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Glutamate metabolism triggered by oxaloacetate in intact plant mitochondria
ARCHIVESOFBIOCHEMISTRYANDBIOPHYSICS Vol. 214, No. 1, March, pp. 366-375, 1982
Glutamate
ETIENNE-PASCAL Physiologic
Cellulaire
Metabolism Triggered by Oxaloacetate Intact Plant Mitochondria’ JOURNET:
WALTER
D. BONNER:
AND
in
ROLAND
V6g&ale, DRF/BV, Centre d’Etud.es NucZaires et Unive-rsitk Scienti$que de Grenoble, 85 X, 880.41 Grenoble Ceo?ex, France
DOUCE et Mbdicale
Received July 10, 1981, and in revised form October 12, 1981 In Percoll-purified potato tuber mitochondria, glutamate metabolism can be triggered by oxaloacetate, in the presence of ADP and thiamine pyrophospbate. There is a lag phase before Ox uptake is initiated. During this lag period, oxaloacetate is rapidly converted into a-ketoglutarate and succinate, or into malate at the expense of the NADH generated by a-ketoglutarate dehydrogenase. The ratio of the flux rates of both pathways is strongly dependent on the glutamate concentration in the medium. When all the oxaloacetate is consumed, a rapid O2 uptake is initiated. The effects of malonate on glutamate metabolism triggered by oxaloacetate and on a-ketoglutarate oxidation are reported. It is concluded that the inhibition of the succinate dehydrogenase by either malonate or oxaloacetate does not affect the rate of a-ketoglutarate dehydrogenase functioning. All the metabolites accumulated are excreted by the mitochondria in the supernatant. Some of them are then reabsorbed. These results emphasize the importance of the anion carriers in the overall process.
Mitochondria isolated from a large variety of plant tissues oxidize glutamate in the presence of malate (for a review, see Hanson and Day (1)). This oxidation, which is not clearly understood, is probably due to glutamate dehydrogenase (EC 1.4.1.3), malate dehydrogenase (EC 1.1.1.37), and glutamate-oxaloacetate transaminase (EC 2.6.1.1) localized in the matrix space. With the aim of clarifying the mechanisms of glutamate metabolism in intact plant mitochondria, this report details the effect of oxaloacetate on glutamate metabolism in potato tuber mitochondria. Some of these findings have been presented previously (2).
market. Tubers (4 kg) were cut into 6 liters of chilled medium containing 0.3 M mannitol, 4 mM cystein, 1 mM EDTA, 20 mM pyrophosphate-10 mM phosphate buffer, pH. 7.5, and 0.2% (w/v) defatted BSA! The fragments were disrupted at low speed for 2 s in a l-gallon Waring Blendor. The brei was squeezed through 8 layers of muslin (Ruby, Voiron, France) and a 50-pm nylon net, and intact mitochondria were prepared as fast as possible according to the method of Bonner (3). Longer grinding times had a deleterious effect on the mitochondrial oxidative and phosphorylative capacities. In order to remove various extra-mitochondrial membranes (amyloplast membranes containing carotenoids, etc.) and vacuolar enzymes, mitochondria thus obtained were purified by centrifugation in a nontoxic silica sol (Percoll, Pharmacia) gradient which maintained isoosmotic conditions throughout the isolation procedure (4). Two centrifuge tubes were filled with 34 ml each of Percoll medium (29% (v/v) Percoll; 300 mM mannitol; 10 mM phosphate buffer; pH 7.2; 1 mM EDTA and 0.1% (w/ v) BSA). Aliquots (2 ml sample, 50 mg protein) of the mitochondria were then layered on the Percoll medium. The tubes were placed in a precooled Sorvall SS 34 angular rotor and centrifuged for 30 min at 18,500 rpm (40,OOOg). Intact purified mitochondria
MATERIALS AND METHODS Preparation of mitoohondtia. Potato tubers (Solanum tuberosum, L.) were obtained from a local 1 Supported in part by Research Grant from the Centre National de la Recherche Scientifique (ERA 847: Interactions Plastes-Cytoplasme-Mitochondries). * To whom correspondence should be addressed. ‘Present address: Department of Biochemistry and Biophysics, University of Pennsylvania, School of Medicine G4, Philadelphia, Pennsylvania 19104. 0003-9861/82/030366-10$02.00/O Copyright All rights
Q 1982 by Academic Press, Inc of reproduction in any form reserved.
4 Abbreviations used: BSA, bovine serum albumin; TPP, thiamine pyrophosphate, Mops, I-morpholineethanesulfonic acid. 366
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were recovered as a broad band near the bottom of the tube, and extra-mitochondrial membrane systems formed a band at the sample-gradient interface, The purified mitochondria were diluted with washing medium (15 vol to 1 vol mitochondrial suspension) and recovered as a loose pellet after centrifugation (10,OOOgfor 10 min at 3°C; SS 34 rotor, Sorvall). The supernatant was removed by aspiration and the pellet of purified mitochondria resuspended in a medium containing 300 mM mannitol, 10 mM phosphate, pH 7.2, and 0.1% (w/v) BSA. The final protein concentration was 40 to 60 mg ml-‘. Integrity assays of the inner and the outer membranes were evaluated as indicated by Deuce et al. (5). 0, uptake measurements. O2 uptake was measured at 25°C using a Clark-type O2 electrode system purchased from Hansatech Ltd, Hardwick Industrial Estate, Kings Lynn, Norfolk, England (6). The reaction medium (medium A) contained: 0.3 M mannitol; 5 mM MgC12;10 mM KCl; 10 mM phosphate buffer, pH 7.2; 0.1% (w/v) defatted BSA, and known amounts of mitochondrial protein in a total volume of 1 ml. The O2 concentration in air-saturated medium A was taken as 240 pM (7). Assay of metabolic products. The products of the glutamate metabolism were routinely assayed with intact mitochondria at 25°C in a cell containing medium A and known amounts of mitochondrial protein under continuous stirring. The reaction was initiated by the addition of oxaloacetate. At various times, 2.5ml aliquots were taken and added to 0.75 ml of cold 20% (v/v) HClO, containing 1 mM EDTA. The samples were quickly neutralized with KOH (up to pH 5.5) and centrifuged for 10 min at 27,OOOg to remove KCIOa. The supernatant was used for oxaloacetate, aspartate, a-ketoglutarate, succinate, and malate determination. Simultaneously, the O2 consumption of l-ml aliquots was measured. Oxaloacetate, after decarboxylation with NiCl*, was determined with lactate dehydrogenase (EC 1.1.1.27) according to Wedding et al. (8). Aspartate was determined with glutamate-oxaloacetate transaminase (EC 2.6.1.1) and malate dehydrogenase (EC 1.1.1.37) according to Bergmeyer et al. (9). a-Ketoglutarate was determined with glutamate dehydrogenase (EC 1.4.1.3) according to Bergmeyer and Bernt (10). Succinate was determined with succinate thiokinase (EC 6.2.1.5), pyruvate kinase (EC 2.7.1.40), and lactate dehydrogenase according to Williamson (11). Malate was determined with malate dehydrogenase in the presence of hydrazine by the method of Gutman and Wahlefeld (12). Assay of enzymic activities. Glutamate-oxaloacetate transaminase and malate dehydrogenase activities were determined by the methods of Bergmeyer and Bernt (13,14). Fumarase activity was determined by the method of Hill and Bradshaw (15). Mitockondrial protein determination. Mitochon-
367
drial protein content was determined by the method of Lowry et al. (16).
RESULTS Effect of oxaloacetate on glutamate oxidation. Figure 1 indicates that, in the presence of 1.5 mM ADP and 300 PM TPP, the oxidation of glutamate by potato tuber mitochondria can be triggered by oxaloacetate. There is a lag period before O2 uptake is initiated. The length of the lag phase is directly proportional to the concentration of oxaloacetate added. The absence of TPP increases considerably this induction period. Furthermore, addition of 3 mM Na-arsenite, a well-known inhibitor of oxidative decarboxylations, extends indefinitely the lag phase. Glutamate alone is poorly oxidized by potato tuber mitochondria. In addition we have verified that potato mitochondria have no appreciable oxaloacetate decarboxylase activity (results not shown). The time course of the formation of intermediates during the oxidation of glutamate triggered by oxaloacetate in the presence of TPP and ADP is shown in Figs. 2B and 3B. During the period before the onset of O2uptake, oxaloacetate is rapidly metabolized and aspartate, succinate, malate, and a-ketoglutarate accumulate. If the medium contains initially a low concentration of glutamate (up to 1 mM), aspartate, succinate, and malate are formed equally (Fig. 2B). Under these conditions, a very small and transient accumulation of a-ketoglutarate occurs and the rate of malate, succinate or aspartate formation is two times lower than that of oxaloacetate disappearance. In contrast, if the medium contains initially higher concentrations of glutamate (above 1 mM), (Yketoglutarate accumulates, and the rate of aspartate production is higher than that of succinate and malate (Fig. 3B). Rapid centrifugation of the mitochondria (lO,OOOg, 40 s, Beckman Microfuge B) shows that the metabolites formed appear in the supernatant. Consequently, this result strongly suggests that aspartate, malate, succinate, and a-ketoglutarate molecules, once formed in the matrix space, are rap-
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FIG. 1. Glutamate oxidation triggered by oxaloacetate in intact purified potato tuber mitocbondria. Incubation medium (see Materials and Methods) contained 1.5 mM ADP and 0.6 mg mitochondrial protein ml-‘. Concentrations given are final concentrations in the reaction medium. Numbers on traces refer to nmol 0s consumed min-’ mg- i of mitochondrial protein. MP, purified mitochondria.
transidly excreted in the medium during the internal glutamate-oxaloacetate course of glutamate oxidation triggered by aminase alone. In addition, since the sum of the rates of a-ketoglutarate and succioxaloacetate. When all the oxaloacetate is metabo- nate accumulation (most of the succinate lized, several phenomena occur (Figs. 2B was formed directly from a-ketoglutarate) and 3B); first, the inhibition of O2 con- is approximately equal to the rate of assumption is released; second, the produc- partate production (Figs. 2B, 3B), it is very tion of aspartate is stopped; third, a-ke- likely that, during the course of oxaloacetate disappearance, the oxidation of suctoglutarate and succinate are rapidly reabsorbed and oxidized by the mitochon- cinate is strongly inhibited and that, condria; fourth, the production of malate is sequently, malate production does not maintained. Finally, when the succinate result from succinate oxidation. As a matconcentration becomes very low, the rates ter of fact, it is known that oxaloacetate of O2 consumption and malate accumula- is a potent inhibitor of succinate dehydrogenase (17). This inhibition is competitive tion decrease. During the course of oxaloacetate dis- with a Ki value of 4 PM (18). In order to make sure that, during the appearance, part of the a-ketoglutarate formed may derive from the action of glu- course of oxaloacetate disappearance, suctamate dehydrogenase. However, this cinate oxidation is fully inhibited, we have studied the effects of malonate (a strong pathway is not important since external glutamate alone is poorly oxidized by po- inhibitor of succinate dehydrogenase) on tato tuber mitochondria (Fig. 1). Conse- glutamate oxidation triggered by oxaloquently, the bulk of cu-ketoglutarate formed acetate. Effect of mabnate on the oxidation of (estimated from the rate of aspartate production) is practically generated by the glutamate triggered by oxaloacetate. The
GLUTAMATE
360
t
FM
METABOLISM
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&I
oxaloacetate
TUBER
360
~.JM
MITOCHONDRIA
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B
oxaloacetate
succinate
FIG. 2. Disappearance of added oxaloacetate (O), production of malate (m), aspartate (0), (Yketoglutarate (A) and succinate (A), and Oa consumption during glutamate (1 mM) oxidation by intact purified potato tuber mitochondria. The standard assay solution (see Materials and Methods) was used with 0.48 mg mitochondrial protein ml-‘, a low concentration of glutamate (1 mM), 0.3 mM TPP, and 1.5 mM ADP. Final pH was 7.2 and final volume of the reaction medium was 23 ml. (Aj Assay in presence of 2 mM The reaction was initiated by addition of 360 pM oxaloacetate. malonate; (B) assay without malonate. a-ket, ol-ketoglutarate.
presence of malonate during the course of glutamate oxidation triggered by oxaloacetate is practically without effect on the rate of oxaloacetate disappearance (Figs. 2A, 3A). In addition, during the period before the onset of Oa uptake, malonate is also practically without effect on malate, aspartate, succinate, and a-ketoglutarate accumulation rates. However, and in contrast with what was observed without malonate (Figs. 2B, 3B), when the oxaloacetate concentration becomes very low, we observe only a discrete and transient increase of the rate of O2 consumption (Figs. 2A, 3A) accompanied by a rapid disappearance of a-ketoglutarate. Under these conditions, the production of malate is stopped. Then, and as expected, when the concentration of a-ketoglutarate becomes very low, succinate reaches a maximum concentration and is not further metabolized. Since malonate is without effect on the rate of succinate production
during the course of oxaloacetate disappearance, the results demonstrate that during this period and in absence of malonate (Figs. 2B, 3B), succinate dehydrogenase is fully inhibited and the bulk of the malate formed does not derive from succinate oxidation. Consequently, under these conditions, it is very likely that part of the oxaloacetate added in the medium is converted to malate and that this conversion is dependent on the reduced pyridine nucleotide generated by the TPPlinked a-ketoglutarate dehydrogenase. In support of this hypothesis we have systematically observed that the rate of succinate production (i.e., the rate of Lu-ketoglutarate oxidation) is equal to the rate of malate formation. All these results strongly emphasize the importance of cu-ketoglutarate oxidation during the course of glutamate oxidation triggered by oxaloacetate. Therefore, we examined also the effect of malonate on
370
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300
20
100
12
3
4
5
min
0I 0
1
2
3
4
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FIG. 3. Disappearance of added oxaloacetate (O), production of malate (m), aspartate (0), crketoglutarate (*), and succinate (A), and O2 consumption during glutamate (5 mM) oxidation by intact purified potato tuber mitochondria. The standard assay solution (see Materials and Methods) was used with 0.67 mg mitochondrial protein ml-‘, a high concentration of glutamate (5 mM), 0.3 mM TPP, and 1.5 mM ADP. Final pH was 7.2 and final volume of the reaction medium was 21 ml. The reaction was initiated by addition of 310 pM oxaloacetate. (A) Assay in presence of 2 mM malonate; (B) assay without malonate. cu-ket,a-ketoglutarate. Note that oxaloacetate concentration is lower (310 pM ) than that of Fig. 2 (360 PM) in order to see succinate disappearance before anaerobiosis. Note the obvious and transient accumulation of a-ketoglutarate in the reaction medium.
accumulated products and O2consumption during the course of cu-ketoglutarate oxidation by potato tuber mitochondria. cz-Ketoglutarate oxidation. The time course of a-ketoglutarate oxidation was followed by assaying several citric acid cycle intermediates quantitatively. Figure 4 shows the results from a representative series of experiments with 200 PM a-ketoglutarate in the presence of 300 PM TPP and 1.5 mM ADP. It is clear from Fig. 4B that cu-ketoglutarate is rapidly oxidized (maximal rate: 60 nmol min-’ mg-l protein). The O2 consumption is constant at 120 natom min-’ mg-’ protein for at least 2 min. Then after 4 min the respiratory rate decreases until the reaction stops completely. In the absence of ADP or TPP, a-ketoglutarate is very poorly oxidized (results not shown). This last finding is in basic agreement with that of Bowman et al. (19). In fact, it is well known that the oxidation of a-ketoglutarate requires
TPP (oxidative decarboxylation) and ADP (phosphorylation at the substrate level). Analysis of the reaction products shows that during the course of a-ketoglutarate oxidation, potato tuber mitochondria excrete malate (Fig. 4B). In addition, a transient accumulation of succinate occurs. Since the rate of natom oxygen uptake is equal to the rate of succinate accumulation plus twice the rate of malate formation, this result demonstrates also that two dehydrogenases are operating, namely, the a-ketoglutarate dehydrogenase and the succinate dehydrogenase. When the same experiment is carried out in the presence of malonate (Fig. 4A), the rate of a-ketoglutarate disappearance remains unchanged and mitochondria excrete almost exclusively succinate. Under these conditions, only one dehydrogenase is operating, namely, the a-ketoglutarate dehydrogenase. We also verified that, upon addition of
GLUTAMATE
371
METABOLISM IN POTATO TUBER MITOCHONDRIA
400 oxygen
3oo (I.
- ketoglutarate
oxygen
f
I
123456
7
min
0
1
2
3
4
5
6
7
mini
FIG. 4. Oxidation of a limiting amount of a-ketoglutarate (0) by intact purified potato tuber mitochondria, appearance of succinate (A) and malate (*), and O2consumption. The standard assay solution (see Materials and Methods) was used with 0.85 mg mitochondrial protein ml-‘, 0.3 ItIM TPP, and 1.5 mM ADP. Final pH was 7.2 and final volume of the reaction medium was 11 ml. The reaction was initiated by addition of 195 PM a-ketoglutarate. (A) Assay in presence of 2 mM malonate; (B) assay without malonate. Note that the rapid disappearance of cY-ketoglutarate is not affected by the presence of malonate.
oxaloacetate to mitochondria oxidizing cY-ketoglutarate, a clear inhibition of the respiratory rate occurs which is gradually reversed (Fig. 5). Enzymatic analysis shows that, during the time of inhibition, the oxaloacetate is converted to malate, a conversion that is dependent on the reduced pyridine nucleotide generated by the Lu-ketoglutarate dehydrogenase. These results demonstrate that the inhibition of succinate oxidation by either oxaloacetate or malonate is without effect on the rate of a-ketoglutarate metabolism. They also indicate that, in the presence of oxaloacetate, the intramitochondrial NADH generated by the a-ketoglutarate dehydrogenase is continuously oxidized by the malate dehydrogenase. Intramitochondrial tamate-oxaloacetate
locaticm of the glutransaminase activity.
Knowledge of the precise locus of glutamate-oxaloacetate transaminase is essential to the interpretation of transport phenomena. Consequently, we have directly
determined the intramitochondrial location of glutamate-oxaloacetate transaminase by selectively breaking the outer and inner membranes and observing which enzymes are thereby released to the medium. Figure 6 gives the activities of succinate:cytochrome c oxidoreductase, malate dehydrogenase, glutamate-oxaloacetate transaminase, and fumarase of purified potato mitochondria as a function of the mannitol concentration of the medium. At concentrations below 200 mOsm, down to 50 mOsm, the progressive increase in succinate:cytochrome c oxidoreductase activity reflects the bursting of the outer membrane of the mitochondria (5). Under these conditions and if the medium contains Me, the true matrix enzymes such as fumarase and malate dehydrogenase are not released. At lower osmolarities, i.e., below 50 mOsm, the inner membrane starts to break and the soluble matrix enzymes, fumarase and malate dehydrogenase, are released. The glutamate-oxalo-
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JOURNET, BONNER, AND DOUCE
acetate transaminase activity appears in the supernatant at the same osmolarities that partially release fumarase and malate dehydrogenase, indicating that the glutamate-oxaloacetate transaminase is a soluble matrix enzyme. No activity is released at osmolarities which break the outer membrane, showing that the glutamate-oxaloacetate transaminase is not present in the intermembrane space. Nevertheless, we cannot rule out, on the basis of those data, the possibility that some glutamate-oxaloacetate transaminase is membrane-bound in situ. In any event, however the glutamate-oxaloacetate transaminase enzyme is bound, it must face the matrix space since, after addition of aspartate and cu-ketoglutarate, a relatively low glutamate-oxaloacetate transaminase activity (i.e., 190 nmol oxaloacetate formed min-’ mg-’ mitochondrial protein) is detected in suspension of intact mitochondria. Addition of 0.02% (v/v) Triton X-100, to break the inner membrane and allow substrates free access to the enzyme, stimulates the glutamate-oxaloacetate transaminase activity approximately three times. In fact, this low activity measured in intact mitochondria could be entirely attributable to the capacity of aspartate, a-ketoglutarate, glutamate, oxaloacetate, and malate to pass through the inner membrane, allowing the transamination reaction inside the matrix space to occur.
300 PM TPP 15mM ADP 1 mM a-ketoglutarate 7’
i
i
158
250 pM oxaloacetate
1 20 PM O2 T
207 -1 II IllI” pti
72
FIG. 5. Effect of oxaloacetate on wketoglutarate oxidation by intact purified potato tuber mitochondria. Incubation medium (see Materials and Methods) contained 0.6 mg mitochondrial protein ml-‘. Concentrations given are final concentrations in the reaction medium. Numbers on trace refer to nmol O2 consumed mini. mg-i mitochondrial protein. MP, purified mitochondria.
succinate, and malate are formed equally (Fig. 2). In addition, since the maximal rate of a-ketoglutarate disappearance (estimated from the rate of a-ketoglutarate oxidation, see Fig. 4) is higher than the rate of a-ketoglutarate production (estimated from the rate of aspartate production, see Fig. 2), cu-ketoglutarate does not accumulate in the incubation medium and the rate of a-ketoglutarate production DISCUSSION through the transaminase governs the overall reaction. Consequently, at low iniThese results demonstrate that, during tial glutamate concentration (up to 1 mM) the course of glutamate oxidation triggered by oxaloacetate, part of the oxalo- and during the period preceding the onset of O2 consumption, the functioning of (Yacetate is metabolized by transamination whereas the other part is rapidly con- ketoglutarate dehydrogenase is in synverted to malate and this conversion is chrony with that of glutamate-oxaloacedependent on the reduced pyridine nu- tate transaminase and malate dehydrocleotide generated by the TPP-linked CY- genase, inasmuch as succinate oxidation ketoglutarate dehydrogenase localized in and O2 consumption are strongly inhibthe matrix space. The ratio of the flux ited. On the other hand, at high glutamate rates of both pathways is strongly dependent on glutamate concentration in the concentration (above 1 mM), a greater permedium. Thus at low glutamate concen- centage of the oxaloacetate is metabolized tration (up to 1 KIM), the flux rates in the through the transaminase pathway (Fig. 3). The excess of cY-ketoglutarate thus two pathways are identical and aspartate,
GLUTAMATE
METABOLISM IN POTATO TUBER MITOCHONDRIA
.
succlnate:
cytochrome
A : glutamate
-0xaloacetate
c
373
oxldoreductase
transamlnase
(mOsm)
FIG. 6. Effect of the osmolarity of the incubation medium on the appearance of succinate: cytochrome c oxidoreductase, glutamate-oxaloacetate transaminase, malate dehydrogenase, and fumarase activities in intact purified potato tuber mitochondria. Aliquots of the mitochondrial suspension (1.2 mg mitochondrial proteins) were added to mannitol solutions of various osmolarities, obtained by mixing in definite proportions of medium I (0.3 M mannitol, 0.1% (w/v) BSA, 2 mM MgClz, and 10 mM phosphate buffer, pH 7.2) and medium II (0.1% (w/v) BSA, 2 mM MgClz, and 2 mM Mops buffer, pH 7.2). After 1 min incubation at 4”C, the osmolarity of the medium was adjusted to 0.3 Osm. The final volume was 3 ml. The succinate: cytochrome c oxidoreductase activity was determined. Simultaneously, part of the mitochondria were centrifuged at 10,OOOgduring 10 min and glutamate-oxaloacetate transaminase, malate dehydrogenase, and fumarase activities were determined in the supernatant. The maximal values of glutamate-oxaloacetate transaminase (580 nmol oxaloacetate formed min-’ mg-’ mitochondrial proteins), malate dehydrogenase (55 rmol NADH consumed min-’ mg-’ mitochondrial proteins) and fumarase (540 nmol fumarate formed min-’ mg-’ mitochondrial protein) activities were obtained after addition of Triton X-100 (0.02%) in the incubation medium. The total succinate: cytochrome c oxidoreductase activity was 880 nmol cytochrome c reduced min-’ mg-’ mitochondrial protein. The enzymatic activities are expressed as percentages of their maximal values.
formed is excreted from the matrix space owing to the a-ketoglutarate carrier. Under these circumstances the a-ketoglutarate dehydrogenase complex is working at full capacity (maximal rate: 100 nmol min-’ mg-’ mitochondrial protein, see Fig. 3) and is presumably the limiting factor of the overall process. The reactions involved under all these experimental conditions are summarized in Fig. ‘7. The translocation of metabolites across the mitochondrial membrane plays an important role in the overall process. The results suggest that, in these mitochondria, glutamate enters the matrix in exchange for aspartate (20,21; see, however, 22) and that oxaloacetate enters very rapidly (23) in exchange for either a-ketoglutarate, succinate, or malate (see Fig. 6). The carrier involved in the oxaloacetate
transport could be the a-ketoglutarate carrier, as suggested before by De Santis et al. (24) and Passarella et al. (25), or a specific carrier (26). It is interesting to note that during the period before the onset of O2 consumption, a rapid transport of succinate and cy-ketoglutarate molecules from the mitochondrial compartment to the external medium occurs. In marked contrast, when all the oxaloacetate added is metabolized, these metabolites are no longer excreted and are rapidly reabsorbed by the mitochondria. Consequently, it is possible that the concentration of a-ketoglutarate and succinate on both sides of the inner mitochondrial membrane governs the movement of these metabolites and that a-ketoglutarate or succinate efflux from mitochondria may occur via a different carrier from that im-
GLU
> ASP
elfen”aC
*pace succ
“-KET
<.m. 0.1.
Y xtu ,naP
a
MAL
I
dP”CP succ
n-KET
b
FIG. 7. Schemes of the metabolic processes involved in the glutamate oxidation triggered by oxaloacetate in potato tuber mitochondria. (a) Added oxaloacetate readily penetrates the matrix space through the inner membrane. Part of oxaloacetate (OAA), at least one-half, reacts with glutamate (GLU) under the control of the glutamate-oxaloacetate transaminase (2); a-ketoglutarate thus produced is either oxidized by the cu-ketoglutarate dehydrogenase (3) or excreted into the external space; since the succinate dehydrogenase (4) is strongly inhibited by oxaloacetate, succinate (SUCC) accumulates in the external space. Simultaneously, the other part of oxaloacetate is converted into malate (MAL) under the control of malate dehydrogenase (l), a conversion that depends on the reduced pyridine nuclotides generated by the TPP-linked ol-ketoglutarate dehydrogenase. In these conditions, Oz consumption by the respiratory chain is inhibited. The activity of the glutamate dehydrogenase (6) is relatively negligible. (b) When all the oxaloacetate is consumed, the inhibition of succinate dehydrogenase by oxaloacetate is released, and the previously accumulated succinate and a-ketoglutarate can be rapidly oxidized. Malate thus produced is excreted from the matrix space. With time, malate is slowly oxidized into oxaloacetate, which reacts immediately with glutamate. Three dehydrogenases are involved in that slow process and this explains the residual O2 consumption observed, when ol-ketoglutarate and succinate are consumed (see Figs. 2B and 3B). 0, Enzymes; 5, fumarase; n , carriers; o.m., outer membrane; im., inner membrane; ASP, aspartate. 374
GLUTAMATE
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IN POTATO
plied in a-ketoglutarate and succinate entry. The results presented here do not exclude the possibility that the transamination of glutamate with oxaloacetate takes place outside the mitochondrial matrix space. If the transamination is localized outside the inner membrane, there is no need to postulate a complicated efflux pathway for cu-ketoglutarate (see Fig. ‘7); instead there is an overall influx of oxaloacetate and a-ketoglutarate, balanced by an overall efflux of succinate and malate. However, this suggestion is unlikely since the bulk of the glutamate-oxaloacetate transaminase activity present in Percollpurified mitochondria suspensions is localized in the matrix space. Finally, the results presented in this paper emphasize the great flexibility exhibited by the plant mitochondria. As a matter of fact, plant mitochondria are able to export or import reducing equivalents in the form of oxaloacetate, aspartate, a-ketoglutarate, malate, and glutamate. ACKNOWLEDGMENTS The authors are indebted to Dr. Jon Hoek and to Dr. Michel Neuburger for helpful remarks. REFERENCES 1. HANSON, J. B., AND DAY, D. A. (1980) in The Biochemistry of Plants, Vol. 1, The Plant Cell (Tolbert, N. E., ed.), pp. 315-358, Academic Press, New York. 2. DOUCE, R., AND BONNER, W. D., JR. (1973) Fed. Proc. 32, 596. 3. BONNER, W. D. (1967) in Methods in Enzymology (Estabrook, R. W., and Pullman, M. E., eds.), Vol. 10, pp. 126-133, Academic Press, New York. 4. JACKSON, C., AND MOORE, A. L. (1979) in Methodological Surveys (B): Biochemistry, Vol. 9, Plant Organelles (Reid, E., ed.), pp. 1-12, Ellis Horwood, Chichester. 5. DOUCE, R., CHRISTENSEN, E. L., AND BONNER, W. D. (1972) Biochim Biophys. Acta 275, 148 160. 6. DELIEU, T., AND WALKER, D, A. (1972) New Phyto1 71, 201-225. 7. ESTABROOK, R. W. (1967) in Methods in Enzymology (Estabrook, R. W., and Pullman, M. E., eds.), Vol. 10, pp. 41-47, Academic Press, New York.
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8. WEDDING, R. T., BLACK, R. K., AND PAP, D. (1976) Plant PhysioL 58, 740-743. 9. BERGMEYER, H. LT., BERNT, E., M~LLERING, H., AND PFLEIDERER, G. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), 2nd Ed., Vol. 4, pp. 1696-1700, Academic Press, New York. 10. BERGMEYER, H. U., ANDBERNT, E. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), 2nd Ed., Vol. 3, pp. 1577-1580, Academic Press, New York. 11. WILLIAMSON, J. R. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), 2nd Ed., Vol. 3, pp. 1616-1621, Academic Press, New York. 12. GUTMANN, I., AND WAHLEFELD, A. W. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), 2nd Ed., Vol. 3, pp. 1585-1589, Academic Press, New York. 13. BERGMEYER, H. U., ANDBERNT, E. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), 2nd Ed., Vol. 2, pp. 727-733, Academic Press, New York. 14. BERGMEYER, H. U., ANDBERNT, E. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), 2nd Ed., Vol. 2, pp. 613-617, Academic Press, New York. 15. HILL, R. L., AND BRADSHAW, R. A. (1969) in Methods in Enzymology (Lowenstein, J. M., ed.), Vol. 13, pp. 91-99, Academic Press, New York. 16. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. BioL Chem. 193, 265-275. 17. SINGER, T. P., OESTREICHER, G., HOGUE, P., CONTREIRAS, J., AND BRANDAO, I. (1973) Plant Physiol. 52, 616-621. 18. ZEYLEMAKER, W. P., KLAASSE, D. M., AND SLATER, E. C. (1969) B&him Biophys. Acta 191, 229-238. 19. BOWMAN, E. J., IKUMA, H., AND STEIN, H. J. (1976) Plant PhysioL 58,426-432. 20. AZZI, A., CHAPPELL, J. B., AND ROBINSON, B. H. (1967) Biochem. Biophys. Res. Commun. 29, 148-152. 21. TISCHLER, M. E., PACHENCE, J., WILLIAMSON, J. R., ANDLANOUE, K. F. (1976) Arch. Biochem. Biophys. 173,448-462. 22. DAY, D. A., AND WISKICH, J. T. (1977) Plant Sci. L&t. 9, 33-36. 23. DOUCE, R., AND BONNER, W. D. (1972) Biochem. Biophys. Res. Cwmmun 47, 619-621. 24. DE SANTIS, A., ARRIGONI, O., AND PALMIERI, F. (1976) Plant Cell PhysioL 17, 1221-1233. 25. PASSARELLA, S., PALMIERI, F., AND QUAGLIARIELLO, E. (1977) Arch. Biochem. Biophys. 180, 160-168. 26. DAY, D. A., AND WISKICH, J. T. (1981) Arch. Biochem. Biophys. 211, 100-107.
Glutamate
ETIENNE-PASCAL Physiologic
Cellulaire
Metabolism Triggered by Oxaloacetate Intact Plant Mitochondria’ JOURNET:
WALTER
D. BONNER:
AND
in
ROLAND
V6g&ale, DRF/BV, Centre d’Etud.es NucZaires et Unive-rsitk Scienti$que de Grenoble, 85 X, 880.41 Grenoble Ceo?ex, France
DOUCE et Mbdicale
Received July 10, 1981, and in revised form October 12, 1981 In Percoll-purified potato tuber mitochondria, glutamate metabolism can be triggered by oxaloacetate, in the presence of ADP and thiamine pyrophospbate. There is a lag phase before Ox uptake is initiated. During this lag period, oxaloacetate is rapidly converted into a-ketoglutarate and succinate, or into malate at the expense of the NADH generated by a-ketoglutarate dehydrogenase. The ratio of the flux rates of both pathways is strongly dependent on the glutamate concentration in the medium. When all the oxaloacetate is consumed, a rapid O2 uptake is initiated. The effects of malonate on glutamate metabolism triggered by oxaloacetate and on a-ketoglutarate oxidation are reported. It is concluded that the inhibition of the succinate dehydrogenase by either malonate or oxaloacetate does not affect the rate of a-ketoglutarate dehydrogenase functioning. All the metabolites accumulated are excreted by the mitochondria in the supernatant. Some of them are then reabsorbed. These results emphasize the importance of the anion carriers in the overall process.
Mitochondria isolated from a large variety of plant tissues oxidize glutamate in the presence of malate (for a review, see Hanson and Day (1)). This oxidation, which is not clearly understood, is probably due to glutamate dehydrogenase (EC 1.4.1.3), malate dehydrogenase (EC 1.1.1.37), and glutamate-oxaloacetate transaminase (EC 2.6.1.1) localized in the matrix space. With the aim of clarifying the mechanisms of glutamate metabolism in intact plant mitochondria, this report details the effect of oxaloacetate on glutamate metabolism in potato tuber mitochondria. Some of these findings have been presented previously (2).
market. Tubers (4 kg) were cut into 6 liters of chilled medium containing 0.3 M mannitol, 4 mM cystein, 1 mM EDTA, 20 mM pyrophosphate-10 mM phosphate buffer, pH. 7.5, and 0.2% (w/v) defatted BSA! The fragments were disrupted at low speed for 2 s in a l-gallon Waring Blendor. The brei was squeezed through 8 layers of muslin (Ruby, Voiron, France) and a 50-pm nylon net, and intact mitochondria were prepared as fast as possible according to the method of Bonner (3). Longer grinding times had a deleterious effect on the mitochondrial oxidative and phosphorylative capacities. In order to remove various extra-mitochondrial membranes (amyloplast membranes containing carotenoids, etc.) and vacuolar enzymes, mitochondria thus obtained were purified by centrifugation in a nontoxic silica sol (Percoll, Pharmacia) gradient which maintained isoosmotic conditions throughout the isolation procedure (4). Two centrifuge tubes were filled with 34 ml each of Percoll medium (29% (v/v) Percoll; 300 mM mannitol; 10 mM phosphate buffer; pH 7.2; 1 mM EDTA and 0.1% (w/ v) BSA). Aliquots (2 ml sample, 50 mg protein) of the mitochondria were then layered on the Percoll medium. The tubes were placed in a precooled Sorvall SS 34 angular rotor and centrifuged for 30 min at 18,500 rpm (40,OOOg). Intact purified mitochondria
MATERIALS AND METHODS Preparation of mitoohondtia. Potato tubers (Solanum tuberosum, L.) were obtained from a local 1 Supported in part by Research Grant from the Centre National de la Recherche Scientifique (ERA 847: Interactions Plastes-Cytoplasme-Mitochondries). * To whom correspondence should be addressed. ‘Present address: Department of Biochemistry and Biophysics, University of Pennsylvania, School of Medicine G4, Philadelphia, Pennsylvania 19104. 0003-9861/82/030366-10$02.00/O Copyright All rights
Q 1982 by Academic Press, Inc of reproduction in any form reserved.
4 Abbreviations used: BSA, bovine serum albumin; TPP, thiamine pyrophosphate, Mops, I-morpholineethanesulfonic acid. 366
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were recovered as a broad band near the bottom of the tube, and extra-mitochondrial membrane systems formed a band at the sample-gradient interface, The purified mitochondria were diluted with washing medium (15 vol to 1 vol mitochondrial suspension) and recovered as a loose pellet after centrifugation (10,OOOgfor 10 min at 3°C; SS 34 rotor, Sorvall). The supernatant was removed by aspiration and the pellet of purified mitochondria resuspended in a medium containing 300 mM mannitol, 10 mM phosphate, pH 7.2, and 0.1% (w/v) BSA. The final protein concentration was 40 to 60 mg ml-‘. Integrity assays of the inner and the outer membranes were evaluated as indicated by Deuce et al. (5). 0, uptake measurements. O2 uptake was measured at 25°C using a Clark-type O2 electrode system purchased from Hansatech Ltd, Hardwick Industrial Estate, Kings Lynn, Norfolk, England (6). The reaction medium (medium A) contained: 0.3 M mannitol; 5 mM MgC12;10 mM KCl; 10 mM phosphate buffer, pH 7.2; 0.1% (w/v) defatted BSA, and known amounts of mitochondrial protein in a total volume of 1 ml. The O2 concentration in air-saturated medium A was taken as 240 pM (7). Assay of metabolic products. The products of the glutamate metabolism were routinely assayed with intact mitochondria at 25°C in a cell containing medium A and known amounts of mitochondrial protein under continuous stirring. The reaction was initiated by the addition of oxaloacetate. At various times, 2.5ml aliquots were taken and added to 0.75 ml of cold 20% (v/v) HClO, containing 1 mM EDTA. The samples were quickly neutralized with KOH (up to pH 5.5) and centrifuged for 10 min at 27,OOOg to remove KCIOa. The supernatant was used for oxaloacetate, aspartate, a-ketoglutarate, succinate, and malate determination. Simultaneously, the O2 consumption of l-ml aliquots was measured. Oxaloacetate, after decarboxylation with NiCl*, was determined with lactate dehydrogenase (EC 1.1.1.27) according to Wedding et al. (8). Aspartate was determined with glutamate-oxaloacetate transaminase (EC 2.6.1.1) and malate dehydrogenase (EC 1.1.1.37) according to Bergmeyer et al. (9). a-Ketoglutarate was determined with glutamate dehydrogenase (EC 1.4.1.3) according to Bergmeyer and Bernt (10). Succinate was determined with succinate thiokinase (EC 6.2.1.5), pyruvate kinase (EC 2.7.1.40), and lactate dehydrogenase according to Williamson (11). Malate was determined with malate dehydrogenase in the presence of hydrazine by the method of Gutman and Wahlefeld (12). Assay of enzymic activities. Glutamate-oxaloacetate transaminase and malate dehydrogenase activities were determined by the methods of Bergmeyer and Bernt (13,14). Fumarase activity was determined by the method of Hill and Bradshaw (15). Mitockondrial protein determination. Mitochon-
367
drial protein content was determined by the method of Lowry et al. (16).
RESULTS Effect of oxaloacetate on glutamate oxidation. Figure 1 indicates that, in the presence of 1.5 mM ADP and 300 PM TPP, the oxidation of glutamate by potato tuber mitochondria can be triggered by oxaloacetate. There is a lag period before O2 uptake is initiated. The length of the lag phase is directly proportional to the concentration of oxaloacetate added. The absence of TPP increases considerably this induction period. Furthermore, addition of 3 mM Na-arsenite, a well-known inhibitor of oxidative decarboxylations, extends indefinitely the lag phase. Glutamate alone is poorly oxidized by potato tuber mitochondria. In addition we have verified that potato mitochondria have no appreciable oxaloacetate decarboxylase activity (results not shown). The time course of the formation of intermediates during the oxidation of glutamate triggered by oxaloacetate in the presence of TPP and ADP is shown in Figs. 2B and 3B. During the period before the onset of O2uptake, oxaloacetate is rapidly metabolized and aspartate, succinate, malate, and a-ketoglutarate accumulate. If the medium contains initially a low concentration of glutamate (up to 1 mM), aspartate, succinate, and malate are formed equally (Fig. 2B). Under these conditions, a very small and transient accumulation of a-ketoglutarate occurs and the rate of malate, succinate or aspartate formation is two times lower than that of oxaloacetate disappearance. In contrast, if the medium contains initially higher concentrations of glutamate (above 1 mM), (Yketoglutarate accumulates, and the rate of aspartate production is higher than that of succinate and malate (Fig. 3B). Rapid centrifugation of the mitochondria (lO,OOOg, 40 s, Beckman Microfuge B) shows that the metabolites formed appear in the supernatant. Consequently, this result strongly suggests that aspartate, malate, succinate, and a-ketoglutarate molecules, once formed in the matrix space, are rap-
368
JOURNET,BONNER,ANDDOUCE
FIG. 1. Glutamate oxidation triggered by oxaloacetate in intact purified potato tuber mitocbondria. Incubation medium (see Materials and Methods) contained 1.5 mM ADP and 0.6 mg mitochondrial protein ml-‘. Concentrations given are final concentrations in the reaction medium. Numbers on traces refer to nmol 0s consumed min-’ mg- i of mitochondrial protein. MP, purified mitochondria.
transidly excreted in the medium during the internal glutamate-oxaloacetate course of glutamate oxidation triggered by aminase alone. In addition, since the sum of the rates of a-ketoglutarate and succioxaloacetate. When all the oxaloacetate is metabo- nate accumulation (most of the succinate lized, several phenomena occur (Figs. 2B was formed directly from a-ketoglutarate) and 3B); first, the inhibition of O2 con- is approximately equal to the rate of assumption is released; second, the produc- partate production (Figs. 2B, 3B), it is very tion of aspartate is stopped; third, a-ke- likely that, during the course of oxaloacetate disappearance, the oxidation of suctoglutarate and succinate are rapidly reabsorbed and oxidized by the mitochon- cinate is strongly inhibited and that, condria; fourth, the production of malate is sequently, malate production does not maintained. Finally, when the succinate result from succinate oxidation. As a matconcentration becomes very low, the rates ter of fact, it is known that oxaloacetate of O2 consumption and malate accumula- is a potent inhibitor of succinate dehydrogenase (17). This inhibition is competitive tion decrease. During the course of oxaloacetate dis- with a Ki value of 4 PM (18). In order to make sure that, during the appearance, part of the a-ketoglutarate formed may derive from the action of glu- course of oxaloacetate disappearance, suctamate dehydrogenase. However, this cinate oxidation is fully inhibited, we have studied the effects of malonate (a strong pathway is not important since external glutamate alone is poorly oxidized by po- inhibitor of succinate dehydrogenase) on tato tuber mitochondria (Fig. 1). Conse- glutamate oxidation triggered by oxaloquently, the bulk of cu-ketoglutarate formed acetate. Effect of mabnate on the oxidation of (estimated from the rate of aspartate production) is practically generated by the glutamate triggered by oxaloacetate. The
GLUTAMATE
360
t
FM
METABOLISM
IN POTATO
&I
oxaloacetate
TUBER
360
~.JM
MITOCHONDRIA
369
B
oxaloacetate
succinate
FIG. 2. Disappearance of added oxaloacetate (O), production of malate (m), aspartate (0), (Yketoglutarate (A) and succinate (A), and Oa consumption during glutamate (1 mM) oxidation by intact purified potato tuber mitochondria. The standard assay solution (see Materials and Methods) was used with 0.48 mg mitochondrial protein ml-‘, a low concentration of glutamate (1 mM), 0.3 mM TPP, and 1.5 mM ADP. Final pH was 7.2 and final volume of the reaction medium was 23 ml. (Aj Assay in presence of 2 mM The reaction was initiated by addition of 360 pM oxaloacetate. malonate; (B) assay without malonate. a-ket, ol-ketoglutarate.
presence of malonate during the course of glutamate oxidation triggered by oxaloacetate is practically without effect on the rate of oxaloacetate disappearance (Figs. 2A, 3A). In addition, during the period before the onset of Oa uptake, malonate is also practically without effect on malate, aspartate, succinate, and a-ketoglutarate accumulation rates. However, and in contrast with what was observed without malonate (Figs. 2B, 3B), when the oxaloacetate concentration becomes very low, we observe only a discrete and transient increase of the rate of O2 consumption (Figs. 2A, 3A) accompanied by a rapid disappearance of a-ketoglutarate. Under these conditions, the production of malate is stopped. Then, and as expected, when the concentration of a-ketoglutarate becomes very low, succinate reaches a maximum concentration and is not further metabolized. Since malonate is without effect on the rate of succinate production
during the course of oxaloacetate disappearance, the results demonstrate that during this period and in absence of malonate (Figs. 2B, 3B), succinate dehydrogenase is fully inhibited and the bulk of the malate formed does not derive from succinate oxidation. Consequently, under these conditions, it is very likely that part of the oxaloacetate added in the medium is converted to malate and that this conversion is dependent on the reduced pyridine nucleotide generated by the TPPlinked a-ketoglutarate dehydrogenase. In support of this hypothesis we have systematically observed that the rate of succinate production (i.e., the rate of Lu-ketoglutarate oxidation) is equal to the rate of malate formation. All these results strongly emphasize the importance of cu-ketoglutarate oxidation during the course of glutamate oxidation triggered by oxaloacetate. Therefore, we examined also the effect of malonate on
370
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300
20
100
12
3
4
5
min
0I 0
1
2
3
4
5
min
FIG. 3. Disappearance of added oxaloacetate (O), production of malate (m), aspartate (0), crketoglutarate (*), and succinate (A), and O2 consumption during glutamate (5 mM) oxidation by intact purified potato tuber mitochondria. The standard assay solution (see Materials and Methods) was used with 0.67 mg mitochondrial protein ml-‘, a high concentration of glutamate (5 mM), 0.3 mM TPP, and 1.5 mM ADP. Final pH was 7.2 and final volume of the reaction medium was 21 ml. The reaction was initiated by addition of 310 pM oxaloacetate. (A) Assay in presence of 2 mM malonate; (B) assay without malonate. cu-ket,a-ketoglutarate. Note that oxaloacetate concentration is lower (310 pM ) than that of Fig. 2 (360 PM) in order to see succinate disappearance before anaerobiosis. Note the obvious and transient accumulation of a-ketoglutarate in the reaction medium.
accumulated products and O2consumption during the course of cu-ketoglutarate oxidation by potato tuber mitochondria. cz-Ketoglutarate oxidation. The time course of a-ketoglutarate oxidation was followed by assaying several citric acid cycle intermediates quantitatively. Figure 4 shows the results from a representative series of experiments with 200 PM a-ketoglutarate in the presence of 300 PM TPP and 1.5 mM ADP. It is clear from Fig. 4B that cu-ketoglutarate is rapidly oxidized (maximal rate: 60 nmol min-’ mg-l protein). The O2 consumption is constant at 120 natom min-’ mg-’ protein for at least 2 min. Then after 4 min the respiratory rate decreases until the reaction stops completely. In the absence of ADP or TPP, a-ketoglutarate is very poorly oxidized (results not shown). This last finding is in basic agreement with that of Bowman et al. (19). In fact, it is well known that the oxidation of a-ketoglutarate requires
TPP (oxidative decarboxylation) and ADP (phosphorylation at the substrate level). Analysis of the reaction products shows that during the course of a-ketoglutarate oxidation, potato tuber mitochondria excrete malate (Fig. 4B). In addition, a transient accumulation of succinate occurs. Since the rate of natom oxygen uptake is equal to the rate of succinate accumulation plus twice the rate of malate formation, this result demonstrates also that two dehydrogenases are operating, namely, the a-ketoglutarate dehydrogenase and the succinate dehydrogenase. When the same experiment is carried out in the presence of malonate (Fig. 4A), the rate of a-ketoglutarate disappearance remains unchanged and mitochondria excrete almost exclusively succinate. Under these conditions, only one dehydrogenase is operating, namely, the a-ketoglutarate dehydrogenase. We also verified that, upon addition of
GLUTAMATE
371
METABOLISM IN POTATO TUBER MITOCHONDRIA
400 oxygen
3oo (I.
- ketoglutarate
oxygen
f
I
123456
7
min
0
1
2
3
4
5
6
7
mini
FIG. 4. Oxidation of a limiting amount of a-ketoglutarate (0) by intact purified potato tuber mitochondria, appearance of succinate (A) and malate (*), and O2consumption. The standard assay solution (see Materials and Methods) was used with 0.85 mg mitochondrial protein ml-‘, 0.3 ItIM TPP, and 1.5 mM ADP. Final pH was 7.2 and final volume of the reaction medium was 11 ml. The reaction was initiated by addition of 195 PM a-ketoglutarate. (A) Assay in presence of 2 mM malonate; (B) assay without malonate. Note that the rapid disappearance of cY-ketoglutarate is not affected by the presence of malonate.
oxaloacetate to mitochondria oxidizing cY-ketoglutarate, a clear inhibition of the respiratory rate occurs which is gradually reversed (Fig. 5). Enzymatic analysis shows that, during the time of inhibition, the oxaloacetate is converted to malate, a conversion that is dependent on the reduced pyridine nucleotide generated by the Lu-ketoglutarate dehydrogenase. These results demonstrate that the inhibition of succinate oxidation by either oxaloacetate or malonate is without effect on the rate of a-ketoglutarate metabolism. They also indicate that, in the presence of oxaloacetate, the intramitochondrial NADH generated by the a-ketoglutarate dehydrogenase is continuously oxidized by the malate dehydrogenase. Intramitochondrial tamate-oxaloacetate
locaticm of the glutransaminase activity.
Knowledge of the precise locus of glutamate-oxaloacetate transaminase is essential to the interpretation of transport phenomena. Consequently, we have directly
determined the intramitochondrial location of glutamate-oxaloacetate transaminase by selectively breaking the outer and inner membranes and observing which enzymes are thereby released to the medium. Figure 6 gives the activities of succinate:cytochrome c oxidoreductase, malate dehydrogenase, glutamate-oxaloacetate transaminase, and fumarase of purified potato mitochondria as a function of the mannitol concentration of the medium. At concentrations below 200 mOsm, down to 50 mOsm, the progressive increase in succinate:cytochrome c oxidoreductase activity reflects the bursting of the outer membrane of the mitochondria (5). Under these conditions and if the medium contains Me, the true matrix enzymes such as fumarase and malate dehydrogenase are not released. At lower osmolarities, i.e., below 50 mOsm, the inner membrane starts to break and the soluble matrix enzymes, fumarase and malate dehydrogenase, are released. The glutamate-oxalo-
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JOURNET, BONNER, AND DOUCE
acetate transaminase activity appears in the supernatant at the same osmolarities that partially release fumarase and malate dehydrogenase, indicating that the glutamate-oxaloacetate transaminase is a soluble matrix enzyme. No activity is released at osmolarities which break the outer membrane, showing that the glutamate-oxaloacetate transaminase is not present in the intermembrane space. Nevertheless, we cannot rule out, on the basis of those data, the possibility that some glutamate-oxaloacetate transaminase is membrane-bound in situ. In any event, however the glutamate-oxaloacetate transaminase enzyme is bound, it must face the matrix space since, after addition of aspartate and cu-ketoglutarate, a relatively low glutamate-oxaloacetate transaminase activity (i.e., 190 nmol oxaloacetate formed min-’ mg-’ mitochondrial protein) is detected in suspension of intact mitochondria. Addition of 0.02% (v/v) Triton X-100, to break the inner membrane and allow substrates free access to the enzyme, stimulates the glutamate-oxaloacetate transaminase activity approximately three times. In fact, this low activity measured in intact mitochondria could be entirely attributable to the capacity of aspartate, a-ketoglutarate, glutamate, oxaloacetate, and malate to pass through the inner membrane, allowing the transamination reaction inside the matrix space to occur.
300 PM TPP 15mM ADP 1 mM a-ketoglutarate 7’
i
i
158
250 pM oxaloacetate
1 20 PM O2 T
207 -1 II IllI” pti
72
FIG. 5. Effect of oxaloacetate on wketoglutarate oxidation by intact purified potato tuber mitochondria. Incubation medium (see Materials and Methods) contained 0.6 mg mitochondrial protein ml-‘. Concentrations given are final concentrations in the reaction medium. Numbers on trace refer to nmol O2 consumed mini. mg-i mitochondrial protein. MP, purified mitochondria.
succinate, and malate are formed equally (Fig. 2). In addition, since the maximal rate of a-ketoglutarate disappearance (estimated from the rate of a-ketoglutarate oxidation, see Fig. 4) is higher than the rate of a-ketoglutarate production (estimated from the rate of aspartate production, see Fig. 2), cu-ketoglutarate does not accumulate in the incubation medium and the rate of a-ketoglutarate production DISCUSSION through the transaminase governs the overall reaction. Consequently, at low iniThese results demonstrate that, during tial glutamate concentration (up to 1 mM) the course of glutamate oxidation triggered by oxaloacetate, part of the oxalo- and during the period preceding the onset of O2 consumption, the functioning of (Yacetate is metabolized by transamination whereas the other part is rapidly con- ketoglutarate dehydrogenase is in synverted to malate and this conversion is chrony with that of glutamate-oxaloacedependent on the reduced pyridine nu- tate transaminase and malate dehydrocleotide generated by the TPP-linked CY- genase, inasmuch as succinate oxidation ketoglutarate dehydrogenase localized in and O2 consumption are strongly inhibthe matrix space. The ratio of the flux ited. On the other hand, at high glutamate rates of both pathways is strongly dependent on glutamate concentration in the concentration (above 1 mM), a greater permedium. Thus at low glutamate concen- centage of the oxaloacetate is metabolized tration (up to 1 KIM), the flux rates in the through the transaminase pathway (Fig. 3). The excess of cY-ketoglutarate thus two pathways are identical and aspartate,
GLUTAMATE
METABOLISM IN POTATO TUBER MITOCHONDRIA
.
succlnate:
cytochrome
A : glutamate
-0xaloacetate
c
373
oxldoreductase
transamlnase
(mOsm)
FIG. 6. Effect of the osmolarity of the incubation medium on the appearance of succinate: cytochrome c oxidoreductase, glutamate-oxaloacetate transaminase, malate dehydrogenase, and fumarase activities in intact purified potato tuber mitochondria. Aliquots of the mitochondrial suspension (1.2 mg mitochondrial proteins) were added to mannitol solutions of various osmolarities, obtained by mixing in definite proportions of medium I (0.3 M mannitol, 0.1% (w/v) BSA, 2 mM MgClz, and 10 mM phosphate buffer, pH 7.2) and medium II (0.1% (w/v) BSA, 2 mM MgClz, and 2 mM Mops buffer, pH 7.2). After 1 min incubation at 4”C, the osmolarity of the medium was adjusted to 0.3 Osm. The final volume was 3 ml. The succinate: cytochrome c oxidoreductase activity was determined. Simultaneously, part of the mitochondria were centrifuged at 10,OOOgduring 10 min and glutamate-oxaloacetate transaminase, malate dehydrogenase, and fumarase activities were determined in the supernatant. The maximal values of glutamate-oxaloacetate transaminase (580 nmol oxaloacetate formed min-’ mg-’ mitochondrial proteins), malate dehydrogenase (55 rmol NADH consumed min-’ mg-’ mitochondrial proteins) and fumarase (540 nmol fumarate formed min-’ mg-’ mitochondrial protein) activities were obtained after addition of Triton X-100 (0.02%) in the incubation medium. The total succinate: cytochrome c oxidoreductase activity was 880 nmol cytochrome c reduced min-’ mg-’ mitochondrial protein. The enzymatic activities are expressed as percentages of their maximal values.
formed is excreted from the matrix space owing to the a-ketoglutarate carrier. Under these circumstances the a-ketoglutarate dehydrogenase complex is working at full capacity (maximal rate: 100 nmol min-’ mg-’ mitochondrial protein, see Fig. 3) and is presumably the limiting factor of the overall process. The reactions involved under all these experimental conditions are summarized in Fig. ‘7. The translocation of metabolites across the mitochondrial membrane plays an important role in the overall process. The results suggest that, in these mitochondria, glutamate enters the matrix in exchange for aspartate (20,21; see, however, 22) and that oxaloacetate enters very rapidly (23) in exchange for either a-ketoglutarate, succinate, or malate (see Fig. 6). The carrier involved in the oxaloacetate
transport could be the a-ketoglutarate carrier, as suggested before by De Santis et al. (24) and Passarella et al. (25), or a specific carrier (26). It is interesting to note that during the period before the onset of O2 consumption, a rapid transport of succinate and cy-ketoglutarate molecules from the mitochondrial compartment to the external medium occurs. In marked contrast, when all the oxaloacetate added is metabolized, these metabolites are no longer excreted and are rapidly reabsorbed by the mitochondria. Consequently, it is possible that the concentration of a-ketoglutarate and succinate on both sides of the inner mitochondrial membrane governs the movement of these metabolites and that a-ketoglutarate or succinate efflux from mitochondria may occur via a different carrier from that im-
GLU
> ASP
elfen”aC
*pace succ
“-KET
<.m. 0.1.
Y xtu ,naP
a
MAL
I
dP”CP succ
n-KET
b
FIG. 7. Schemes of the metabolic processes involved in the glutamate oxidation triggered by oxaloacetate in potato tuber mitochondria. (a) Added oxaloacetate readily penetrates the matrix space through the inner membrane. Part of oxaloacetate (OAA), at least one-half, reacts with glutamate (GLU) under the control of the glutamate-oxaloacetate transaminase (2); a-ketoglutarate thus produced is either oxidized by the cu-ketoglutarate dehydrogenase (3) or excreted into the external space; since the succinate dehydrogenase (4) is strongly inhibited by oxaloacetate, succinate (SUCC) accumulates in the external space. Simultaneously, the other part of oxaloacetate is converted into malate (MAL) under the control of malate dehydrogenase (l), a conversion that depends on the reduced pyridine nuclotides generated by the TPP-linked ol-ketoglutarate dehydrogenase. In these conditions, Oz consumption by the respiratory chain is inhibited. The activity of the glutamate dehydrogenase (6) is relatively negligible. (b) When all the oxaloacetate is consumed, the inhibition of succinate dehydrogenase by oxaloacetate is released, and the previously accumulated succinate and a-ketoglutarate can be rapidly oxidized. Malate thus produced is excreted from the matrix space. With time, malate is slowly oxidized into oxaloacetate, which reacts immediately with glutamate. Three dehydrogenases are involved in that slow process and this explains the residual O2 consumption observed, when ol-ketoglutarate and succinate are consumed (see Figs. 2B and 3B). 0, Enzymes; 5, fumarase; n , carriers; o.m., outer membrane; im., inner membrane; ASP, aspartate. 374
GLUTAMATE
METABOLISM
IN POTATO
plied in a-ketoglutarate and succinate entry. The results presented here do not exclude the possibility that the transamination of glutamate with oxaloacetate takes place outside the mitochondrial matrix space. If the transamination is localized outside the inner membrane, there is no need to postulate a complicated efflux pathway for cu-ketoglutarate (see Fig. ‘7); instead there is an overall influx of oxaloacetate and a-ketoglutarate, balanced by an overall efflux of succinate and malate. However, this suggestion is unlikely since the bulk of the glutamate-oxaloacetate transaminase activity present in Percollpurified mitochondria suspensions is localized in the matrix space. Finally, the results presented in this paper emphasize the great flexibility exhibited by the plant mitochondria. As a matter of fact, plant mitochondria are able to export or import reducing equivalents in the form of oxaloacetate, aspartate, a-ketoglutarate, malate, and glutamate. ACKNOWLEDGMENTS The authors are indebted to Dr. Jon Hoek and to Dr. Michel Neuburger for helpful remarks. REFERENCES 1. HANSON, J. B., AND DAY, D. A. (1980) in The Biochemistry of Plants, Vol. 1, The Plant Cell (Tolbert, N. E., ed.), pp. 315-358, Academic Press, New York. 2. DOUCE, R., AND BONNER, W. D., JR. (1973) Fed. Proc. 32, 596. 3. BONNER, W. D. (1967) in Methods in Enzymology (Estabrook, R. W., and Pullman, M. E., eds.), Vol. 10, pp. 126-133, Academic Press, New York. 4. JACKSON, C., AND MOORE, A. L. (1979) in Methodological Surveys (B): Biochemistry, Vol. 9, Plant Organelles (Reid, E., ed.), pp. 1-12, Ellis Horwood, Chichester. 5. DOUCE, R., CHRISTENSEN, E. L., AND BONNER, W. D. (1972) Biochim Biophys. Acta 275, 148 160. 6. DELIEU, T., AND WALKER, D, A. (1972) New Phyto1 71, 201-225. 7. ESTABROOK, R. W. (1967) in Methods in Enzymology (Estabrook, R. W., and Pullman, M. E., eds.), Vol. 10, pp. 41-47, Academic Press, New York.
TUBER
MITOCHONDRIA
375
8. WEDDING, R. T., BLACK, R. K., AND PAP, D. (1976) Plant PhysioL 58, 740-743. 9. BERGMEYER, H. LT., BERNT, E., M~LLERING, H., AND PFLEIDERER, G. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), 2nd Ed., Vol. 4, pp. 1696-1700, Academic Press, New York. 10. BERGMEYER, H. U., ANDBERNT, E. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), 2nd Ed., Vol. 3, pp. 1577-1580, Academic Press, New York. 11. WILLIAMSON, J. R. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), 2nd Ed., Vol. 3, pp. 1616-1621, Academic Press, New York. 12. GUTMANN, I., AND WAHLEFELD, A. W. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), 2nd Ed., Vol. 3, pp. 1585-1589, Academic Press, New York. 13. BERGMEYER, H. U., ANDBERNT, E. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), 2nd Ed., Vol. 2, pp. 727-733, Academic Press, New York. 14. BERGMEYER, H. U., ANDBERNT, E. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), 2nd Ed., Vol. 2, pp. 613-617, Academic Press, New York. 15. HILL, R. L., AND BRADSHAW, R. A. (1969) in Methods in Enzymology (Lowenstein, J. M., ed.), Vol. 13, pp. 91-99, Academic Press, New York. 16. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. BioL Chem. 193, 265-275. 17. SINGER, T. P., OESTREICHER, G., HOGUE, P., CONTREIRAS, J., AND BRANDAO, I. (1973) Plant Physiol. 52, 616-621. 18. ZEYLEMAKER, W. P., KLAASSE, D. M., AND SLATER, E. C. (1969) B&him Biophys. Acta 191, 229-238. 19. BOWMAN, E. J., IKUMA, H., AND STEIN, H. J. (1976) Plant PhysioL 58,426-432. 20. AZZI, A., CHAPPELL, J. B., AND ROBINSON, B. H. (1967) Biochem. Biophys. Res. Commun. 29, 148-152. 21. TISCHLER, M. E., PACHENCE, J., WILLIAMSON, J. R., ANDLANOUE, K. F. (1976) Arch. Biochem. Biophys. 173,448-462. 22. DAY, D. A., AND WISKICH, J. T. (1977) Plant Sci. L&t. 9, 33-36. 23. DOUCE, R., AND BONNER, W. D. (1972) Biochem. Biophys. Res. Cwmmun 47, 619-621. 24. DE SANTIS, A., ARRIGONI, O., AND PALMIERI, F. (1976) Plant Cell PhysioL 17, 1221-1233. 25. PASSARELLA, S., PALMIERI, F., AND QUAGLIARIELLO, E. (1977) Arch. Biochem. Biophys. 180, 160-168. 26. DAY, D. A., AND WISKICH, J. T. (1981) Arch. Biochem. Biophys. 211, 100-107.