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THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular BiologY, h

Val. 267, No. 15, Issue of May 25, pp. 10411-10422,1992 Printed in U.S.A.

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Glutamate-Malate Metabolism in Liver Mitochondria A MODEL CONSTRUCTED ON THE BASIS OF MITOCHONDRIAL LEVELS OF ENZYMES, SPECIFICITY, DISSOCIATION CONSTANTS, AND STOICHIOMETRY OF HETERO-ENZYME COMPLEXES* (Received for publication, October 4, 1991)

Leonard A. Fahien$ and JanK. Tellers From the Department of Pharmacology, University of Wisconsin Medical School,Madison, Wisconsin 53706

The level of aspartate aminotransferasein liver mi- protein-binding sites. Furthermore, citrate synthase can astochondria was found to be-140 PM, or 2-3 orders of sociate with and receive oxalacetate from malate dehydrogenmagnitude higher than its dissociation constant in comase by a channeling mechanism (8-10). This could favor a plexes with the inner mitochondrial membrane and the transfer of oxalacetate from malate dehydrogenase to citrate high molecular weight enzymes(Mr= 1.6 X 10‘ to 2.7 synthase because malate dehydrogenase does not readily asx 10‘) carbamyl-phosphate synthase I, glutamate de- sociate with or channel oxalacetate into theaminotransferase hydrogenase, and the a-ketoglutarate dehydrogenase alone (11-14). Therefore, a transfer of oxalacetate from macomplex. The total concentration of aminotransferaselate dehydrogenase to the aminotransferase is possibly facilibinding sites on these structures inliver mitochondria tated by mitochondrial structures that place these two enwas more than sufficient to accommodate all of the zymes in close proximity to one another. Consequently, maaminotransferase.Therefore,in liver mitochondria, late dehydrogenase may not be completely free in the matrix, the aminotransferase could be associatedwith the inner mitochondrial membrane and/or these high molec-but may, in part, be localized by these mitochondrial structures. This is consistent with previous results that demonular weight enzymes. The aminotransferase in these hetero-enzyme com- strated that when mitochondria are rendered permeable to plexes could be supplied with oxalacetate because bind-molecules even larger than malate dehydrogenase, malate ing of aminotransferase to the high molecular weight dehydrogenase and several other Krebs cycle enzymes are enzymes can enhance binding of malate dehydrogen- retained (15). Although malate dehydrogenase has a high affinity for the inner mitochondrial membrane and Complex ase, and binding of both malate dehydrogenase and the I (4, 16-18) and the aminotransferase has a high affinity for aminotransferasefacilitated binding of fumarase. The level of malate dehydrogenasewas found to be a unique site on the membrane (4), the inner mitochondrial so high (140 MM) in liver mitochondria, comparedwith membrane may not be capable of placing these two enzymes that of citrate synthase (25 PM) and the pyruvate de- in close proximity to one another. The aminotransferase, hydrogenase complex (0.3 PM), that there would also citrate, andhigh ionic strength displace malate dehydrogenase be asufficientsupply of oxalacetate citrate to synthase- from the membrane (4,16). Furthermore, oxidation of malate pyruvate dehydrogenase. by malate dehydrogenase may not readily take place on Complex I because NAD and malate displace malate dehydrogenase fromComplex I (18). However, we have shown, using several different methods, that the aminotransferase forms a The well-documented ability of exogenous malate to en- binary complex with the a-ketoglutaric dehydrogenase (EC hance aspartate aminotransferase (EC 2.6.1.1) activity in liver 1.2.4.2) complex and that even in the presence of NAD, mitochondria (1) would be difficult to explain unless at least malate, and high ionic strength, malate dehydrogenase can some of the malate dehydrogenase ((S)-malate:NAD+ oxido- associate with the binary complex (11,12). Thus, in the reductase, EC 1.1.1.37) is localized in mitochondria in close ternary complex, malate dehydrogenase and the aminotransproximity to aspartate aminotransferase. At a physiological ferase could be in close proximity to one another; and consepH, equilibrium of the malate dehydrogenase reaction is quite quently, the ternary complex maybe able to catalyze the unfavorable (2), and malate dehydrogenase itself has a high combined malate dehydrogenase-aminotransferase reactions affinity for oxalacetate (3). Therefore, since the level of malate in liver mitochondria. Consistent with this, we found that dehydrogenase is quite high in liver mitochondria (-280 ~ L M adding the a-ketoglutarate dehydrogenase complex to the with respect to oxalacetate-binding sites) (4), a significant combined malate dehydrogenase-aminotransferase reaction fraction of the oxalacetate generated would remain bound to markedly decreases the K,,, of malate (11). Another potential malate dehydrogenase. In addition, the level of free oxalace- advantage of the ternarycomplex is that kinetic experiments tate is quite low and is considerably lower than thehigh level (12) indicated that a-ketoglutarate can be directly transferred of matrix proteins (5-7) so thatthere can be competing (19) from the aminotransferase to the a-ketoglutarate dehy* This work was supported by National Institutes of Health Grant drogenase complex or that binding of the a-ketoglutarate CA 40445. The costs of publication of this article were defrayed in dehydrogenase complex to theaminotransferase canenhance part by the payment of page charges. This article must therefore be dissociation of a-ketoglutarate from the aminotransferase so hereby marked “aduertisement” in accordance with 18U.S.C. Section that a-ketoglutarate canrapidly react with the dehydrogenase 1734 solely to indicate this fact. complex without having to diffuse from the hetero-enzyme $ To whom reprint requests should be addressed Dept. of Pharsystem. macology, University of Wisconsin Medical School, 1300 University Glutamate dehydrogenase (L-glutamate:NAD(P)+ oxidoreAve., Madison, WI 53706. I Present address: Dept. of Molecular Biology and Biotechnology, ductase (deaminating), EC 1.4.1.3) and carbamyl-phosphate synthase (ammonia) I (EC 6.3.4.16) are present in liver miUniversity of Sheffield, Sheffield S10 2TN, Great Britain.

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Glutamate-Malate Metabolism in Liver Mitochondria

tochondria in high levels (20, 21) and can also associate with aminotransferase alone and malate dehydrogenase alone (13, 14, 22). Therefore, glutamate dehydrogenase and carbamylphosphate synthase I may also place malate dehydrogenase and the aminotransferase in close proximity to one another. Another potential advantage of a malate dehydrogenase-glutamate dehydrogenase-aminotransferase complex is that previous kinetic results (12) are also consistent with a direct transfer type of mechanism for delivery of a-ketoglutarate from the aminotransferase to glutamate dehydrogenase and delivery of NADH from glutamate dehydrogenase to malate dehydrogenase. Dissociation of NADH from glutamate dehydrogenase is rate-limiting in the glutamate dehydrogenase reaction (23). Therefore, when glutamate dehydrogenase is reacting with glutamate instead of a-ketoglutarate, malate dehydrogenase (by facilitating removal of NADH from glutamate dehydrogenase) can increase NH: production. Consequently, if carbamyl-phosphate synthase I associates with this ternary complex, then it would be localized where NH: is produced by glutamate dehydrogenase at an accelerated rate. Therefore, in this paper, we have determined if malate dehydrogenase and the aminotransferase can simultaneously associate with glutamate dehydrogenase and if carbamylphosphate synthase Ican associate with the resultant ternary complex. Additional information is required to determine whether the kinetic and binding interactions described above, which were found with pure enzymes, could also occur in liver mitochondria. Therefore, in this paper, we have determined if in liver mitochondria the concentrations of malate dehydrogenase- and aminotransferase-binding sites on the high molecular weight enzymes are in the range of the levels of malate dehydrogenase and the aminotransferase and if the levels of malate dehydrogenase and the aminotransferase are considerably higher than their dissociation constants from the hetero-enzyme complexes. In addition, to evaluate the possibility that other matrix enzymes can compete with the enzyme components of these hetero-enzyme complexes, we have also measured the levels of these potential competing enzymes in liver mitochondria and, in some cases, studied the affinity of potential competing enzymes for the hetero-enzyme complexes. Special focus was placed on citrate synthase and fumarase, which are known to also interact with malate dehydrogenase and the aminotransferase (8,9, 24-26).

isolated from the liver of control male buffalo rats (-200 g) (which had been fasted for 17 h) in H-medium (220 mM mannitol, 70 mM sucrose, 2 mM potassium Bes' (pH 7.5), 1 mM potassium EGTA, and 0.1% defatted bovine serum albumin) as described previously (40). The low-speed (3 min a t 1100 X g) residues were repeatedly rehomogenized according to the protocol of Bustamante et al. (40).After the first washing at 20,000 X g for 10 min, the loose material on the surface of the pellet was discarded. The mitochondria were suspended (-5 mg/ml) in H-medium (minus albumin and EGTA) and stirred briefly (2min) at 0 "C with 0.1 mg digitonin/rng of protein to disrupt lysosomes (40).H-medium (minus albumin and EGTA at 0 "C) was then added, and the mitochondria were centrifuged for 20 min at 20,000 X g. (An aliquot of this suspension was centrifuged separately for electron microscopy.) Thesupernatantand fluffy layer were removed, andthe mitochondria were suspended manually inHmedium (minus albumin and EGTA, plus 20 pg/ml leupeptin). Mitochondria removed by centrifugation of this suspension were incubated for 30 min at 30 "C in KC1 medium containing an uncoupling agent and 1 mM dichloroacetate to activate the pyruvate dehydrogenase complex as described previously (41).Finally, the mitochondria were recovered by centrifugation, suspended to give a final concentration of -32 mg/ml in H-medium (minus albumin and EGTA, plus 20 pg/ml leupeptin), and immediately extracted or stored in small aliquots at -80 "C. Similar enzyme levels were found when the mitochondria were immediately extracted insteadof stored at -80 "C prior to extraction. With the exception of the a-ketoglutarate dehydrogenase complex, all enzyme assays were performed with mitochondrial extracts prepared (Method I) as described below. In addition, each enzyme was assayed in a separately prepared extract that was designed to take advantage of previously known properties of the enzyme. The specific activities of the enzymes were essentially the same when extracted with the specific method or when extracted with Method I. Both protein and enzyme assays were performed with aliquots of the total uncentrifuged extracts, and the same assay medium was used for extracts prepared with Method I and the specific extracts prepared for a given enzyme. Except where indicated, enzyme assays were performed at 30 "C by measuring the change in absorbance at 340 nm, and specific activities are expressed as micromoles of product/minute/milligram of protein. The specific activities reported are maximal as determined by varying the level of substrates and/or activators. Methods similar to Method I have been found previously to give complete extraction of the pyruvate dehydrogenase complex, glutamate dehydrogenase, and several other enzymes (42).In Method I, the mitochondria were suspended with a small homogenizer in an 50 M sodium Hepes (pH 7.5), equal volume of 1.0% Triton X-100, m 0.2 mM EDTA, 2 mM dithiothreitol, and 40 pg/ml leupeptin. The extract was incubated for 20 min at 20 "C. An additional freeze-thaw cycle did not increase the activity of the enzymes. Specific extraction of a given enzyme was performed in the same manner, except a different extraction medium (described below with the specific enzyme) was employed. MATERIALS AND METHODS For specific extraction of the pyruvate dehydrogenase complex, the Enzymes and Reagents-Bovine heart pyruvate dehydrogenase and extraction medium was an equal volume of 50 mM potassium MOPS a-ketoglutarate dehydrogenase complexes, bovine and rat liver mi- (pH 6.9), 20 mM MgCl,, 20 mM calcium EGTA, 2 mM dichloroacetate, tochondrial glutamate dehydrogenases andaspartate aminotrans- 2 mM azide, 40 pg/ml leupeptin, 1.0% Triton X-100, and 2 mM ferase, and rat liver carbamyl-phosphate synthase I and ornithine dithiothreitol. transcarbamylase were prepared as described previously (12, 20, 27-For the assay of the pyruvate dehydrogenase complex (based upon 31).Pig heart mitochondrial malate dehydrogenase, citrate synthase, the assay of Linn etal. (43),with the additions of inhibitors of NADH and fumarase were obtained from Boehringer Mannheim. Other oxidase and lactate dehydrogenase), a 10-50-pl aliquot of the extract enzymes, coenzymes, substrates, and reagents were obtained from was added to 1 mlof 0.5% Triton X-100,50 mM sodium Hepes (pH mg/ml albumin, 0.1 mM EDTA, 0.1 mM dithiothreitol, 1 mM Sigma. Stock solutions of all reagents used in assays were adjusted to 7.5), 0.1 the pH of the assay. KCN, 5 p~ rotenone, 10 pg/ml leupeptin, 5 mM NAD, 0.2 mM CoA, Concentrations of Enzyme and Protein-The concentrations of 0.2 mM thiamine pyrophosphate, 1.25 mMMgC12, 1 mM pyruvate, pure pyruvate dehydrogenase and a-ketoglutarate dehydrogenase and 10 mM oxalate. The reaction was started by the addition of complexes were measured as described previously with bovine serum pyruvate after the absorbance had reached a constant value. Oxalate (10 mM) reduced the activity of lactate dehydrogenase (measured albumin as a standard (32).The concentrations of pure glutamate dehydrogenase, fumarase, carbamyl-phosphate synthase I, malate with thiamine pyrophosphate and coenzyme A omitted and 40 p M dehydrogenase, aspartate aminotransferase, succinate thiokinase, and NADH added to the pyruvate dehydrogenase complex assay to l.Thus, in the presence of malate dehydrogenase (1.4 nmol), glutamate dehydrogenase (0.3 nmol), and aminotransferase (1.1 nmol), the results (Table I, line 3) were actually consistent with no binary complexes, but with 68% of the bound glutamate dehydrogenase in a malate dehydrogenase-glutamate dehydrogenase-aminotransferase complex and 32% of the bound glutamate dehydrogenase in a malate dehydrogenase-glutamate dehydrogenase-(aminotransferase)2complex. When the amount of glutamate dehydrogenase added was increased to 1.2 nmol, the results were consistent with no binary complex, but with 94% of the bound glutamate dehydrogenase in a malate dehydrogenase-glutamate dehydrogenase-aminotransferase complex and only 6% of the bound glutamate dehydrogenase in a malate dehydrogenase-glutamate dehydrogenase(aminotransferase)p complex. Since the glutamate dehydrogenase hexamers over this concentration range can undergo a concentration-dependent association into higher molecular weight forms (68), these results suggest that the amount of glutamate dehydrogenase-(aminotransferase)nbecomes lower as glutamate dehydrogenase becomes more polymerized.(For the sake of simplicity, the proposed complexes are designated as malate dehydrogenase-glutamate dehydrogenase-aminotransferase. A more accurate designation might be (malate dehydrogenase),-(glutamate dehydrogenase),-(aminotransferase),, where n equals the number of glutamate dehydrogenase hexamers associated with each other.) The two ternary complexes containing the two dimers and either glutamate dehydrogenase or the a-ketoglutarate dehydrogenase complex can also associate with each other toform a quaternary complex (12). These interactionsmay take place because one subunit of a malate dehydrogenase or aminotransferase dimer attaches to one high molecular weight enzyme, and the other subunit attaches to the other (12). In addition, glutamate dehydrogenase can also associate with the a-ketoglutarate dehydrogenase complex (Fig. 3). Thus, the quarternary complex could also be stabilized by bonds between vacant sites on glutamate dehydrogenase and the aketoglutarate dehydrogenase complex. When experiments similar to those described above were performed by adding malate dehydrogenase alone (data not shown) to carbamyl-phosphate synthase I (1.9 nmol), the results were consistent with a KOof 2.5W M and -1.1 carbamylphosphate synthase I monomers bound per malate dehydrogenase dimer. When similar experiments were performed with the aminotransferase instead of malate dehydrogenase, the results could be closely approximated by a KD of 0.5 W M and 2.6 carbamyl-phosphate synthase Imonomers bound per aminotransferase dimer. Carbamyl-phosphate synthase I can be a mixture of monomers and dimers at the concentrations employed in these experiments (69). Therefore, the almost

0.10

0.05

0.15

[KDHC] Addednmol

FIG. 3. Plot of nanomoles of glutamate dehydrogenase bound versus nanomoles of a-ketoglutarate dehydrogenase complex added. In these experiments, glutamate dehydrogenase (GDH) (0.3 nmol) was incubated in a volume of 1 mlunder the conditions described in the legend to Table I with the indicated nanomoles of the a-ketoglutarate dehydrogenase complex (KDHC). The amount of glutamate dehydrogenase bound was determined as described under “Materials and Methods.” The results shown have been corrected bysubtracting the amount of glutamate dehydrogenase bound (0.02 nmol) in the absence of the a-ketoglutarate dehydrogenase complex. Experimental conditions are given inthe legend to Table I.

TABLEI1 Interactions among carbamyl-phosphate synthase I, malate dehydrogenase, aspartate aminotransferase, and glutamate dehydrogenase Experimental conditions are described in the legend to Table I. The amounts of enzymes in the 1-ml incubation mixture were as follows: glutamate dehydrogenase (GDH), 0.3 nmol; aspartate aminotransferase (AspAT), 1.1 nmol; malate dehydrogenase (MDH), 1.4 nmol; and carbamyl-phosphate synthase I (CPS),1.9 nmol. Methods for determining the amount of enzyme bound are described under “Materials and Methods.” Enzyme precipitated Additions

MDH AspAT CPS GDH ~-~~ nmol

MDH, CPS AspAT, CPS AspAT, CPS, MDH AspAT, CPS, MDH, GDH AspAT, GDH, MDH CPS, GDH CPS

%

nmol

%

nmol

%

nmol

0.51 27 0.43 39 1.1 58 0.70 50 0.43 39 1.3 68 0.85 61 0.59 1.7 54 89 0.3

%

0.46 33

0.25

18 0.33 30

0.25 0.24 13 0.02 0.11 6

100 83 7

1:l stoichiometry (Table 11, line 1) observed with malate dehydrogenase indicates that essentially all of the bound malate dehydrogenase is in malate dehydrogenase-carbamylphosphate synthase I and/or malate dehydrogenase-(carbamyl-phosphate synthase I)z-malate dehydrogenase complexes. Alternatively, the 2.6:l stoichiometry (line 2) observed with the aminotransferase is consistent with most (86%) of the bound aminotransferase being in a carbamyl-phosphate synthaseI-aminotransferase-(carbamyl-phosphatesynthase I ) z complex and the remainder in an aminotransferase-(carbamyl-phosphate synthase I)zcomplex. The results obtained when malate dehydrogenase and the aminotransferase were both added to carbamyl-phosphate synthase I are shown in Table I1 (line 3). As was the case when glutamate dehydrogenase was the high molecular weight supporting enzyme, adding aminotransferase to malate dehydrogenase plus carbamyl-phosphate synthase I (lines l and 3) increased (almost doubled) the binding of malate dehydrogenase. Also similar to the case with glutamate dehydrogenase, the amount of aminotransferase andcarbamyl-phosphate synthase I bound was essentially the same ineither the

Glutamate-Malate Metabolism

Mitochondria in Liver

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TABLEI11 presence or absence of malate dehydrogenase (line 2). These Zncorporation of fumarase into hetero-enzyme complexes experiments were performed under the same conditions as for Experimental conditions are described in the legend to Table I. Table I, where as mentioned above, there was no evidence of a binary malate dehydrogenase-aminotransferase complex. The amounts of enzymesin the 1-ml incubation mixture were as follows: fumarase(FUM), 0.26 nmol; glutamate dehydrogenase Thus, the only explanation as to why the aminotransferase (GDH), 0.6 nmol; aspartateaminotransferase (AspAT), 1.1 nmol; could enhance binding of malate dehydrogenase in the pres- malate dehydrogenase (MDH), 1.4 nmol; and a-ketoglutarate dehyence of carbamyl-phosphate synthase Iwould be that malate drogenase complex (KDHC), 0.037 nmol. Methods for determining dehydrogenase associates with complexes between the ami- the amount of enzyme bound are described under “Materials and notransferase and carbamyl-phosphate synthase I. The actual Methods.” Enzyme precipitated stoichiometry under these conditions (line 3) was 1.6 malate dehydrogenase dimers and 2.6 carbamyl-phosphate synthase FUM MDH AspAT GDH Additions I monomers bound per aminotransferase dimer. This stoichinmol % nmol % nmol % nmol % ometry was consistent with no binary complexes containing 0.016 0.14 6 10 malate dehydrogenase and with 63% of the bound aminotrans- FUM, MDH 0.039 15 12 0.13 ferase being in the form of malate dehydrogenase-carbamyl- FUM, AspAT 0.039 0.14 15 0.13 10 12 FUM, AspAT, MDH phosphate synthase I-aminotransferase-(carbamyl-phosphate FUM 0.041 16 synthase I)*-malate dehydrogenase and the remainder in the FUM, 2 GDH 0.12 0.010 4 form of aminotransferase-(carbamyl-phosphate synthase I)*- FUM, GDH, MDH, 0.11 0.49 0.66 43 0.47 35 60 78 AspAT malate dehydrogenase. 0.042 16 When carbamyl-phosphate synthase I was incubated with KDHC, FUM 0.13 50 0.24 0.28 17 25 KDHC, FUM, MDH, glutamate dehydrogenase alone (Table 11, line 6), only 13% AspAT of the carbamyl-phosphate synthase I and 7% of the glutamate dehydrogenase were precipitated uersus 6% of the carbamylphosphate synthase I (Table 11, line 7) and 7% of the gluta- 1-4), there wasno significant increase in precipitation of mate dehydrogenase (Table I, line 5 ) when each of these two fumarase when incubated with malate dehydrogenase and/or enzymes was incubated alone. Thus, there is no significant aminotransferase over that found when fumarase was incuinteraction between these two enzymes. Even lesser amounts bated alone. Similarly, precipitation of malate dehydrogenase and theaminotransferase was only slightly higher when these of a binary complex between these two enzymes would be expected when both malate dehydrogenase and the amino- two enzymes were incubated with fumarase than when incutransferase are present because these enzymes can associate bated separately or together in theabsence of fumarase (Table with 83% of the glutamate dehydrogenase alone and 68% of 111, lines 1-3; uersus Table I,lines 4, 6,and 7). Thus, fumarase the carbamyl-phosphate synthaseI alone (Table I, line 3; was a high molecular weightenzyme that did not readily same as Table11, line 5; and Table11, line 3). However, adding support binding of malate dehydrogenase or the aminotranscarbamyl-phosphate synthase Ito thetwo dimers plus gluta- ferase. Fumarase also did not associate with glutamate dehymate dehydrogenase (Table 11, line 4 uersus 5) enhanced drogenase alone (Table 111, line 5). However, when glutamate binding of the two dimers, and therewas more binding of the dehydrogenase was added to the other three enzymes (Table two high molecular weight enzymes than when they were 111, line 6), malate dehydrogenase and the aminotransferase incubated alone with the two dimers. Since there is no signif- were again bound with a stoichiometry similar to that found icantinteraction between the two high molecular weight in the absence of fumarase, and fumarase was also bound. enzymes in the absence of the dimers and thereshould be less Thus, since there was no evidence of binding of fumarase in glutamate dehydrogenase-carbamyl-phosphate synthase I in the presence of dimers and absence of glutamate dehydrogenthe presence of the dimers, these results indicate that a ase or the presence of glutamate dehydrogenase and absence complex can be formed among all four enzymes. The stoichi- of dimers, fumarase apparently associates with the ternary ometry of the precipitate in the presence of all four enzymes complex. When fumarase, aminotransferase, malate dehydrogenase, was consistent with the formation of 0.3 nmol of carbamylphosphatesynthaseI-malate dehydrogenase-glutamate de- and glutamate dehydrogenase were incubated with the divahydrogenase-aminotransferase-carbamyl-phosphate synthase lent cross-linker in the absence of polyethylene glycol and I or about the same amount of malate dehydrogenase-gluta- chromatographed on a Sephadex G-200 column (Fig. 4), a mate dehydrogenase-aminotransferase as formed (0.25 nmol) significant amount of the other three enzymes was eluted in (Table 11, line 5) when carbamyl-phosphate synthase I was the void volume (fraction 20 as measured with blue dextran) omitted. The amount of this ternary complex was not de- with glutamate dehydrogenase. The decrease in the elution creased by carbamyl-phosphate synthase I apparently because volume of these enzymes was not due to inter-or intramolecglutamate dehydrogenase has ahigher affinity than carbamyl- ular cross-linking of the aminotransferase, malate dehydrophosphate synthase I for the dimers. Glutamate dehydrogen- genase, or fumarase. We have shown previously that under ase did not decrease binding of carbamyl-phosphate synthase these experimental conditions, there is essentially no intraI apparently because carbamyl-phosphate synthase I associ- molecular cross-linking of malate dehydrogenase or the amiated with this ternary complex. In addition, since binding of notransferase alone or intermolecular cross-linking of malate glutamate dehydrogenase to the dimers does not markedly dehydrogenase plus aminotransferase (Ref. 13; see also Ref. decrease the amount of free dimers (Table 11, line 5), the 67). Furthermore, when albumin was substituted for glutastoichiometry of the precipitate in the presence of all four mate dehydrogenase, the other threeenzymes were eluted as enzymes was also consistent with the formation of the same single peaks, and only a small amount of the other three amount (0.3 nmol) of malate dehydrogenase-carbamyl-phos- enzymes was in the void volume (Fig.4). These results cannot phate synthase I-aminotransferase-(carbamyl-phosphate syn-be utilized to estimate stoichiometry because the cross-linker thase Ibrnalate dehydrogenase as formed when glutamate decreases the specific activity of the enzyme (67). These dehydrogenase was omitted. results also do not prove that the aminotransferase was reIncorporation of Fumarase-As shown in Table I11 (lines quired for binding of malate dehydrogenase to glutamate ~~~~

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TABLEIV Effect of ligands on binding of fumarase Experimental conditions are described in the legend to Table I. The amounts of enzymes in the 1-ml incubation mixture were as follows: glutamate dehydrogenase (GDH), 1.2 nmol; aspartate aminotransferase (AspAT), 1.1 nmol; malate dehydrogenase (MDH), 1.4 nmol; and fumarase (FUM), 0.26 nmol. Methods for determining the amount of enzyme bound are described under “Materials and Methods.” 40

t

301

A

A / \

i -I

Enzyme precipitated Additions

MDH ~

AspAT

_

GDH

_

nmol % nmol % nmol % nmol %

None Fumarate, 10 mM Citrate, 1.0 mM a-Ketoglutarate, 1.0mM

I\

FUM ~

0.13 50 0.77 55 0.90 82 0.82 68 0.09 36 0.62 44 0.61 56 0.84 70 0.05 19 0.20 14 0.58 53 0.42 35 0.11 41 0.70 50 0.78 71 0.61 51

1

ever, fumarate and/or malate (malate is readily generated by the fumarase present in these experiments) had unique effects a t these high levels in that binding of glutamate dehydrogenase was not decreased and the aminotransferase was not FRACTION FIG.4. Sephadex G-200chromatography of mitochondrial bound in excess over malate dehydrogenase. The stoichiometry in the presence of added fumarate was consistent with malate dehydrogenase, aspartate aminotransferase, fumarase, and glutamate dehydrogenase. In these experiments, 6.0 mg/ equal (26% or 0.22 nmol) amounts of the bound glutamate ml glutamate dehydrogenase ( G D H ) (0)or 6.0 mg/ml bovine serum dehydrogenase being in glutamate dehydrogenase-malate dealbumin (0)was incubated with 2.0 mg/ml dimethyl 3,3’-dithiobis- hydrogenase and glutamate dehydrogenase-aminotransferase propionimidate, 1.0 mg/ml aspartate aminotransferase (Asp-AT),1.0 mg/ml malate dehydrogenase ( M D H ) , and 1.0 mg/ml fumarase and the remainder or 0.39 nmol in a malate dehydrogenase(FUM) for 2 h and chromatographed on a column of Sephadex G- glutamate dehydrogenase-aminotransferase complex. This 200 (2.5 X 27 cm). The void volume was determined by finding the contrasts with a stoichiometry consistent with 94% or 0.77 blue dextran peak (fraction 20) a t 625 nm as described previously nmol of the bound glutamate dehydrogenase being in a malate (13, 67). Enzyme activities are in units of change in absorbance/ dehydrogenase-glutamate dehydrogenase-aminotransferase minute/milliliter of fraction from the column. Assay conditions are complex and the remainder or 0.05 nmol in a glutamate described under “Materials and Methods.” The elutions of glutamate dehydrogenase, fumarase, malate dehydrogenase, and aspartateami- dehydrogenase-(aminotransferase)2complex in the absence of notransferase are shown in the top, second, third, and bottom panels, fumarate (Table IV, line 1).Thus, high levels of fumarate respectively. The incubation was performed in 0.25 M sodium arse- and/or malate apparently decreased (but did not abolish) the nate, 0.1 mM EDTA (pH 8.0) at 25 “C; and the column was equili- ternary complex and equalized the affinity of both dimers for brated and eluted with the same buffer. The volume of fractions from glutamate dehydrogenase. the column was 1.9 ml. Levels of Enzymes in Mitochondria-When Method I (see “Materials and Methods”)was employed,we recovered, based dehydrogenase. At the high levels of enzymes used in these upon the ornithine transcarbamylase assay, 43 mgof mitoexperiments (6.0 mg/mlglutamate dehydrogenase and 1.0 mg/ chondrial protein/g of liver, wet weight, or 148 mg of mitoml malate dehydrogenase), malate dehydrogenase is cross- chondrial protein/g of homogenate protein with a 66% yield linked to glutamate dehydrogenase in the absence of the of mitochondria. This gives a value of 65 mg of mitochondrial aminotransferase (13). These results, however, are consistent protein/g of liver or 224mgof mitochondrial protein/g of with those obtained in polyethylene glycol in that they dem- homogenate protein. The yield was higher (81%)when it was onstratethatinthe presence (unlike inthe absence) of based on the citrate synthase assay. However, -20-fold more glutamate dehydrogenase, the three enzymes are sufficiently protein was required for the citratesynthase assay compared close to one another to permit cfoss-linkingby a cross-linker with the ornithine transcarbamylase assay; and in the orniwith a bridge length of only 12 A (70). thine transcarbamylase assay, the control (no ornithine) rate Although the a-ketoglutarate dehydrogenase complex also was negligible. did not associate with fumarase alone (Table 111, line 7), it When mitochondria and mitochondrial extracts were precould substitute for glutamate dehydrogenase in promoting pared with Method I, comparable (fl.2-fold) enzyme levels interaction between the dimers and fumarase (line 8). were found when the mitochondria were immediately exEffect of Ligands-We have previously shown that physio- tractedor were stored at -80 “C, thawed, and extracted. logical levels of citrate enhance dissociation of the amino- Comparable levels were also found for the indicated enzymes transferase from glutamate dehydrogenase (71). As shown in when Method I1 and the additionalextraction procedures Table IV (line 3), 1.0 mM levels of citrate decreased (but did described under “Materials andMethods” were employed. not completely eliminate) binding of the aminotransferase, A major purpose of this paper was to determine if the but decreased binding of fumarase and malatedehydrogenase apparent stoichiometries and dissociation constants of the essentially to the level observed in the absence of glutamate hetero-enzyme complexes were compatible with their formadehydrogenase (Table 111, line 3). Theseeffects of citrate were tion in liver mitochondria. Table V shows the levels of enspecific in that adding a-ketoglutarate at 1.0 mM levels only zymes that can play a role in glutamate-malate metabolism slightly (1.1-1.3-fold) decreased the amount of each enzyme calculated on the basis of the level of their activities in liver bound (Table 111, line 4),and adding fumarate at even 10 mM mitochondrial extractsas described under “Materialsand levels (Table IV, line 2) produced only a 1.2-1.4-fold decrease Methods.” in binding of the enzymes to glutamate dehydrogenase. HowAs shown in Table V, the concentrations of aspartate

_

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Glutamate-Malate Metabolism in Liver Mitochondria TABLEV Levels of enzymes in liver mitochondria The methods used to calculate enzyme levels are described under “Materials and Methods.” The assay of malate dehydrogenase with malate as a substrate was performed by coupling the reactions with added acetyl-coA and citrate synthase as described previously (11). Methods I and I1 refer to the method used to prepare the mitochondrial extract as described under “Materials and Methods” and in Refs. 3, 20, 27, 29, respectively. Assay Enzyme

Method

pH

Substrate T “C

MDH“

Pure enzyme

Mitochondrial conc mdml

PM

pmol productlminlmgprotein

Oxalacetate 700 7 10 140 Malate 0.79 80 10 140 I Oxalacetate 12.2 1200 10 140 AspAT 25 I1 Aspartate 2.1 180 11.6 130 I1 Glutamate* 265 3.1 130 I Aspartate 250 3.3 146 NADPIDH I NADP 0.16 30 5.3 88 GDH 25 I1 NADH 1.3 76 50 26 25 NADb3.5 0.09 I Leucine/NADH 70 1.7 71 OTC I Ornithine 3.4 34 885 3.8 cs 25 I1 Oxalacetate 0.41 25 165 2.5 I Oxalacetate 124 0.31 2.5 25 PC I Pyruvate 34 0.30 18 Fumarase 25 I1 Malate 0.22 140 1.6 8 25 I1 Fumarate 0.12 74 1.6 8 AlaAT I 8.6 Alanine213 0.16 NAD:IDH I NAD 43 0.05 1.1 4 KDHC I NAD 25 0.13 2 PDHC I NAD 0.3 2.00.041 20 a MDH, malate dehydrogenase;AspAT, aspartate aminotransferase; NADPIDH, NADPisocitrate dehydrogenase; GDH, glutamate dehydrogenase;OTC, ornithine transcarbamylase; CS, citrate synthase; PC, pyruvate carboxylase;AlaAT, alanine aminotransferase; NAD:IDH, NAD:isocitrate dehydrogenase;KDHC, a-ketoglutarate dehydrogenase complex; PDHC, pyruvate dehydrogenase complex. Assays were not actually performed with extracts, but were calculated by multiplying the activity of the reverse reaction in the extract by the ratio of the activity of the forward to the reverse reaction with the pure enzymes. 25 25

I1 I1

Specific activity Liver

7.8 7.8 7.6 30 7.8 7.8 25 11.6 7.6 3013.2 7.5 30 7.8 7.8 7.0 30 24 7.0 30 7.8 8.1 30 7.5 9 30 7.8 7.8 7.8 0.70 30 7.2 30 7.5 5.2 30 7.5 30

aminotransferase, glutamate dehydrogenase, and a-ketoglutarate dehydrogenase complexes are 140, 70, and 2 p M , respectively. According to the dataof others, the concentration of carbamyl-phosphate synthase I monomer would be 400p~ (21). According to the results of this paper, -1.0, 10, and 0.4 aminotransferase molecules can associate, respectively, with 1 molecule of glutamate dehydrogenase hexamer, a-ketoglutarate dehydrogenase complex, and carbamyl-phosphate synthase I monomer. Therefore, the sum of the concentrations of aminotransferase-binding sites on these three high molecular weight enzymes in liver mitochondria would be almost twice the level of aminotransferase. In addition, the level of aminotransferase and the level of the three enzymes that associate with aminotransferase areconsiderably higher than the apparent dissociation constants of the binary complexes (0.5,0.1, and 0.4 p~ for carbamyl-phosphate synthase I, glutamate dehydrogenase, and a-ketoglutaratedehydrogenase complex, respectively). Thus, essentially all of the aminotransferase could be associated with these three enzymes in liver mitochondria. Instead of being competitive with malate dehydrogenase, the aminotransferase can enhance binding of malate dehydrogenase in the presence of each of the three high molecular weight enzymes. In the presence of aminotransferase, the apparent dissociation constants of malate dehydrogenase for glutamate dehydrogenase and the a-ketoglutarate dehydrogenase complex were 0.4 and 1.1p ~ respectively; , and glutamate dehydrogenase and the a-ketoglutarate dehydrogenase complex had 1 and 10 malate dehydrogenase-binding sites, respectively. Thus, since the mitochondrial level of malate dehydrogenase in liver is 140 p ~ a , significant fraction of malate dehydrogenase could also be incorporated into these complexes.

Exclusion of Other Enzymes-The hetero-enzyme interactions described above are specific. With the exception of the interactions noted below, other proteins tested did not associate with carbamyl-phosphatesynthase I, glutamate dehydrogenase, a-ketoglutarate dehydrogenase complex, malate dehydrogenase, aminotransferase, glutamate dehydrogenase-aminotransferase, aminotransferase-carbamyl-phosphate synthate I, or aminotransferase-malate dehydrogenase-a-ketoglutarate dehydrogenase complex (8,11-14, 22, 26, 71, 72). Citrate synthasealso associates with malate dehydrogenase (8) and the aminotransferase (11,26). In addition, succinate thiokinase, NAD:isocitrate dehydrogenase, and, to a lesser extent, citrate synthase (11, 72,73) also associate with the aketoglutarate dehydrogenase complex. Although these interactions could take place in liver mitochondria, citrate synthase would not be expected to markedly displace the three high molecular weight enzymes from the dimers, and succinate thiokinase andNAD:isocitrate dehydrogenase would not be expected to markedly displace the dimers from the aketoglutarate dehydrogenase complex. This is because in liver mitochondria, the levels of glutamate dehydrogenase (70 p ~ (Table V) and carbamyl-phosphate synthase I (400 p ~ (21) ) are significantly greater than thatof citrate synthase (25 p ~ (Table V); and the levels of succinate thiokinase (60 p ~ (calculated from Ref. 37), citratesynthase,and NAD: ) V) are considerably isocitrate dehydrogenase (4 p ~ (Table ) V). Furtherlower than those of the dimers (140 p ~ (Table more, the interactions mentioned above with citrate synthase, succinate thiokinase, and NAD:isocitrate dehydrogenase are weaker (11, 26) and therefore require levels of enzymes in excess of the 0.1 mg/ml levels generally used in our experiments. Consequently, in the presence of 0.1 mg/ml levels of

) ) )

0

10420

Glutamate-MalateMetabolism in Liver Mitochondria

malate dehydrogenase, aminotransferase,citratesynthase, and succinate thiokinase, there was little binding of citrate synthase or succinate thiokinase in either the presence or absence of the three high molecular weight enzymes (Table VI). The aminotransferase canalso associate with the pyruvate dehydrogenase complex (11). However, in liver mitochondria, the level of the pyruvate dehydrogenase complex (0.3 p ~ (Table V) would be too low compared with that of the other high molecular weight enzymes for it to compete for a significant amount of the aminotransferase. Other knownhetero-enzyme interactionswiththe high molecular weight enzymes, such as ornithine transcarbamylase-carbamyl-phosphate synthase I (74) and glutamate dehydrogenase-alanine aminotransferase (75), could be formed in liver mitochondria, but would not be expected to inhibit binding of malate dehydrogenase and aspartate aminotransferase because of the comparativelylower levels of ornithine transcarbamylase (34 p ~ (Table ) V) and alanine aminotransferase (4 p ~ (Table ) V). Although NADP:isocitrate dehydro) V) in liver genase is present a t high levels (88 p ~ (Table mitochondria, it does not interact with the enzymes of the hetero-enzyme system invitro (11,73,76) and apparentlyalso doesnotchannela-ketoglutarateintothea-ketoglutarate dehydrogenase complex in intact mitochondria(77, 78).

ase enhances its affinity for malatedehydrogenase. Alternatively, the restriction in the rotationalmobility of the bound aminotransferase may enhance the affinity of the aminotransferase for malate dehydrogenase. It is known that malate dehydrogenase associateswith immobilized fumarase,the aminotransferase associates with the malate dehydrogenase moiety of this complex, and the aminotransferaseis bound to )immobilized malate dehydrogenase (25). Consequently, it is conceivable that in a similar manner, binding of the aminotransferase to the quite high molecular weight enzymes could enhance the affinity of the aminotransferase for malate dehydrogenase. In the case of the a-ketoglutaratedehydrogenase complex, binding of malate dehydrogenase and the aminotransferase to malate dehydrogenase or aminotransferase already bound to the a-ketoglutarate dehydrogenase complex may, in part, account for the large number of dimer-binding sites found in this ternarycomplex. According to our results, the apparent dissociation constants and stoichiometries of these hetero-enzyme complexes as well as the levels of these enzymes in liver mitochondria are compatible with all of the aminotransferases anda significant fraction of the malate dehydrogenase being associated withthese highmolecularweightenzymes. Furthermore, higher degrees of organization of the hetero-enzyme system are possible because malate dehydrogenase-glutamate dehydrogenase-aminotransferase can associate with the other two DISCUSSION high molecular weight enzymes. In previous assays of the coupled malate dehydrogenaseAccording to previous results (11,12), the aminotransferase aminotransferase reaction performed in the presence of NAD, is not competitivewith malate dehydrogenasefor t h e a (ll), levels of the a-ketoglutarate malate, and glutamate ketoglutarate dehydrogenase complex, but can enhance binddehydrogenase complex as low as 0.003 p~ markedlydeing of malate dehydrogenase. According to our results, similar types of interactions take place between these two dimers and creased the K , of malate in the malate dehydrogenase reaccarbamyl-phosphate synthaseI and glutamatedehydrogenase. tion. According to the binding experiments described in this As discussed in the Introduction, malatedehydrogenase may paper, theKDof malate dehydrogenase for aminotransferase, there be capable of supplying the aminotransferasewith oxalacetate a-ketoglutarate dehydrogenase complex is -1 p ~ and are about 10 malate dehydrogenase-binding sites/a-ketogluin mitochondria only in structures of this type, which would place these two enzymes in close proximity to one another. tarate dehydrogenase complex. This would indicate that the It is known that binding of the aminotransferase to gluta- level of ternary complex would be quite low in the previous mate dehydrogenase alters the fluorescence of a fluorescent assays. However, the KD of malate dehydrogenase for pyridoxal-P-aminotransferase-a-ketoglutaratedehydrogenase probeonglutamate dehydrogenase(79) andresultsinan This increase in thedegree of polarization of a covalently attached complex is measured in direct binding experiments. could group on the aminotransferase (80, 81). Consequently, the be higher than the KO of NADH-malate dehydrogenase-oxaminotransferase could facilitate binding of malate dehydro- alacetate forpyridoxamine-P-aminotransferase-a-ketoglutarate dehydrogenase complex that was estimated in previous genase in the presence of glutamate dehydrogenase because the conformational changeinduced in glutamate dehydrogen- kinetic assays. Another difference is that the level of malate dehydrogenase was considerably lower in the kinetic assays. However, the high level of malate (1 mM) used in the kinetic TABLEVI assays would prevent malatedehydrogenase from dissociating Exclusion of succinate thiokinaseand citrate synthase from heterointo monomers(82). The polyethylene glycol used in the direct enzyme interactions These experiments were performed as described in the legend to binding experimentscould result in a KO slightly higher than Table I. With the exception offumarase and carbamyl-phosphate the actual value because in the case of the citrate synthasesynthase I (present at 0.05 and 0.3 mg, respectively), the amount of malate dehydrogenase complex, polyethylene glycol failed to each enzyme in the 1-ml incubation mixture was 0.1 mg. In terms of precipitate a smallfraction of extremelyhighmolecular nanomoles, the amounts of enzymes added were as follows: aspartate weight complex (83). However, the KO of citrate synthaseaminotransferase (AspAT), 1.1 nmol; malate dehydrogenase (MDH), 1.4 nmol; citrate synthase (CS),1.0 nmol;fumarase (FUM), 0.26 pyruvate dehydrogenase complex was the same in either the nmol; succinate thiokinase (STK), 1.1 nmol; and, where indicated, presence or absenceof polyethylene glycol (66). glutamate dehydrogenase (GDH), 0.3 nmol; a-ketoglutarate dehydroPossible Role of Inner Mitochondrial Membrane-In liver genase complex (KDHC), 0.037 nmol; and carbamyl-phosphate syn- mitochondria, the aminotransferase may be localized in the thase I (CPS), 1.9 nmol. vicinity of the inner mitochondrial membranebecause when Enzyme precipitated aspartate is generated by the aminotransferase, it is transAdditions ported into thecytosol without mixing with the total pool of FUM MDH AspAT GDH CS S T K CPS aspartate in the matrix (84, 85). Furthermore, the liver inner % mitochondrial membrane hasa specific aspartate aminotransNone 15 9 12 10 8 ferase-bindingsite (4).The a-ketoglutarate dehydrogenase 50 KDHC 30 15 8 GDH, KDHC 46 45 59 88 16 8 complex can also associate with the membrane andComplex CPS, GDH 40 10 70 100 54 8 90 I (17, 18).In both cases, the number of enzyme-binding sites

Glutamate-Malate Metabolism in Liver Mitochondria on the membrane is sufficiently high and the dissociation constant sufficiently low so that a high fraction of the total aminotransferaseanda-ketoglutarate dehydrogenase complex could be membrane-bound. Thus, in liver mitochondria, i t may be membrane-bound aminotransferase and a-ketoglutarate dehydrogenase complex that associate with malate dehydrogenase and glutamate dehydrogenase. It is also possible that thehetero-enzyme complex is localized in the vicinity of the membrane as a result of alternating bonds between aminotransferase-a-ketoglutarate dehydrogenase complex and either the membrane or the other enzyme constituent of the hetero-enzyme system. The concentration of specific ligands could determine the fraction of enzyme bound to the membrane or to the other enzyme components. Citrate, for example, dissociates malate dehydrogenase and, to a lesser extent, the aminotransferase from glutamate dehydrogenase, but does not dissociate the aminotransferase from the membrane (4,22,71).M%+ has an opposite effect (4,71). However, neither citrate nor M$+ would be expected to displace the hetero-enzyme complex from the vicinity of the membrane or displace one enzyme in the complex from the vicinity of another. Diffusion of an enzyme away from another or from the vicinity of the membrane wouldbe quite slow in the viscous mitochondrial matrix (6), and not all of the heteroenzymes bound are displaced by either citrate or M e (11). Specific ligands would, however, enable the system to meet the previously described (86) requirement to be flexible and dynamic enough to undergo ultrastructural transformations. Citrate Synthase-Pyruvate Dehydrogenase Complex-Malate dehydrogenase would be less rigorously localized than the aminotransferase in the membrane-hetero-enzyme system described above. As mentioned inthe Introduction, several factors dissociate malate dehydrogenase (but not the aminotransferase) from the membrane. Furthermore, malate dehydrogenase has a high affinity for glutamate dehydrogenase only when aminotransferase is bound to glutamate dehydrogenase, and malate dehydrogenase has a lower affinity than the aminotransferase for the a-ketoglutarate dehydrogenase complex. These factors plus the high mitochondrial level of malate dehydrogenase could enable it to supply oxalacetate to thepreviously described complex between citrate synthase and the pyruvate dehydrogenase complex (66) that can associate with both the membrane (17) and malate dehydrogenase (11).Association of aminotransferase-a-ketoglutarate dehydrogenase complex with the membrane would not be expected to inhibit binding of citrate synthase-pyruvatedehydrogenase complex to the membrane or Complex I. There is more than sufficient Complex I to accommodate both the a-ketoglutarate dehydrogenase and pyruvate dehydrogenase complexes (17, 87), and aminotransferase does not block binding of citrate synthase to the membrane (4). Mitochondrial Levels of Enzymes-Factors that could alter our estimates of enzyme levels in mitochondria ((a)there may be modifiers of enzyme activity in the extract; ( b ) the volume of mitochondrial water is not constant at 1 pl/mg of mitochondrial protein (56); and (c) all of the mitochondrial water may not be free (88))would not be of sufficient magnitude to invalidate our proposed model of organization of the aminotransferase, malate dehydrogenase, and the high molecular weight enzymes because the estimated levels of enzymes are orders of magnitude higher than their dissociation constants in the hetero-enzyme complexes. Furthermore, the mitochondrial extractswere diluted severalfold for most enzyme assays, which would minimize the effect of modifiers. In addition, we found that the kinetic properties of several rat liver mitochondrial enzymes were about the same in mitochondrial

10421

extracts compared with the pure rat liver enzymes. These include the several kinetic properties of glutamate dehydrogenase (89), malate dehydrogenase (ratio of velocity of forward to reverse reaction, K , of malate, K; of a-ketoglutarate, K i of citrate),andaspartate aminotransferase (datanot shown) that we investigated. Therefore, the diluted mitochondrial extracts apparently do not contain factors that significantly modify the activities of these enzymes. The specific activities of enzymes (including the markers citratesynthase and ornithinetranscarbamylase) in liver mitochondrial extracts prepared with the methods we employed were, in several cases, 1.5-2-fold higher than those found by other investigators. Consequently, our value of 65 mg of mitochondrial protein/g of liver was also 1.4-foldhigher than previous estimates (90). Our 2-3-fold higher value for the specific activity of the a-ketoglutarate dehydrogenase complex in liver mitochondria (91) could be due, in part, to reconstituting the extracted complex with thiamine pyrophosphate. Our considerably higher specific activity of alanine aminotransferase (49) could, in part, be due to adding leupeptin. In the absence of leupeptin, we found this enzyme to be quite labile. Over 20 years ago, Sols and Marco (92) estimated the levels of aminotransferase and malate dehydrogenase to be -3- and %fold lower,respectively, than thevalues we found. However, these estimates were based upon the assumption that the mitochondrial components of these enzymes corresponded with and were kinetically similar to their cytosolic counterparts. Their estimate of the level of citrate synthesis, which is exclusively a mitochondrial enzyme, was 35 pM, which is only slightly higher than our value of 25 pM. REFERENCES 1. Wanders, R. J. A., Meijer, A. J., Groen, A. K., and Tager, J. M. (1983)Eur. J. Biochern. 133,245-254 2. Burton, K., and Wilson, T. H. (1953)Biochern. J. 54,86-92 3. Fahien, L. A., and Strmecki, M. (1969)Arch. Biochern. Biophys. 130,478-487 4. Teller, J. K., Fahien, L. A., and Valdivia, E. (1990)J. Biol. Chern. 265,19486-19494 5. Srere, P. A. (1972)in Energy Metabolism and the Regulation of Metabolic Processes in Mitochondria (Mehlman, M., and Hanson, R. W., eds) pp. 79-91,Academic Press, New York 6. Srere, P. A. (1987)Annu. Reu. Biochern. 56,89-124 7. Lopes, C., Klazingnor, W., and van den Bergh, S. G. (1970)Eur. J. Biochern. 83,635-640 8. Halper, L. A., and Srere, P. A. (1977)Arch. Biochern. Biophys. 184,529-534 9. Datta, A., Merz, J. M., and Spivey, H. 0.(1985)J. Biol. Chem. 260,15008-15012 10. Spivey, H. O., and Merz, J. M. (1989)Bioessays 10,127-130 11. Fahien, L. A., Kmiotek, E. H., MacDonald, M. J., Fibich, B., and Mandic, M. (1988)J. Biol. Chem. 263,10687-10697 12. Fahien, L. A., MacDonald, M. J., Teller, J. K., Fibich, B., and Fahien, C. M. (1989)J. Biol. Chern. 264, 12303-12312 13. Fahien, L. A., Kmiotek, E.H., and Smith, L. E. (1979)Arch. Biochern. Bi0ph.y~.192,33-46 14. Fahien, L. A., and Kmiotek, E. H. (1979)J. Biol. Chern. 254, 5983-5990 15. Robinson, J. B., Jr., and Srere, P.A. (1985)J. Biol. Chern. 260, 10800-10805 16. D’Souza, S. F., and Srere, P. A. (1983)J . Biol. Chern. 258,47064709 17. Sumegi, B., and Srere, P. A. (1984)J. Bwl. Chern. 259,1504015045 18. Fukushima, T., Decker, R. V., Anderson, W. M., and Spivey, H. 0.(1989)J. Biol. Chem. 264,16483-16488 19. Srivastava, D.K., and Bernhard, S. A. (1986)Curr. Top. Cell. Regul. 28,l-35 20. Fahien, L. A,,Strmecki, M., and Smith, S. E. (1969)Arch. Biochern. Biophys. 130,449-455

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