyme Substrates mM N-Isopropyl oxamate mM Inhibition %

Ž . Ž .LDH-1 Pyruvate 0.5 qNADH 0.115 10 60.0Ž . Ž .LDH-5 Pyruvate 1.5 qNADH 0.115 10 70.0Ž . Ž .LDH-C4 Pyruvate 0.2 qNADH 0.115 0.1 72.0Ž . Ž .Malate dehydrogenase L-Malate 8.5 qNAD 2.5 10 7.5

Ž . Ž .Glutamate dehydrogenase a-Ketoglutarate 8 qNADH 0.115 10 13.3Ž .Succinate dehydrogenase Succinate 20 10 15.7Ž . Ž .Isocitrate dehydrogenase Isocitrate 1.33 qNADP 0.1 10 0.0

Initial velocities were determined in the presence and absence of N-isopropyl oxamate. Maximum enzymatic activity was attained at theconcentrations of substrates and coenzymes indicated for each dehydrogenase.

( )C. Wong et al.rBiochimica et Biophysica Acta 1343 1997 16–2220

malate and a-hydroxy glutarate were not utilized bymouse LDH-C4; while a-keto glutarate gave signifi-

w xcant activity only at very high concentrations 9 .These peculiar catalytic properties of mouse LDH-C4strongly indicate the presence of a hydrophobic re-gion in or near the substrate binding site of thisisozyme. This would explain its increased affinity forthose a-keto and a-hydroxy acids containing nonpo-lar side carbon chains, and its lack of, or much lesseraffinity for those a-keto and a-hydroxy acids with apolar group at the end of the side carbon chain. Inkeeping with this, it has been pointed out that theincrease in the effectiveness of substrates and in-hibitors induced by nonpolar substituents can be dueonly to a hydrophobic bonding in an enzyme–inhibi-

w xtor or enzyme–substrate complex 27,47 . In addition,it was found that the glycine, threonine and leucinecontent of mouse LDH-C4 was greater than in the

w xother isozymes 48 . Leucine, with its nonpolar Rgroup formed by a branched carbon chain, is excep-tionally high in mouse LDH-C4, which contains 40more residues of leucine than LDH-1 and LDH-5w x48 . It is probable that some of these residues ofleucine are forming part of the hydrophobic regionpresent only at the active site of mouse LDH-C4. Itseems that this hydrophobic region allows the en-zyme to discriminate between a-keto and a-hydroxyacids with different side chains, facilitating, throughhydrophobic interactions, the proper binding of thosesubstrates with nonpolar side chains and rejectingthose with polar side chains.

To obtain the possible selective inhibitor of mouseLDH-C4, we selected a competitive inhibitor of LDHisozymes with a small molecular volume, such asoxamate, that could permit us to increase the lengthof the inhibitor by the attachment of the nonpolarisopropylic carbon chain, trying to reach the hy-drophobic region of LDH-C4, to create an additionalbinding point that could increase the affinity andselectivity of this inhibitor for LDH-C4. Therefore,we assumed that N-isopropyl oxamate would fulfilthe structural and steric requirements to be a competi-tive and selective inhibitor of mouse lactate dehydro-genase isozyme-C4. The oxamate, a well known

w xcompetitive inhibitor of LDH isozymes 27 , willdirect the N-isopropyl oxamate molecule against theactive site of LDH isozymes, and the attachment ofthe nonpolar isopropylic branched chain to the nitro-

gen of oxamate will give N-isopropyl oxamate moreaffinity and selectivity for mouse LDH-C4 isozyme,due to its possible hydrophobic interaction with thenonpolar region of this isozyme, on account of theclose chemical structure of this inhibitor to that of the

Ž .substrate a-ketoisocaproate see Fig. 3 . Therefore,we started this investigation with the synthesis andcharacterization of N-isopropyl oxamate. The kineticstudies showed that this substance was really a pow-erful inhibitor of mouse LDH-C4. The inhibition wasshown to be competitive with respect to the substratepyruvate.

The kinetic studies also showed that the introduc-tion of the nonpolar isopropylic branched chain in thenitrogen of the oxamate molecule significantly in-creased the affinity of the N-isopropyl oxamatemolecule for LDH-C4 isozyme. Consistent with thiswas the 17-fold better binding of N-isopropyl oxam-ate to LDH-C4, indicated by the K values in thei

Žpyruvate competitive inhibition by oxamate K si. Ž0.25 mM and N-isopropyl oxamate K s0.014i

.mM , respectively.Our experiments strongly suggested that nonpolar

interactions played a central role in the differentialbinding of N-isopropyl oxamate to the active site ofthe mouse LDH isozymes because the nonpolar iso-propylic branched chain turned N-isopropyl oxamate

Žmore specific and selective for LDH-C4 K s0.014i. Ž .mM , compared with LDH-1 K s0.4 mM andi

Ž .LDH-5 0.8 mM using pyruvate as a substrate. Theseexperiments showed that N-isopropyl oxamate was a28-fold better inhibitor for LDH-C4 compared withLDH-1 and a 42-fold better inhibitor in relation toLDH-5.

The comparative study of the K values obtainedi

with the LDH isozymes, using oxamate or N-isopro-

Fig. 3. Chemical and structural relationships between LDH-C4substrates, pyruvate and a-ketoisocaproate, and the inhibitors,oxamate and N-isopropyl oxamate.

( )C. Wong et al.rBiochimica et Biophysica Acta 1343 1997 16–22 21

pyl oxamate on the pyruvate to lactate reaction alsoshowed that the nonpolar isopropylic branched chainconferred on N-isopropyl oxamate more affinity andselectivity for mouse LDH-C4, since oxamate showed

Ž .more inhibitory effect on LDH-1 K s0.060 mMiŽ .and LDH-5 K s0.080 mM and less inhibitoryi

Ž .effect on LDH-C4 K 0.25 mM ; whereas, N-iso-i

propyl oxamate showed the highest inhibitory effectŽ .on LDH-C4 K 0.014 mM and the poorest in-i

Ž .hibitory effect on LDH-1 K s0.4 mM and LDH-5iŽ .K s0.8 mM . These results strongly suggest thati

the inactivation proceeded through a reversible bind-ing of N-isopropyl oxamate facilitated by nonpolarinteractions with the hydrophobic region present onlyin the active site of mouse LDH-C4 isozyme, result-ing in a selective inhibition of this isozyme in com-parison with the other LDH isozymes. This selectiv-ity was confirmed by the observation that N-isopro-pyl oxamate, at a concentration of 0.1 mM, inhibitedLDH-C4 up to 70% without affecting the activity ofLDH-1 and LDH-5.

The poor inhibitory effect on LDH-1 and LDH-5cannot be attributed to lack of space in the active site

w xof these isozymes because Baker 27 has shown thatspace is available in their active sites for bulkyinhibitors such as N-phenyl oxamate and salicylate.An example of these bulky inhibitors was the salicy-

Ž .late derivative N- 4-carboxy-2-hydroxyphenylmaleimide, a competitive inhibitor of LDH isozymes,with K s9.2=10y3 mM for LDH-5, 12 mM fori

w xLDH-1, and 4.4 mM for LDH-C4 43 .At very high concentrations of N-isopropyl oxam-Ž .ate 10 mM , LDH-1 and LDH-5 were also inhibited;

whereas, glutamate, malate and succinate dehydroge-nases were only slightly inhibited, and isocitrate de-hydrogenase was not affected by this substance. Theseresults also indicated that N-isopropyl oxamate was aselective inhibitor of mouse LDH-C4.

The powerful inhibitory activity of N-isopropyloxamate on LDH-C4 also was shown when a-keto-isocaproate was used as a substrate. This inhibitionwas also competitive and again the nonpolar iso-propyl branched chain increased the affinity of oxam-ate for LDH-C4 indicated by the K values obtainedi

Ž .with oxamate K s0.35 mM and with N-isopropyliŽ .oxamate K s0.015 mM .i

The present study clearly showed that the attach-ment of the nonpolar isopropylic branched chain in

the nitrogen of oxamate increased the affinity andselectivity of this inhibitor for mouse LDH-C4. Theseresults suggest the presence of a hydrophobic pocketthat can accommodate the nonpolar, isopropylic groupof N-isopropyl oxamate in the active site of mouseLDH-C4, which might explain the selective inhibitionof this isozyme in comparison with the other LDHisozymes.

According to the non-classic antimetabolite theoryw xadvanced by Baker 27 , the inhibition of the pyru-

vate-LDH-C4 reaction by N-isopropyl oxamate repre-sents an example of a non-classic antimetabolite thathas been chemically modified with greater bulk togive more specificity and selectivity to the inhibitor.Whereas, in the inhibition of the a-ketoisocaproate–LDH-C4 reaction, N-isopropyl oxamate represents anexample of a classic antimetabolite with a chemicalstructure close to the metabolite.

Since gossypol inhibits sperm motility and showsantifertility properties due to the selective inhibition

w xof LDH-C4 24,25 , similar effects may be expectedfrom other selective inhibitors of LDH-C4 such asN-isopropyl oxamate. However, the toxicology andpharmacological studies required to substantiate thepossible antifertility effect of N-isopropyl oxamate,are beyond the scope of this investigation.

Acknowledgements

This work was partially supported by researchgrants from the Direccion de Estudios de Posgrado e´Investigacion del Instituto Politecnico Nacional´ ´Ž .DEPI, IPN , Mexico. C.W., L.R.P., B.N. and I.B.´are fellows of the COFAA, IPN, and A.P. is a fellowof the PIFI, IPN. We thank professor Kenneth E.Davis Gaines for correction of the manuscript.

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