Plant Physiol. (1983) 72, 802-8080032-0889/83/72/0802/07/$00.50/0

Mechanisms of Citrate Oxidation by Percoll-PurifiedMitochondria from Potato Tuber1

Received for publication January 19, 1983 and in revised form March 25, 1983

ETIENNE-PASCAL JOURNET AND ROLAND DOUCEPhysiologie Cellulaire Vigeitale, Departement de Recherche Fondamentale, Centre d'Etudes Nucleaires etUniversite Scientfifque et Midicale de Grenoble, 85 X-38041 Grenoble Cedex, France

ABSTRACT

The mechanisms and accurate control of citrate oxidation by Percoll-purified potato (Solanum tuberosum) tuber mitochondria were character-ized in various metabolic conditions by recording time course evolution ofthe citric acid cycle related intermediates and 02 consumption. Intactpotato tuber mitochondria showed good rates of citrate oxidation, providedthat nonlimiting amounts of NAD+ and thiamine pyrophosphate werepresent in the matrix space. Addition of ATP increased initial oxidationrates, by activation of the energy-dependent net citrate uptake, and stim-ulated succinate and malate formation. When the intramitochondrialNADH to NAD+ ratio was high, a-ketoglutarate only was excreted fromthe matrix space. After addition ofADP, aspartate, or oxaloacetate, whichdecreased the NADH to NAD+ ratio, flux rates through the Krebs cycledehydrogenases were strongly increased and a-ketoglutarate, succinate,and malate accumulated up to steady-state concentrations in the reactionmedium. It was concluded that NADH to NAD+ ratio could be the primarysignal for coordination of fluxes through electron transport chain or malatedehydrogenase and NAD+-linked Krebs cycle dehydrogenases. In addition,these results clearly showed that the tricarboxylic acid cycle could serveas an important source of carbon skeletons for extra-mitochondrial syn-thetic processes, according to supply and demand of metabolites.

The Krebs cycle is a conversion block coupling catabolism ofacetyl units to generation of redox equivalents. It also serves as animportant source of carbon skeletons for synthetic processes suchas amino acid synthesis (15). The velocity with which this cycleturns is mainly determined by three quantities: availability ofsubstrates, redox state (NADH/NAD+), and energy state (ATP/ADP, ATP/AMP, energy charge). These three controlling factorsinteract in a highly coordinated and cooperative manner (23). Inaddition, these controls are complemented by input and outputthrough the inner mitochondrial membrane anions translocators,according to demand and offer of metabolites.

In contrast with several extensive studies on metabolic controlof citric acid cycle activity in mammalian mitochondria (23),attempts to examine the over-all Krebs cycle functioning ofhigherplant mitochondria have been sparse in recent years (24). In thiscommunication, we report the mechanisms of citrate oxidation inpurified potato tuber mitochondria.

' Supported in part by Research Grant from the 'Centre National de laRecherche Scientifique' (E.R.A. 847: Interactions Plastes-Cytoplasme-Mi-tochondries).

MATERIALS AND METHODS

Preparation of Mitochondria. Potato tubers (Solanum tubero-sum L.) were obtained from a local market. The mitochondriawere prepared and purified by methods previously described (16).The purification step consisted of separating mitochondria fromcontaminating organelles and membrane vesicles on self-gener-ated Percoll gradients and in a relatively short time. The Percoll-purified mitochondria devoid of carotenoids and galactolipidsshowed no contamination with intact plastids, microbodies, orvacuolar enzymes. Percoll-purified mitochondria exhibited intactmembranes and a dense matrix. The intactness of purified mito-chondrial preparations was ascertained by the measurement ofKCN-sensitive ascorbate Cyt c-dependent 02 uptake (16). Whencompared with washed mitochondria, Percoll-purified mitochon-dria showed improved rates of substrate oxidation, respiratorycontrol, and ADP:O ratios. The recovery of the Cyt oxidase was70 to 90%o and on a Cyt oxidase basis the rate of succinateoxidation by unpurified mitochondria was equal to that recordedfor Percoll-purified mitochondria (16).02 Uptake Measurements. 02 uptake was measured at 25°C

using a Clark-type electrode system purchased from HansatechLtd, Hardwick Industrial Estate, King's Lynn, Norfolk, England.The reaction medium (medium A) contained: 0.3 M mannitol; 5mM MgCl2; 10 mM KC1; 10 mm phosphate buffer, pH 7.2; 0.1%(w/v) defatted BSA and known amounts of mitochondrial proteinin a total volume of 1 ml. The 02 concentration in air-saturatedmedium A was taken as 240 ,UM.

Assay of Metabolic Products. The products of the citrate me-tabolism were routinely assayed with intact mitochondria at 25°Cin a cell containing medium A and known amounts of mitochon-drial protein and of various cofactors or inhibitors, under contin-uous stirring. Each reaction was initiated by the addition of 5 mmcitrate. At various times, 2-ml aliquots were taken and added to0.6 ml of ice-cold 20%o (v/v) HC104 containing 1 mm EDTA. Thesamples were quickly neutralized with 300 pd 5 N KOH (up to pH5.5) and centrifuged for 5 min at 27,000g to remove KC104. Thesupernatant was used for a-ketoglutarate, succinate, malate, ox-aloacetate, pyruvate, and glutamate determination.

Simultaneously, the 02 consumption of a l-ml aliquot wasmeasured. a-Ketoglutarate was determined with glutamate dehy-drogenase (EC 1.4.1.3) according to Bergmeyer and Bernt (1).Succinate was determined with succinyl-CoA synthetase (EC6.2.1.5), pyruvate kinase (EC 2.7.1.40), and lactate dehydrogenase(EC 1.1.1.27), according to Williamson (22) except that ITP wasused instead of GTP. Malate was determined with malate dehy-drogenase (EC 1.1.1.37) in the presence of hydrazine by themethod of Gutman and Wahlefeld (7). Oxaloacetate, after decar-boxylation with NiCl2, and pyruvate were determined with lactatedehydrogenase according to Wedding et aL (21). Glutamate wasdetermined with glutamate dehydrogenase in the presence of 3-acetylpyridine adenine dinucleotide (APAD) according to Witt

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CITRATE OXIDATION IN PLANT MITOCHONDRIA

(26).Mitochondrial Protein Determination. Mitochondrial protein

content was determined by the Folin-Ciocalteu phenol reagent(14).

RESULTS

Respiratory Measurements. Figure 1 indicates that, in the pres-ence of 300 AM TPP2, potato tuber mitochondria oxidize citrate.This figure also indicates that during the course of citrate oxidationpotato tuber mitochondria display depressed initial state 3 rates ofrespiration which rise to a maximum with several consecutive state3/state 4 cycles. This development of state 3 respiration has beentermed 'conditioning' (10, 19). By preincubating the mitochondriawith ATP (Fig. 1) or with a small amount of dicarboxylate suchas malate (result not shown), the maximal rate of 02 consumptionis attained more rapidly. The absence of TPP (result not shown)or the addition of 3 mm Na-arsenite, a well known inhibitor ofoxidative decarboxylations, decreases the final rate of 02 con-sumption in state 3 (Fig. 1).

In a previous paper, we demonstrated that the total amount ofNAD+ present in freshly prepared potato mitochondria was vari-able (20). Values between 0.3 and 2 mm have been found. IfNAD+-deficient mitochondria were incubated with 0.5 mm NAD+at 25°C, the internal pyridine nucleotide pool size increased at arate of 2 + 0.5 nmol/mg protein min. The rate of NAD+ accu-mulation under these conditions depended on NAD+ concentra-tion and temperature (20). Whenever the total amount of mito-chondrial NAD+ is low (i.e. below 0.6 mM), the rate of citrateoxidation by potato tuber mitochondria is considerably stimulatedby exogenous NAD+ (Fig. 2).These results demonstrate that the rate of citrate oxidation by

potato tuber mitochondria may be limited by a lack ofendogenousNAD+ (5) and TPP (3, 10). These results also suggest that in plant

2Abbreviation: TPP, thiamine pyrophosphate.

mitochondria NAD+ and TPP are not firmly bound to the innermembrane and/or to the various NAD+-linked dehydrogenases.Time Course Changes of Products of Citrate Oxidation in State

4. The time course of the formation of intermediates during theoxidation of citrate in the presence of TPP, ATP, NAD+, andvarious inhibitors is shown in Figure 3. In the absence of ADP,mitochondria produce almost exclusively a-ketoglutarate (Fig.3A). When the same experiment is carried out in the presence ofmalonate (Fig. 3B) or Na-arsenite (Fig. 3C), the rate of a-ketoglu-tarate accumulation remains unchanged, about 70 nmol/min-mmg- protein. Inasmuch as the rate of 02 uptake (75 natom.min- Pmg- protein) is approximately equal to the rate of a-ketoglutarate accumulation, these results demonstrate that mainlyone dehydrogenase is operating, namely the isocitrate dehydro-genase. Rapid centrifugation (10,000g, 40 s, Beckman MicrofugeB) showed that more than 95% of a-ketoglutarate was present inthe incubation medium. These results strongly suggest that, instate 4, a-ketoglutarate molecules once formed in the matrix spaceare rapidly excreted in the medium. The very low succinateaccumulation on the one hand, and the low flux through theisocitrate dehydrogenase step on the other hand, are probablyattributable to the fact that NADH is an inhibitor of NAD+-linked isocitrate dehydrogenase (4, 6) and a-ketoglutarate dehy-drogenase (23). Consequently, high levels ofNADH in the absenceof ADP may turn off the accumulation of succinate. Therefore,with the aim of decreasing NADH/NAD+ ratio in state 4, weexamined the effect of aspartate on accumulated products and 02consumption during the course of citrate oxidation by potato tubermitochondria. In this process, the aspartate transaminates witha-ketoglutarate in the matrix space and the oxaloacetate thusformed is immediately converted into malate by the malate de-hydrogenase at the expense of intramitochondrial NADH (8).

Figure 3A shows the results from a representative series ofexperiments. After addition of aspartate to mitochondria, a clearinhibition of the respiration rate is observed. Enzymic analysisshowed that, during the time of inhibition, several phenomena

FIG. 1. Oxidation of citrate by intact purified potato tuber mitochondria. Incubation medium A contained 0.39 mg of mitochondrial protein. ml-'.The concentrations given are the final concentrations in the reaction medium. The arrows on the traces indicate successive additions of 100 ,UM ADP.The numbers on the traces refer to nmol 02 consumed.min-'.mg-g protein. No increase of 02 uptake rates could be observed with this sample ofmitochondria when I mM NAD+ was added in preincubation. The mitochondrial NAD' concentration was 1.2 mM.

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JOURNET AND DOUCE

FIG. 2. Effect ofNAD+ on citrate oxidation by NAD+-depleted potatotuber mitochondria. Incubation medium A contained 0.72 mg of mito-chondrial protein-l'. The concentrations given are the final concentra-tions in the reaction medium. The arrows on the traces indicate successiveadditions of 150 lAM ADP. The numbers on the traces refer to rmol 02

consumed-min1 .mg-' protein. Note the strong increase of state 3 02uptake rates and the decrease of the lag period when NAD+ is added.Inasmuch as the medium contains 3 mm malonate, the succinate dehydro-genase is fully inhibited. The mitochondrial NAD+ concentration was 0.5mM.

occur: first, a-ketoglutarate is partially reabsorbed and metabo-lized by the mitochondria, a steady-state is rapidly attained andthereafter the size of the a-ketoglutarate pool remains approxi-mately constant at 100 AM. Second, a transient accumulation ofsuccinate occurs. Third, malate and glutamate accumulate at theinitial rate of 170 nmol/min-.mg-' protein. This indicates thataspartate transaminates with a-ketoglutarate and the reduction ofoxaloacetate thus formed depends on the reduced pyridine nu-cleotide generated by the isocitrate dehydrogenase and perhapsthe TPP-linked a-ketoglutarate dehydrogenase. Since the rate ofa-ketoglutarate production without aspartate (Fig. 3A) is 2.5 timeslower than that of glutamate or malate production in the presenceof aspartate (i.e. the rate of a-ketoglutarate transamination), it ispossible that flux through isocitrate dehydrogenase in state 4 issubject to coarse regulation by NADH/NAD+ ratio. In support ofthis hypothesis, Figure 3C shows that, in the presence of Na-arsenite, the rate of glutamate or malate production after aspartateaddition (120 nmol.min-'.mg- protein) is still higher than thatof a-ketoglutarate without aspartate. However, in this case, theinhibition of 02 consumption is powerful, probably because lessreducing equivalents are generated by the Krebs cycle.

Likewise, the transient accumulation (without malonate, Fig.3A) or the steady accumulation (with malonate, Fig. 3B) ofsuccinate after the addition of aspartate also indicate that theinhibition of a-ketoglutarate dehydrogenase is partially releasedwhen the intramitochondrial concentration of NADH decreases.The consecutive oxidation of the succinate formed in the absenceof malonate explains why more malate than glutamate accumu-lates in the incubation medium (Fig. 3A). Inasmuch as the rate ofsuccinate production (30 nmol. min-' mg-1 protein, Fig. 3B) inthe presence of malonate and 2 mm aspartate is much lower thanthat of glutamate (130 nmol-min-1.mg-' protein), these resultsalso demonstrate that, in state 4, a greater percentage of the a-

ketoglutarate formed is metabolized through the transaminase

pathway. It is noteworthy that oxaloacetate, as a substitute foraspartate, leads also to an acceleration of a-ketoglutarate andsuccinate formation (results not shown; see also Fig. 6). It seemslikely therefore that, in plant mitochondria as well as in animalmitochondria (23), the restricted dehydrogenase activities in theabsence of ADP are mainly caused by a high ratio of NADH toNAD+.

Effect of ATP on Time Course Changes of Products of CitrateOxidation in State 4. It is obvious that oxidation ofa-ketoglutaraterequires nucleotides. As a matter of fact, the very low rate of state4 oxidation of a-ketoglutarate (Fig. 3A) is due also to the control-ling influence at the substrate level site (25). Furthermore, succi-nate dehydrogenase in mitochondria isolated from plant tissues isusually found to be in a deactivated state and preincubation ofthe mitochondria with ATP overcomes this problem (17). There-fore, we examined also the effect ofATP on accumulated productsand 02 consumption during the course of citrate oxidation in thepresence of aspartate by potato tuber mitochondria (Fig. 4). Inthe absence of ATP, addition of aspartate to mitochondria oxidiz-ing citrate results in a powerful inhibition of 02 consumption(compate with Fig. 3A). This inhibition is comparable to thatpreviously observed in the presence of Na-arsenite (Fig. 3C).Under these conditions, the accumulation of a-ketoglutarate (at80 nmol.min' mg-' protein) is stopped and its pool size declinesslightly. Glutamate and malate are formed equally (at 120 nmol.minml-mg-I protein) and a very small amount of oxaloacetateappears in the reaction medium. These observations demonstratethat, in the absence of ATP, the totality of a-ketoglutarate formedis metabolized through the transaminase pathway in so far as themedium contains aspartate.The addition of ATP to the medium triggers a cascade of

reactions. First, after a lag phase, 02 uptake is initiated; second,before and after the onset of 02 uptake, the pool size of a-ketoglutarate declines markedly until a new steady-state is ob-tained; third, a small and transient accumulation of succinateoccurs, which is accompanied by the disappearance of oxaloace-tate; fourth, malate accumulates more rapidly than glutamate.These results demonstrate that, during the course of citrate oxi-dation in the presence of aspartate, ATP releases the inhibition ofthe e-ketoglutarate dehydrogenase and succinate dehydrogenase.Therefore, the restarting of 02 consumption can be explained bytwo concurring phenomena: first, the increase in the amount ofreducing equivalents generated by the mitochondrial dehydrogen-ases after ATP addition; second, a decrease in a-ketoglutarateconcentration, which shifts the glutamate oxaloacetate transami-nase equilibrium.Time Course Changes of Products of Citrate Oxidation in State

3. The time course of the formation of intermediates during theoxidation of citrate in the presence of TPP, NAD+, ADP, andvarious inhibitors is shown in Figure 5. Under state 3 conditions,potato tuber mitochondria produce a-ketoglutarate, succinate, andmalate (Fig. 5A). This is in contrast with what was previouslyobserved in the absence ofADP (Fig. 3A). Consequently, in vitro,the reactions of the citric acid cycle operate essentially not as acycle, because of carbon loss as a-ketoglutarate and succinate.After 3 min of incubation in state 3, loss of a-ketoglutarate ceasesbut loss of succinate continues so that less malate is formed. Inaddition, even up to 10 min of incubation in state 3, malateformation, in contrast with succinate accumulation, showed nosign of reaching a maximum (result not shown). Because the rateof 02 uptake (390 natom min-'. mg-' protein) is approximatelyequal to the mean rate of a-ketoglutarate accumulation plus twicethe rate of succinate formation and three times the rate of malateformation, these results demonstrate that three dehydrogenasesare operating during the course of citrate oxidation in state 3,namely the isocitrate dehydrogenase, the a-ketoglutarate dehydro-genase, and the succinate dehydrogenase. When the same experi-

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CITRATE OXIDATION IN PLANT MITOCHONDRIA 805

FIG. 3. Time course products accumulation and 02 consumption during citrate oxidation in the absence of ADP by purified potato tubermitochondria. The standard assay solution was used with 0.80 mg mitochondrial proteinml-', 0.3 mm ATP, I mm NAD+ plus 0.3 mm TPP (A) or plus0.3 mm TPP and 3 mM malonate (B) or plus 0.3 mM TPP and 3 mM Na-arsenite (C). Final pH was 7.2 and final volume of the reaction medium was 20ml. The reaction was initiated by addition of 5 mm citrate, after 5 min preincubation of mitochondria in the reaction medium. No pyruvate could bedetected in any sample, and no oxaloacetate in A and B. a-Ketoglutarate (0); succinate (K); malate (V); glutamate (1); oxaloacetate (0).

ment is carried out in the presence of malonate (Fig. 5B), potatomitochondria produce a-ketoglutarate and succinate. Again, evenup to 10 min of incubation in state 3, succinate formation, incontrast with a-ketoglutarate loss, showed no sign of reaching amaximum (result not shown). Under these circumstances, inas-much as the rate of 02 uptake (220 natom min-'.mg-1 protein)is approximately equal to the rate of a-ketoglutarate accumulationplus twice the rate of succinate formation, two dehydrogenases areoperating, namely the isocitrate dehydrogenase and the a-ketoglu-tarate dehydrogenase. Finally, in the presence of Na-arsenite, onlyone dehydrogenase is operating, namely the isocitrate dehydro-genase (at 110 nmol.min-' mg-' protein, Fig. 5C). These figuresalso indicate that, with citrate, state 3 rates ofpotato mitochondriaincrease by a factor of 1.7 if malonate is omitted, and by a factorof 3.4 if Na-arsenite is omitted (10).The addition of aspartate in state 3 to potato mitochondria

oxidizing citrate is practically without effect on the rate of 02

consumption (Fig. SA). The same thing holds true in the presenceof malonate (Fig. 5B). Under these conditions, the small size poolof a-ketoglutarate does not decrease and the rate of glutamateproduction is low (45 [Fig. 3A] and 25 [Fig. 3B] nmol.min-u.mg-1 protein). These results demonstrate that, in state 3, in contrastwith what was observed in state 4, flux through a-ketoglutaratedehydrogenase is much higher than that through glutamate oxal-oacetate transaminase. It is possible therefore that the concentra-tion of a-ketoglutarate in the medium is not sufficient enough topush the glutamate oxaloacetate transaminase in the desired di-rection, i.e. towards the formation of oxaloacetate. In support ofthis suggestion, we have observed that, upon addition of oxaloac-etate to mitochondria oxidizing citrate in state 3, a clear inhibitionof the respiratory rate occurs which is gradually reversed, becauseof the competition for NADH by the malate dehydrogenase (Fig.6). Likewise, upon addition of aspartate to mitochondria oxidizingcitrate in the presence of Na-arsenite, a clear inhibition of thestate 3 respiratory rate also occurs because the concentration ofa-ketoglutarate in the incubation medium is much higher (Fig.SC) than that observed in the absence of Na-arsenite. In this case,

the most part of a-ketoglutarate molecules thus formed is divertedtowards the formation of oxaloacetate, at the rate of 100 nmol-min- mg-' protein.

DISCUSSION

The results presented in this article demonstrate that potatotuber mitochondria purified by centrifugation in density gradientsof modified silica sol (Percoll) retain a good rate of citrate-de-pendent 02 consumption insofar as the medium contains TPP andNAD+. ATP has a strong action upon citrate oxidation. First, bypreincubating the mitochondria with ATP, the maximal rate ofcitrate-dependent 02 uptake is attained more rapidly. In contrastto mammalian mitochondria, freshly isolated potato tuber mito-chondria contain very low levels of endogenous exchangeablemetabolites. Consequently, citrate must be taken up in mitochon-dria by energy-dependent net anion accumulation. It is cleartherefore that uptake and retention of citrate are probably de-pendent upon the proton-motive force arising from ATP hydrol-ysis (2, 10). Second, ATP alone, i.e. in the absence ofADP, triggersa-ketoglutarate and succinate oxidation and increases the amountof reducing equivalents generated by the mitochondrial dehydro-genases during the course of citrate oxidation. In fact, it is wellestablished now that the inactive form of succinate dehydrogenasecan be converted in the active form by incubation with activationligand such as ATP (17). The stimulation of a-ketoglutaratedehydrogenase by ATP is not clear since this enzyme requiresADP (phosphorylation at the substrate level). It is possible that,under our experimental conditions, ATP is slowly hydrolyzed bythe mitochondrial ATPase, and ADP thus formed is recycled backto ATP by the slow functioning of a-ketoglutarate dehydrogenase.In support of this suggestion, we observed that, in the presence ofmalonate, a potent inhibitor of succinate dehydrogenase, a-keto-glutarate oxidation is not fully inhibited under state 4 conditions(result not shown).

It is of interest that aspartate, as well as oxaloacetate, increasedthe rate of citrate oxidation in state 4. In the presence of aspartate,NADH is recycled back to NAD+ via malate dehydrogenase and

600 y mal 600 600

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400 + 2 mM as 400 +2 mM asp Y mal 400 + 2 mM asp

E 300 -/oxygen 300 300

00~~~~~~~~~~

B, c 200 0 A e-00ll° 200

2 I46 8 0 2 4 6 8 0 2 4 6 8

time (min) time (min) time (min)

10 12

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JOURNET AND DOUCE

FIG. 4. Effect of 2 mM aspartate and 300 pM ATP on time course products accumulation and 02 consumption during citrate oxidation by purifiedpotato tuber mitochondria. The standard assay solution was used with 0.62 mg mitochondrial protein * ml-', 1 mm NAD, and 0.3 mM TPP. Final pH was7.2 and final volume of the reaction medium was 20 ml. The reaction was initiated by addition of 5 mm citrate, after 5 min preincubation ofmitochondriain the reaction medium. No pyruvate could be detected in any sample. a-Ketoglutarate (0); succinate (5); malate (V); glutamate (5); oxaloacetate(0).

this causes an increased flux through isocitrate dehydrogenase,because plant NAD-linked isocitrate dehydrogenase is inhibitedby NADH (Ki of 190 ,UM) competitively with NAD+ (4) and isvery sensitive to the mole fraction of NAD in the reduced form(6). Likewise, flux through a-ketoglutarate dehydrogenase withcitrate as substrate became significant in the presence of aspartate,provided that the medium contained ATP. Again, this was causedmainly by the decreased NADH concentration as a result of theintramitochondrial supply of oxaloacetate via glutamate oxaloac-etate transaminase. As a matter of fact, studies with a-ketoglutar-ate dehydrogenase showed that inhibition byNADH was noncom-

petitive with respect to NAD+ (23). Thus, in plant mitochondria,changes of the intramitochondrial NADH/NAD+ ratio appear toprovide the primary signal for coordination of flux through theelectron transport chain with those enzymes which are associatedwith a large negative free energy change such as isocitrate dehy-drogenase and a-ketoglutarate dehydrogenase.During the course of citrate oxidation in state 3, potato tuber

mitochondria excrete a-ketoglutarate, succinate, and malate. Itseems possible therefore that the activity of a-ketoglutarate de-hydrogenase and succinate dehydrogenase could influence therate of a-ketoglutarate and succinate effluxes from the mitochon-dria. For example, if the a-ketoglutarate dehydrogenase is in-hibited (in state 4 or in the presence of Na-arsenite), a-ketoglutar-ate diffuses out into the bulk medium along the steep concentra-tion gradient. As long as a-ketoglutarate is excreted, we have

shown that 02 consumption keeps going. In contrast, if a-ketoglu-tarate dehydrogenase is fully activated (in state 3), a steady-stateconcentration of extramitochondrial a-ketoglutarate is rapidlyachieved. We demonstrated that, when the ratio of mitochondrialvolume to suspending medium volume is increased, steady-stateconcentrations of a-ketoglutarate and succinate are attained in themedium more rapidly, and vice versa. In fact, the practical con-sequence of a large suspending medium volume is that a-ketoglu-tarate and succinate lost from isolated mitochondria will notaccumulate in the medium very rapidly; the medium acts as buffervolume (11). It is clear that the situation is entirely different invivo, as in this case the ratio of mitochondrial volume to cytoplasmvolume is high. Consequently, during the course of citrate oxida-tion, there is probably a competition for intramitochondrial a-ketoglutarate or succinate between efflux from the mitochondriaand further metabolism in the citric acid cycle. These resultsclearly show that the tricarboxylic acid cycle could serve as animportant source of carbon skeletons for synthetic processes,especially the formation of amino acids which take place mainlyin the plastidial compartment (13) and that input and output arevery likely regulated according to supply and demand of metabo-lites.

These results suggest that there also exists a kind of competitionfor a-ketoglutarate between glutamate oxaloacetate transaminaseand a-ketoglutarate dehydrogenase, in so far as the mediumcontains aspartate. Under state 4 conditions, in the presence of

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CITRATE OXIDATION IN PLANT MITOCHONDRIA

A I B500 500 500

0 2rnMsa 400 xy2 400

300 _ , /_oxygen + 2 mb sp 300

11,,, 200 - 200 ; 200

100 . 100 100

-.-ket n

0 0 00 5 0 5 10

rn (MM) Un (min)

C

a oxygen

I MOAAI s$Cc

5 10 1S 20

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FIG. 5. Time course products accumulation and 02 consumption during citrate oxidation in the presence of ADP by purified potato tubermitochondria. The standard assay solution was used with 0.185 mg mitochondrial protein.ml-', 1.5 mm ADP, 0.3 mm ATP, 1 mM NAD plus 0.3 mMTPP (A) or plus 0.3 mM TPP and 3 mM malonate (B) or plus 3 mM Na-arsenite (C). Final pH was 7.2 and final volume of the reaction medium was 20ml. The reaction was initiated by addition of 5 mm citrate, after 5 min preincubation of mitochondria in the reaction medium. No pyruvate could bedetected in any sample, and no oxaloacetate in A and B. a-Ketoglutarate (0); succinate (U); malate (V); glutamate (El); oxaloacetate (0).

FIG. 6. Effect of limiting amounts of oxaloacetate on citrate oxidation by purified potato tuber mitochondria. Incubation medium A contained 0.72mg mitochondrial protein ml-1, 1 mm NAD, and 0.3 mm ATP. The concentrations given are the final concentrations in the reaction medium. Thenumbers on the traces refer to nmol 02 consumed-min-'. mg-' protein.

In the absence of Na-arsenite, the inhibition of 02 consumption by oxaloacetate is also severe because oxaloacetate is a potent inhibitor of succinatedehydrogenase.Note that, after each addition of oxaloacetate, the calculated NADH consumption rate values during the inhibition phase of 02 uptake are,

respectively: 220 and 266 (state 3 + TPP), 126 and 140 (state 3 + Na-arsenite), 205 and 233 (state 4 + TPP), and 146 and 138 (state 4 + Na-arsenite)rmol NADH consumed. min-' mg-' protein. If these calculated rates are compared with that of 02 uptake, expressed in natom * min-'*mg-' protein,after the reverse of the inhibition, it can be concluded that, in state 3, the addition of oxaloacetate has practically no effect on the flux rates throughNAD+-linked dehydrogenases. On the contrary, in state 4, the addition of oxaloacetate strongly increases the flux rates through NAD+-linkeddehydrogenases, approximately up to the rates attained in state 3.

aspartate, most of the a-ketoglutarate generated from citrate oxi-dation is diverted towards glutamate oxaloacetate transaminase,whereas in state 3, a-ketoglutarate is diverted towards a-ketoglu-tarate dehydrogenase. In addition, the concentration of a-ketoglu-tarate attained in the medium presumably exerts a strong effect

on the flux rate through glutamate oxaloacetate transaminase anda-ketoglutarate dehydrogenase. Conversely, inasmuch as a-keto-glutarate is generated intramitochondrially from citrate by isocit-rate dehydrogenase, its steady-state concentrations will be affectedby those factors that influence flux through glutamate oxaloacetate

807

3 mM arsente

1.5 mMA l

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808 JOURNET AND DOUCE

transaminase, a-ketoglutarate dehydrogenase, and diffusion outinto the bulk medium.

Finally, the results presented in this paper emphasize the prob-lem of aspartate transport into plant mitochondria: in animalmitochondria, the entry of aspartate in exchange for glutamate isvery slow and does not in fact occur in energized mitochondria(12). However, our results indicate that plant mitochondria are

able to use extramitochondrial aspartate as a source of oxaloace-tate (9). In fact, according to the work of Proudlove and Moore(18), aspartate appears to permeate the inner plant mitochondrialmembrane on a diffusional way. Consequently, in contrast withthe situation observed in animal mitochondria, aspartate can enterplant mitochondria sufficiently rapidly to sustain a rapid transferof reducing equivalents out of the mitochondria.

LITERATURE CITED

1. BERGMEYER HU, E BERNT 1974 2-Oxoglutarate: UV spectrophotometric deter-mination. In HU Bergmeyer, ed, Methods of Enzymatic Analysis, Ed 2, Vol 3.Academic Press, New York, pp 1577-1580

2. BIRNBERG PR, DL JAYROE, JB HANSON 1982 Citrate transport in corn mitochon-dria. Plant Physiol 70: 511-516

3. BOWMAN EJ, H IKUMA, HJ STEIN 1976 Citric acid cycle activity in mitochondriaisolated from Mung bean hypocotyls. Plant Physiol 58: 426-432

4. Cox GF, DD DAVIES 1967 Nicotinamide adenine dinucleotide-specific isocitratedehydrogenase from pea mitochondria-purification and properties. BiochemJ 105: 729-734

5. DAY D, T WISKICH 1974 The effect of exogenous nicotinamide adenine dinu-cleotide on the oxidation of nicotinamide adenine dinucleotide-linked sub-strates by isolated plant mitochondria. Plant Physiol 54: 360-363

6. DUGGLEBY RG, DT DENNIS 1970 Regulation of the nicotinamide adeninenucleotide-specific isocitrate dehydrogenase from a higher plant-the effect ofreduced nicotinamide adenine dinucleotide and mixtures of citrate and isocit-rate. J Biol Chem 245: 3751-3754

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