IN IiRNAIIONAI. JOURNAL OF LEPROSY^ Volume 60, Number IPrinted in the U.S.A.

Energy Generation Mechanisms in the in Vitro-grownMycobacterium lepraemurium'

Muhammad Ishaque 2

Mycobacterium lepraemurium. the etio-logic agent of murine leprosy, was first dis-covered by Stefansky in 1903 ( 23 ). Despitenumerous attempts for its in vitro cultiva-tion, the mycobacterium remained an ob-ligate intracellular parasite until 1970 whenOgawa and Motomura ( 20) reported thegrowth of Al. lepraemurium on Ogawa egg-yolk medium. These findings have subse-quently been confirmed ( 7 .' 8 . 2 ' ).

It has long been established that adeno-sine triphosphate (ATP) is the most impor-tant metabolically available form of energyin living cells. Energy coupling reactions thataccompany biological oxidation of sub-strates have been studied extensively inmany bacterial systems ( 3 . 6 ). We have re-ported ( 1 ") such mechanisms in in vivo-grownAl. lepraemurium. However, when in vivo-grown cells are used for metabolic studies,it is always argued whether the results re-ported are not due to the presence of hosttissues, although in very small amounts, inthe purified preparations used during suchstudies. Although M. lepraemurium nearly70 years after its discovery has been culti-vated in vitro, energy conservation mecha-nisms using in vitro-grown cells have notyet been studied. Therefore, we investigatedthe mechanisms of electron transport andenergy generation in in vitro-grown M. lep-meontritem. The experimental data showthat this mycobacterium is capable of con-serving oxidative energy by means of threeenergy coupling sites observed to be presentand operative in the electron transport chain.

MATERIALS AND METHODSMicroorganism. The Hawaiian strain of

AI. lepraemurium used in these studies was

' Received for publication on 22 February 1991; ac-cepted for publication in revised form on 9 October1991.

M. Ishaque, Ph.D., Associate Professor, AppliedMicrobiology Research Center, Institut Armand-Frap-pier, Universitê du Quebec, Case Postale 100, Laval,Québec, Canada I - 17V 1137.

originally obtained from Dr. P. Greenberg(La Roche Research Laboratory, Nutley,New Jersey, U.S.A.). It was maintained inSprague Dawley rats or C3H mice by sub-cutaneous serial passages every 4-5 months.

In vitro cultivation of M. lepraenzuriunt.Primary as well as successive subcultures ofA/. lepraemurium were obtained on Ogawaegg-yolk medium as described earlier ( 7 ).Since malachite green interferes with thespectral studies, it was added in the egg-yolkmedium only to obtain primary cultures andwas omitted from the medium used to ob-tain subcultures.

Preparation of cell suspensions. Thegrowth of the fourth subcultures from thesurface of Ogawa egg-yolk medium was re-moved and suspended in 50 mM sodium-potassium phosphate buffer, pH 6.5. Thecell suspension was centrifuged at 10,000 xg x 10 min at 4°C in a Beckman J-21 Bcentrifuge. The pellet was washed twicemore, and cell-free extracts were preparedas described below.

Preparation of cell-free extracts. To pre-pare cell-free extracts, the cells were sus-pended 1:4 w/v (wet weight) in a sonicationmedium containing 50 mM Tris (hydroxy-methyl) aminomethane buffer, pH 6.5, 300mM sucrose, 5 mM ethylenediamine tetra-acetic acid (EDTA-Na,) and 50 mM MgC1 2 •6H2O. The cell suspensions were passedtwice through a chilled Aminco-FrenchPressure Cell at 18,000 psi (1 psi = 703.07kg/m 2). The homogenate was centrifuged at20,000 x g x 30 min in a Beckman J-21Brefrigerated centrifuge. The resulting super-natant was used as the cell-free extract. Toobtain particulate and supernatant frac-tions, 20,000 x g cell-free extracts were fur-ther centrifuged at 150,000 x g x 120 minin a Beckman model L-65 ultracentrifugeusing the rotor type 50 Ti at 2°C. The re-sulting supernatant was used as the 150,000x g supernatant fraction, and the pellet afterwashing once was suspended in an appro-priate volume of sonication medium andused as 150,000 x g particulate fraction.

61

62^ International Journal of Leprosy^ 1992

Oxidation and coupled phosphorylation.Oxygen consumption by various substrateswas measured at 32°C using a Y.S.I. oxygenpolarograph (Yellow Springs Instrument Co.Inc., Yellow Springs, Ohio, U.S.A.) in a re-action mixture containing 50 mM sucrose,5 mM MgC1,•6H2O, 5 mM KF, 2 mMadenosine diphosphate (ADP), 4 mMK,HPO., and 50 mM Tris-HC1 buffer, pH6.5. The reaction mixture containing the cellfree extracts, particulate, or supernatantfraction (3-4.5 mg protein) was incubatedfor 3 min and then the reaction, in a totalvolume of 3 ml, was started either by gen-erating NADH or by adding 3.3 mMNADH, 10 mM succinate, ascorbate, glyc-erol, 3 mM dithiothreitol or DL penicilla-mine. NADH generating system contained12 units of alcohol dehydrogenase, 10 mMsemicarbazide, 40 mM ethanol, and 1 mMNAD+. The reaction was carried out for atleast 10 min.

For adenosine triphosphate (ATP) deter-mination, the reaction in a total volume of2 ml was carried out in the same reactionmixture used for oxygen consumption at32°C for 10 min. The reaction was termi-nated by adding 0.1 ml of 0.1 N HCI. TheATP formed was extracted with 15% per-chloric acid, neutralized according to theprocedure of Gibson and Morita ( 5), and theamount of ATP in the neutralized super-natant was determined by the luciferin-lu-ciferase method as described by Strehler ( 24 ).All possible controls, such as reactionmixtures with or without substrate as wellas reaction mixtures containing bacterialfractions boiled for 5 min to exclude nonen-zymatic oxidation, were included. Thephosphorus moles incorporated into high-energy bonds to oxygen atoms utilized (P/0) ratios were corrected by subtracting theendogenous values of 0, consumed and theamount of ATP exhibited by the cell-freeextracts.

Uncouplers and inhibitors. The follow-ing uncouplers and inhibitors were usedduring this study: 2,4-dinitrophenol (DNP),2,6-dibromophenol (DBP), pentachloro-phenol (PCP), m,chlorocarbonylcyanidephenylhydrazone (CCCP), dicoumarol,gramicidin, rotenone, amytal, thenoyltri-fluoroacetone (TTFA), antimycin A, 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQ-

NO), cyanide, p-chloromercuribenzoate(PCMB), and N-ethylmaleimide.

Cyanide was dissolved in water. PCMBwas dissolved in 0.1 M glycyl glycine buffer(pH 7.5). All other uncouplers and inhibi-tors were dissolved in ethanol. The volumeof ethanol used did not affect oxidative orphosphorylative activity of the cell-freepreparation.

Difference spectra. The reduced minusoxidized difference spectra were obtainedby means of a Cary model 118C dual-beamrecording spectrophotometer equipped withan intense light source. To study the effectof inhibitors on the difference spectra, anappropriate concentration of inhibitors wasadded in both cuvettes, incubated for about5 min, and then the substrate was added inthe sample cuvette and the spectra weretaken.

Protein determination. Protein concen-tration in the cell-free preparations was de-termined using the Folin-Ciocalteau reagentby the method of Lowry, et a!. (u). Crys-talline bovine serum albumin was used asa standard.

Chemicals. ATP, ADP, alcohol dehy-drogenase, NAD+, and NADH, as well asall the inhibitors and uncouplers were pur-chased from the Sigma Chemical Co., St.Louis, Missouri, U.S.A. All other chemicalswere of the highest grade available.

RESULTSPhosphorylation coupled to the oxidation

of various substrates. Reaction mixtureswithout substrate and heat-inactivated bac-terial fractions did not show any oxidation.The data in Table 1 represent the rates ofoxidation of various substrates and the as-sociated ATP generation by the cell-freeextracts (20,000 x g) prepared from in vitro-grown M. lepraennerium. The rates of oxi-dation of generated NADH or exogenouslyadded NADH were the same, but theamount of ATP formed with generatedNADH was always higher than that ob-tained with added NADH in the same ex-periment. The generated system was, there-fore, used in all subsequent experimentswhen NADH was the substrate. When suc-cinate was used as the substrate, P/O ratiosof 0.36 were obtained. Although the rate ofoxidation of ascorbate was higher than

60, I^Ishaque: Energy Generation Mechanisms in M. lepraemurium^63

TABLE 1. A TP generation coupled to theoxidation of various electron donors by cell-free extracts (20,000 x g) of in vitro-grownM. lepraemurium.

Substrate

0, con-sumed

(natoms)

ATPformed

(nmoles)

P/0

NADH generated 95 71 0.75NADH added 97 50 0.52Succinate 52 19 0.36Ascorbate 158 0 0.00Ascorbate + 0.01 mM

TMPD" 410 0 0.00Ascorbatc + 0.01 mM

cytochrome c 300 36 0.12Glycerol 110 0 0.00Dithiothreitol 360 0 0.00DL penicillamine 455 0 0.00

" NADH = reduced form of NAD (nicotinamide-ad-enine dinucleotide).

" TM PD = tetramethyl-p-phenyline-d 'amine.

NADH and succinate, its oxidation did notyield any ATP. Ascorbate was very rapidlyoxidized in the presence of added tetra-methyl-p-phenyline-diamine (TM PD) (0.01mM) but the oxidation was not coupled tophosphorylation. However, in the presenceof 0.01 mM cytochrome c, ascorbate wasalso oxidized readily and yielded a P/O ratioof 0.12. Cytochrome c in the in vitro-grownM. lepraemurium ( 9) is present in very smallamounts. Added cytochrome c seems tocouple with the endogenous cytochrome c,since the rate of ascorbate oxidation is en-hanced in the presence of added cytochromec. The rate of oxidation of glycerol was thesame as obtained with NADH but no phos-phate esterification occurred during glyceroloxidation. Sulfhydryl compounds, dithio-threitol and DL penicillamine were activelyoxidized but without any associated ATPproduction. These results suggest that all ofthe phosphorylation sites were operative inthe respiratory chain of in vitro-grown AI.lepraeinurium.

Requirements for ATP production. Thedata in Table 2 show that with generatedNADH as the substrate, although the ratesof oxygen uptake were not affected, the P/Oratios decreased considerably when eitherADP, Pi, or MgC1, were omitted from thereaction mixture. The results suggest thatAMP could replace ADP as a phosphate

TABLE 2. Requirements for ATP produc-tion associated with NADH oxidation by cell-ji•e extracts of in vitro-grown M. leprae-murium.

S stemy

0, con-sumed

(natoms)

ATPformed

(nmoles)

P/O

Complete' 96 69 0.72Plus NAD"+ 1 mM 95 67 0.70Minus ADP 96 3 0.03Minus ADP + AMR'

2 mM 96 69 0.72Minus Pi 96 5 0.05Minus MgCI, 92 17 0.18Minus KF 99 15 0.15Plus EGTA' 0.1 mM 95 65 0.68Minus sucrose 94 66 0.70

The complete reaction mixture is outlined in Ma-terials and Methods.

NAD = nicotinamide-adenine dinuucleotide.ADP = adenosine diphosphate.AMP = adenosine monophosphate.EGTA = ethylene glycol tetra-acetic acid.

acceptor possibly due to the presence of en-zyme pyrophosphatase in the cell-free ex-tract. The omission of KF from the reactionmixture caused a slight stimulation ofNADH oxidation but it caused 79% inhi-bition of the coupled phosphorylation. Theaddition of NAD+ or ethylene glycol tetra-acetic acid (EGTA) to the complete reactionmixture did not enhance the phosphoryla-tion efficiency, and the omission of sucrosecaused no appreciable decrease in NADHoxidation as well as the associated ATP pro-duction. In order to obtain good P/O ratios,it was necessary to use freshly harvested cellsto prepare cell-free extracts. Although thecell-free extracts prepared from frozen cellsdid not lose any oxidative activity, theirphosphorylative activity was completelylost. Similar results were obtained with invivo -grown AI. lepracinurium (I).

ATP production by various cell-free frac-tionated systems. The relative rates ofoxidation and coupled phosphorylation bycell-free extracts, particulate and solublefractions of in vitro-grown M. lepraeinu-Ilin are shown in Table 3. Although therates of oxidation of NADH and succinateby the particulate fraction were higher thanthose obtained with cell-free extracts, theamounts of ATP formed were compara-

64^ International Journal of Leprosy^ 1992

TABLE 3. Energy generation coupled to the oxidation of NADI! and succinate by ddlerentfractions of in vitro-grown M. lepraemurium.

Electron donor

FractionNAME Succinate

O.consumed(n atoms)

A.UPformed

(n moles)P/0

O.consumed(n atoms)

ATPformed

(n moles)P/0

Cell free extracts(20,000 x g) 96 70 0.73 53 20 0.38

Particulate fraction(150,000 x c;) 130 58 0.45 65 13 0.20

Soluble fraction(150,000 x g) 15 0 0 0 0 0

Particulate + solublefraction (1:1) 130 94 0.72 64 24 0.38

NADH = reduced form of NAD (nicotinamide-adenine dinucleotide).

lively lower, resulting in lower P/O ratios.NADH was oxidized at a very slow rate bythe soluble fraction without any ATP for-mation. The soluble fraction failed to oxi-dize succinate. When particulate and solu-ble fractions were combined 1:1 based onprotein, the rates of oxidation of NADHand succinate were similar to those obtainedby the particulate fraction alone, but theamounts of coupled ATP produced werehigher and thus the P/O ratios with bothsubstrates were the same as obtained withcell-free extracts. These results show that

both particulate and soluble fractions arenecessary for efficient ATP production byin vitro-grown A/. lepraemurium. In all sub-sequent experiments, crude cell-free ex-tracts (20,000 x g) were used during thisstudy.

Effect of uncouplers on ATP production.In almost all cases low concentrations ofvarious uncouplers of oxidative phosphor-ylation, i.e., DNP, DBP, PCP, CCCP, anddicoumarol, abolished ADP phosphoryla-tion without causing any inhibition of theoxidation of either NADH or succinate (Ta-

TABLE 4. Elect of uncouplers on phosphorylation coupled to NADII and succinateoxidation by cell-free extracts of in vitro-grown M. lepraemurium.

Substrate Uncoupler Concentration (mM) 0, uptake(n atoms)

ATPformed

(n moles)13/0

NADH" None - 95 67 0.71DNP'' 0.01 95 0 0.00IMP' 0.01 94 0 0.00PCP'' 0.02 95 5 0.05CCCP 0.01 96 4 0.04Dicoumarol 0.02 94 0 0.00Gramicidin 12 pg/mg protein 95 0 0.00

Succinate None - 51 19 0.37DNP 0.01 50 0 0.00DBP 0.01 50 3 0.06PCP 0.02 49 0 0.00CCCP 0.01 52 0 0.00Dicoumarol 0.02 48 3 0.06Gramicidin 12 pg/mg protein 50 0 0.00

" NADH = reduced form of NAD (nicotinamide-adenine dinucleotide)." DNP = 2,4-dinitrophcnol.

DBP = 2,6-dibromophenol.PCP = pentachlorophenol.CCCP = rn,chlorocarboncyanide phenylhydrazone.

60, I^Ishaque: Ener,try Generation Mechanisms . in M. lepraemurium^65

TABLE 5. Effect of respirators' chain inhibitors on oxidative phosphoulation by cell-freeextracts of in vitro-grown M. lepraemurium.

Substrate InhibitorConcen-tration(mM)

O.consumed(n atoms)

ATPformed

(n moles)P/0

NADI+ - - 94 68 0.72Rotenone 0.01 0 0 0.00Amytal 0.10 4 0 0.00TTFA'' 0.10 95 68 0.72Antimycin A 0.10 5 0 0.00HQNO 0.20 4 0 0.00Cyanide 1.00 0 0 0.00PCMB'' 0.01 4 0 0.00N-ethylmaleimide 0.01 5 0 0.00

Succinate - 52 20 0.38Rotenone 0.01 53 20 0.38Amytal 0.10 52 20 0.38TTFA 0.10 0 0 0.00Antimycin A 0.10 4 0 0.00HQNO 0.20 0 0 0.00Cyanide 1.00 0 0 0.00PCM 11 0.01 3 0 0.00N-ethylmaleimide 0.01 4 0 0.00

Ascorbate + cytochrome c - - 300 35 0.12Rotenone 0.01 295 35 0.12Antimycin A 0.20 297 35 0.12IIQNO 0.20 298 35 0.12Cyanide 1.00 0 0 0.00

NADH = reduced form of NAD (nicotinamide-adenine dinucleotide).TTFA = thenoyltrilluoroacetone.HQNO = 2-n-hepty1-4-hydroxyquinoline-N-oxide.

d PCMB = p-chloromercuri benzoate.

ble 4). In addition, dicoumarol and grami-cidin (12 pg/mg protein) effectively inhib-ited ATP synthesis linked with oxidativephosphorylation. The energy is thus gen-erated through the process of oxidativephosphorylation by the in vitro-grown Al.lepraemurium.

Effect of respiratory chain inhibitors. Thedata in Table 5 show that NADH oxidationas well as the associated phosphorylationwere markedly sensitive to the flavoproteininhibitors rotenone and amytal. However,neither the oxidation of succinate nor thecoupled ATP synthesis was affected by theseinhibitors. Interestingly, the oxidation ofNADH and the accompanying ATP for-mation is not inhibited by 0.1 mM TTFA,but oxidation of the succinate and the con-comitant ATP generation were completelyinhibited. Although the phosphorylationscoupled to NADH and succinate oxidationwere completely inhibited by antimycin Aor HQNO, these inhibitors did not affectATP generation coupled to ascorbate oxi-

dation. However, complete inhibition of thephosphate esterification was caused by cy-anide when NADH, succinate, or ascorbateserved as the electron donor. The thiol-binding agents, PCMB and N-ethylmaleim-ide, were effective inhibitors of both NADHand succinate oxidations as well as the con-comitant ATP generation (Table 5).

The effect of various inhibitors on theelectron transfer reactions when NADH andsuccinate served as electron donors is shownin Figures 1 and 2, respectively. Cell-freeextracts treated with NADH (Fig. I, traceA) revealed absorption peaks at 562 and530 nm as well as at 550 and 523 nm whichare indicative of alpha and beta peaks of b-and c-type cytochromes, respectively. Thegamma peaks of these cytochromes are fusedtogether in a single peak at 428 nm. Theabsorption peak at 625 nm is of the d typecytochrome. When the cell-free extracts werepreincubated with 0.01 mM rotenone for 5min, no cytochrome was reduced upon ad-dition of NADH (Fig. 1, trace D); whereas

66^ International Journal of Leprosy^ 1992

I0 01 ABSORBANCE

A

C

400^440^480^520^560^600^640WAVELENGTH (nm)

FIG. 1. Effect of different inhibitors on the cyto-chrome system of in vitro-grown .1/. lepraernurium re-duced with NADH. The reaction mixture in a totalvolume of 2 ml contained cell-free extracts (3 mg pro-tein) in 500 mM potassium phosphate buffer, pH 6.5.Trace A = NADH reduced minus oxidized differencespectrum; Traces B, C, and D = changes in absorptionspectra in presence of 0.1 mM tenoyltrifluoroacetone(TTFA), 0.1 mM antimycin A, and 0.01 mM rotenone,respectively.

in the presence of the same concentrationsof rotenone, the cytochromes were com-pletely reduced by succinate (Fig. 2, traceB). On the other hand, 0.1 mM TTFA com-pletely prevented the reduction of respira-tory components by succinate (Fig. 2, traceD), but the same system was not affected byTTFA when NADH served as the electrondonor (Fig. 1, trace B). When the cell-freeextracts containing antimycin A were treat-ed with NADH or succinate, cytochrome bwas reduced but cytochrome c remained inthe oxidized state (Figs. 1 and 2, trace C).

DISCUSSIONAlthough components of the respiratory

chain, mechanisms of electron transport andthe energy production processes have beenstudied extensively in several bacterial sys-tems, relatively very little is known aboutsuch systems in leprosy-causing mycobac-teria, namely, M. leprac and Al. leprae-murium. The reason for this has undoubt-edly been the inability to cultivate these

IO 01 ABSORBANCE

A

400^440^480 520^560^600^640WAVELENGTH (nm)

Fla 2. Effect of different inhibitors on cytochromesystem of in vitro-grown .1/. lepraemurium reduced withsuccinate. Reaction mixture was the same as in Figure1. Trace A = succinate reduced minus oxidized differ-ence spectrum; Traces B, C, and D = changes in ab-sorption spectra in presence of 0.1 mM rotenone, 0.1mM antimycin A, and 0.1 mM tenoyltrifluoroacetone(TTFA), respectively.

mycobacteria in vitro. Al. lepractintrium cannow be cultivated in vitro and an adequateamount of pure cells can be obtained formetabolic studies using the mass bacterialinoculation technique described by Ishaque( 7). It has been shown previously (9) that invitro-grown M. lepraemurium containedflavoproteins and cytochromes of the b, c,(1, and o types. It has also been shown ( 6)that glycerol, succinate, Tween 80, and somesulfhydryl compounds were oxidized by thecell-free extracts prepared from in vitro-grown M. lepraemurium. Energy couplingmechanisms in in vivo-grown M. leprae-murium have previously been reported ( 10 ).The results presented in this report show forthe first time that cell-free preparations fromin vitro-grown M. lepraennerium catalyzedphosphorylation coupled to the oxidationof generated NADH, added NADH, suc-cinate, and ascorbate (plus cytochrome c),yielding P/O ratios of 0.75, 0.52, 0.36 and0.12, respectively (Table 1). That the phos-phate esterification resulted from oxidativephosphorylation is supported by the data

60, 1^Ishaque: Energy Generation Mechanisms' in M. lepraemurium^67

which show that the classical uncouplingagents, at concentrations which do not in-hibit oxidation, completely uncoupled theprocess when either NADH or succinateserved as the electron donor (Table 4).

Although the rate of oxidation of addedNADH was nearly the same as that ob-tained by the generated NADH, consis-tently much lower P/O ratios resulted withexogenously added NADH. Such a phe-nomenon has also been observed in the invivo-grown M. lepraenntriton (I 0). AddedNADH was possibly partly oxidized by thecell-free extracts of Al. lepraenturitimthrough the nonphosphorylative pathway assuggested in AI. phlei ( 2 ' 4 ' 19 ). Similar results,about NADH oxidation, have been report-ed in other bacterial systems ( 5 ' 12 ) and inmammalian mitochondria (''' ' 6 ). The sol-uble fraction alone failed to carry out ATPsynthesis with any substrate. Thus, similarto the in vivo-grown bacilli (I"), the gener-ation of high energy phosphate bonds by thein vitro-grown AI. lepraemurium requiredthe combined participation of both partic-ulate and soluble components (Table 3).

The localization of the phosphorylationsites in the electron transport chain by theuse of respiratory chain inhibitors in con-junction with substrates which enter thechain at different but specific loci indicatesthe presence of three functional energy-cou-pling sites in in vitro-grown AI. lepraemu-rhon. The high sensitivity of phosphoryla-tion coupled to the oxidation of NADH, butnot to succinate, to rotenone or to amytal,is an accepted indication of a first energycoupling site. Antimycin A or HQNO, spe-cific inhibitors of the second energy cou-pling site, are both very effective inhibitorsof phosphorylations coupled to NADH orsuccinate oxidations in this organism (Table5). Moreover, the presence of the first andsecond energy coupling sites is also sup-ported by the observed effect of rotenoneand antimycin A (Figs. 1 and 2) on cyto-chrome reduction.

Quinones or vitamin K have been re-ported to be involved in the electron trans-port chain of M. phlei ( 1 ) and other bacteria(1 1. 22 , .) The marked inhibitory effect of di-coumarol on NADH and succinate-linkedATP synthesis in the present (Table 4) aswell as in the earlier studies ( 10) indicates

the involvement of menaquinone (a vita-min K derivative) in the electron transportchain of Al. lepraemitrittin.

The electron transport chain of in vitro-grown M. lepraemitrium is very similar tothe in vivo-grown mycobacterium ( 10 ) in be-ing sensitive to inhibitors of the electrontransfer reactions and to uncouplers of ox-idative phosphorylation. Also as in in vitro-grown M. lepraeinterhon, the first two en-ergy conservation sites were found to beoperative in the in vivo-grown Al. leprae-minium. However, of interest is the obser-vation that, contrary to in vivo-grown Al.lepraeinurizn, ATP generation coupled toascorbate oxidation in the in vitro-grownmycobacterium occurred in the presence ofadded cytochrome c (Table 1). The failureof the cell-f•ee preparations from in vivo-grown Al. lepraenturium to generate ATP( 10) in the presence of added cytochrome cled us to conclude that this mycobacteriumis deficient in the terminal energy couplingsite. This variation in the in vivo- versus invitro-grown cells could possibly be due tothe difference in the respiratory compo-nent(s) in the cytochrome c: 0, oxido-re-ductase region of the electron transportchain. It may be emphasized that cyto-chrome a+a 3 is present in in vivo-grown Al.lepraemitrizon ("); whereas in vitro-growncells were found to be deficient in cyto-chrome a+a 3 and instead contained cyto-chrome d, (9). These findings might havesome significance to the fact that a func-tional third site was detected in the in vitro-but not in the in vivo-grown Al. Lem -acquit-limn. More detailed studies are necessaryto explain this variation but it is temptingto speculate that the presence of an d,-typecytochrome in the in vitro-grown Al. leprae-murium may be the reason for the presenceof the terminal energy coupling site. Thepresence of a third phosphorylation site isalso supported by the effect of inhibitors(Table 5) on ascorbate oxidation. Cyto-chrome o has been shown to be present inin vitro-grown Al. lepraenntrium (9 ). Wheth-er cytochrome o is involved in energy con-servation remains to be investigated.

We have shown previously that a numberof sulfhydryl compounds were oxidized byin vivo- ( 14 ) as well as by in vitro-grown ( 8 )Al. lepraemurium. Similar to host-grown Al.

68^ International Journal of Leprosy^ 1992

SUCICINATE

FPS

A SC OR B AT E+ cyt c

Site I

RotenoneAmytal

— — TTFA

Site 2

Cy t b

Menaquinone^IIAntimycin AHONG

Site 3Cyt c >̂Cyt 0, d if^'02

Cyanide

NADH >̂ FPD

FIG. 3. Proposed pathways of energy and electron transfer reactions in cultivated Al. lepraemurizn. Theelectron transport from NADI I to 0 2 involved entire respiratory chain. Electrons derived from oxidation of thesuccinate couple with the respiratory chain mainly at cytochrome e level. The entry of electrons from ascorbateoccurs at cytochrome c level. The third site has been found functional only in the presence of added cytochromec. TTFA = tenoyltrilluoroacetone; HQNO = 2-n-heptyl-4-hydroxyquinolone-N-oxide.

lepraemurium( 1 "), the oxidations of NADHand succinate as well as the associated phos-phorylations catalyzed by the cell-free ex-tracts of in vitro-grown cells were markedlyinhibited by the thiol-binding agents PCMBand N-ethylmaleimide (Table 5). These re-sults suggested the involvement of sulfhy-dryl groups in the enzymatic oxidation ofthese substrates. The present results ob-tained by in vitro-grown cells clearly indi-cate that the results obtained in earlier stud-ies with in vivo-grown M. lepraemurium (m)were indeed due to pure cells, devoid of anyhost tissues.

Based on experimental observations theprobable pathways of electron and energytransfer reactions are presented in Figure 3.

SUMMARYMycobacterium lepracmurium was culti-

vated on Ogawa egg-yolk medium and itsenergy coupling mechanisms were investi-gated. Cell-free extracts prepared from invitro-grown cells catalyzed phosphorylationcoupled to the oxidation of generatedNADH, added NADH, and succinate-yielding ratios of phosphorus moles incor-porated into high-energy bonds to oxygenatoms utilized (P/O ratios) of 0.75, 0.52,and 0.36, respectively. Ascorbate oxidationalone or in the presence of tetramethyl-p-phenyline-diamine (TMPD) did not yieldany adenosine triphosphate (ATP). How-ever, ascorbate in the presence of added cy-tochrome c was coupled to ATP synthesisand yielded a P/O ratio of 0.12. The oxi-

dative phosphorylation was uncoupled byall of the uncouplers used without any in-hibition of oxygen consumption. ATP gen-eration coupled to NADH oxidation wascompletely inhibited by the flavoprotein in-hibitors, such as rotenone and amytal; theseinhibitors had no effect, however, on ATPsynthesis associated with succinate oxida-tion. Antimycin A or 2-n-heptyl-4-hy-droxy-quinoline-N-oxide (HQNO) and cy-anide inhibited markedly the oxidations ofNADH and succinate as well as the coupledATP generation. The phosphorylation cou-pled to ascorbate plus cytochrome c was notaffected by either of the flavoprotein inhib-itors or by antimycin A or HQNO, but wascompletely inhibited by cyanide. The thiol-bearing agents p-chloromercuribenzoate(PCMB) and N-ethylmaleimide were thepotent inhibitors of the phosphorylation as-sociated with the oxidation of NADH andsuccinate. The results indicate that the threeenergy-coupling sites are functional in therespiratory chain of in vitro-grown M. lep-raemurium.

RESUMENSe estudiaron los mecanismos acopladores de ener-

gia del Mycobacterium lepraemurium crecido en el me-dio de Ogawa-yema de huevo. Los extractor libres decélulas de las micobacterias cultivadas in vitro catali-zaron la fosforilaciOn acoplada a la oxidaciOn del NADHgenerado y del NADH y succinato adicionados, ex-hibiendo indices de incorporaciOn de fOsforo (en moles)en enlaces de alts energia/atomos de oxigeno utilizados(indices P/O) de 0.75, 0.52, y 0.36, respectivamente.La oxidación del ascorbato solo o en presencia de te-

60, 1^Ishaque: Energy Generation Mechanisms in M. lepraemurium^69

trametil-p-fenilen diamina (TMFD) no produjo nadade trifosfato de adenosine (ATP). Sin embargo, en pre-sencia de citocromo c exOgeno, el ascorbato fue aco-plado a la sintesis de ATP y produjo un indite P/0 de0.12. La fosforilación oxidativa fuc desacoplada portodos los desacopladores usados sin que ocurriera nin-guna inhibición del consumo de oxigeno. La genera-tion de ATP acoplada a la oxidación del NADH fuecornpletamente inhibida por los inhibidores de flavo-proteinas, retenona y amital; sin embargo, estos inhi-bidores no tuvieron efecto sobre la sintesis del ATPasociada a la oxidación del succinato. La antimicinaA o Oxido de la 2-n-heptil-4-hidroxiquinolina (HQNO)y el cianuro, inhibieron marcadamente las oxidacionesdel NADH y del succinato, asi como la generaciónacoplada del ATP. La fosforilación acoplada al ascor-bato en presencia de citocromo c, no fue afectada nipor los inhibidores de llavoproteinas ni por la anti-micina A o HQNO, Pero fue completamentc inhibidapor cianuro. Los agentes tiolados p-cloromercuriben-zoato (PCMB) y N-etilmaleimida, fueron potentes in-hibidores de la fosforilación oxidativa del NADH ysuccinato. Los resultados indican que los 3 sitios aco-pladores de energia son funcionales en la cadena sitiosacopladores de energia son funcionales en la cadenarespiratoria del Al. lepraemurium cultivado in vitro.

RESUMEMycobacterhun lepraemurium a été cultivé sur mi-

lieu de jaune d'oeuf d'Ogawa et ses mécanismes decouplage d'energie ont été étudiés. Des extraits acel-lulaires prepares a partir de cellules cultivées in vitroont catalyse la phosphorilation couplée a l'oxydationdu NADH endogene, NADH exogène, et des rapportsde production de succinate des moles de phosphoreincorporés dans des liens a haute energia a des atomesd'oxygène utilises (rapport P/O) de respectivement0, 75, 0, 52, et 0, 36. L'oxydation de l'ascorbate seulou en presence de diamine de tetraméthyl-p-phenyline(TMPD) n'a pas d'adenosine triphosphate (ATP). Ce-pendant, de l'ascorbate en presence de cytochrome cadditionnel etait couplée 0 la synthese d'ATP et pro-duisait un rapport P/O de 0, 12. La phosphorilationoxydative était decouplée par tons les découpleurs uti-lises sans inhibition de la consommation d'oxygène.La production d'ATP couplée a l'oxydation du NADHétait completement inhibée par les inhibiteurs de lallavoprotéine, telle que la rotenone et l'amytal; ces in-hibiteurs n'avaicnt pas d'effet, cependant, sur la syn-these d'ATP associée a l'oxydation du succinate.L'antimycinc A ou le 2-n-heptyle-4-hydroxiquinoline-n-oxyde (HQNO) et la cyanide inhibaient de fawnmarquee les oxydations du NADH et du succinate ainsique le couplage de la production d'ATP couple. Laphosphorilation couplet a l'ascorbate plus cytochromec n'était affectée par aucun des inhibiteurs de la fla-voproteine ni par I'antimycine A ou le HQNO, maisétait completement inhibée par la cyanide. Les agentsp-chloromercuribenzoate (PCMB) et N-ethylmaleim-

ide et porteurs de thiol étaient les inhibiteurs puissantsde la phosphorilation associée a l'oxydation du NADHet du succinate. Les résultats indiquent que les troissites de couplage d'énergie sont fonctionnels dans lachaine respiratoire de M. lepraemurium cultivé in - vitro.

Acknowledgment. This investigation was generous-ly supported by Le Secours aux Lépreux, Canada, Inc.

REFERENCES1. ASANO, A. and BRODIE, A. F. Oxidative phos-

phorylation in fractionated bacterial systems.XVIII. Phosphorylation coupled to different seg-ments of the respiratory chains of MycobacteriumWei. J. Biol. Chem. 240 (1965) 4002-4010.

2. BOo1N, E., HIGASIII, T. and BRODIE, A. F. Exog-enous NADH oxidation and particulate fumaratereductase in Mycobacterium phlei. Arch. Biochem.Biophys. 129 (1969) 211-220.

3. Boos, W. Bacterial transport. Ann. Rev. Bio-chem. 43 (1974) 123-146.

4. BRODIE, A. F. Oxidative phosphorylation in frac-tionated bacterial systems. 1. Role of soluble fac-tors. J. Biol. Chem. 234 (1959) 398-404.

5. GIBSON, J. and MORITA, S. Changes in adenosinenucleotides of intact Chromatium D produced byillumination. J. Bacteriol. 93 (1967) 1544-1550.

6. HAROLD, F. NI. Conservation and transformationof energy by bacterial membranes. Bacteriol. Rev.36 (1972) 172-230.

7. ISHAQUE, M. In vitro cultivation of Mycobacte-rium lepraemurium and its identification by ani-mal inoculation. Can. J. Microbiol. 27 (1981) 788-794.

8. ISHAQUE, NI. Respiratory activities of in vitro-grown Mycobacterium lepraemurium. Int. J. Lepr.51 (1983) 64-71.

9. Isiii■QuE, M. Cytochrome system in cultivatedMycobacterium lepraemurium. Cytobios 39 (1984)165-170.

10. ISHAQUE, M., ADAPOE, C. and KATO, L. Energycoupling mechanisms in host grown Alycobacte-rium lepraemurium. Can. J. Biochem. 59 (1981)75-82.

11. ISHAQUE, M. and ALEEM, M. I. H. Energy couplingin Hydrogenomonas eutropha. Biochim. Biophys.Acta 223 (1970) 388-397.

12. ISHAQUE, M., DONAWA, A. and ALEEM, M. I. H.Electron transport and energy generation in Pseu-domonassaccharophila. Can. J. Biochem. 49 (1971)1175-1182.

13. ISHAQUE, M. and KATO, L. The cytochrome sys-tem in Mycobacterium lepraemurium. Can. J. Mi-crobiol. 20 (1974) 943-947.

14. KATO, L., ADAQUE, C. and ISHAQUE, M. The re-spiratory metabolism of Mycobacterium leprae-murium. Can. J. Microbiol. 22 (1976) 1293-1299.

15. LEHNINGER, A. L. Phosphorylation coupled to ox-idation of dihydrodiphosphopyridine nucleotide.J. Biol. Chem. 190 (1951) 345-359.

70^ International Journal of Leprosy^ 1992

16. LESTER, R. L., ZIEGLER, D. M. and GREEN, D. E.

Studies on the mechanism of oxidative phos-phorylation. II. Role of bound pyridine nucleotide

in phosphorylation. 24 (1957) 155-160.

17. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L.

and RANDALL, R. J. Protein measurement withFolin phenol reagent. J. Biol. Chem. 193 (1951)

265-275.

18. Mom, T. Biochemical properties of cultivated

Mycobacterium lepracmurizun. Int. J. Lepr. 43(1975) 210-217.

19. Mulmw, I'. S. and BuormE, A. F. Oxidative phos-

phorylation, in fractionated bacterial systems. XV.

Reduced nicotinamide adenine dinucleotide phos-

phate-linked phosphorylation. J. Biol. Chem. 239(1964) 4292-4297.

20. OGAWA, T. and MOTOMURA, K. Studies of murine

leprosy bacillus. 1. Attempt to cultivate in vitro

the Hawaiian strain of Mycobacterium leprae-Kitasato, Arch. Exp. Med. 43 (1970) 21-

36.

21. PATTYN, S. R. and PORTAELS, F. In vitro culti-vation and characterization of llycobacterhonpraemurizon. Int. J. Lepr. 48 (1980) 7-14.

22. SCROLLS, I'. B. and SMITH, L. Composition andproperties of membrane-bound respiratory chainsystem of Micrococcus^Biochem.13iophys. Acta 153 (1968) 363-375.

23. STEFANSKY, W. K. Eine lepra-iihnliche Erkran-kung der Haut and der lymphdrusen bei wander-

ratten. Zentrallel. l3akteriol. parasitenkd. Nick-tionskr. Hyg. Abt. I: Orig. 33 (1903) 481-487.

24. STREIILER, B. M. Adenosine-5'-triphosphate and

creatine phosphate; determination with luciferase.

In: Methods of Enzymatic Analysis. Bergmeyer, H.U., ed. New York: Academic Press, 1965, p. 563.

Page 1Page 2Page 3Page 4Page 5Page 6Page 7Page 8Page 9Page 10