THE JOURNAL OF BIOLOGICAL Val. 267, No. 15, Issue May · PDF fileTHE JOURNAL OF BIOLOGICAL CHEMISTRY Val. 267, No. 15, Issue of May 25, pp. 10411-10422,1992 0 1992 by The American

THE JOURNAL OF BIOLOGICAL CHEMISTRY Val. 267, No. 15, Issue of May 25, pp. 10411-10422,1992 0 1992 by The American Society for Biochemistry and Molecular BiologY, h . Printed in U. S. A.

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 Jan K. Tellers From the Department of Pharmacology, University of Wisconsin Medical School, Madison, Wisconsin 53706

The level of aspartate aminotransferase in liver mitochondria was found to be -140 PM, or 2-3 orders of magnitude higher than its dissociation constant in complexes with the inner mitochondrial membrane and the high molecular weight enzymes (Mr = 1.6 X 10‘ to 2.7 x 10‘) carbamyl-phosphate synthase I, glutamate dehydrogenase, and the a-ketoglutarate dehydrogenase complex. The total concentration of aminotransferase- binding sites on these structures in liver mitochondria was more than sufficient to accommodate all of the aminotransferase. Therefore, in liver mitochondria, the aminotransferase could be associated with the inner mitochondrial membrane and/or these high molecular weight enzymes.

The aminotransferase in these hetero-enzyme complexes could be supplied with oxalacetate because binding of aminotransferase to the high molecular weight enzymes can enhance binding of malate dehydrogenase, and binding of both malate dehydrogenase and the aminotransferase facilitated binding of fumarase.

The level of malate dehydrogenase was found to be so high (140 MM) in liver mitochondria, compared with that of citrate synthase (25 PM) and the pyruvate dehydrogenase complex (0.3 PM), that there would also be a sufficient supply of oxalacetate to citrate synthase- pyruvate dehydrogenase.

The well-documented ability of exogenous malate to enhance aspartate aminotransferase (EC 2.6.1.1) activity in liver mitochondria (1) would be difficult to explain unless at least some of the malate dehydrogenase ((S)-malate:NAD+ oxido- reductase, EC 1.1.1.37) is localized in mitochondria in close proximity to aspartate aminotransferase. At a physiological pH, equilibrium of the malate dehydrogenase reaction is quite unfavorable (2), and malate dehydrogenase itself has a high affinity for oxalacetate (3). Therefore, since the level of malate dehydrogenase is quite high in liver mitochondria (-280 ~ L M with respect to oxalacetate-binding sites) (4), a significant fraction of the oxalacetate generated would remain bound to malate dehydrogenase. In addition, the level of free oxalacetate is quite low and is considerably lower than the high level of matrix proteins (5-7) so that there can be competing

* This work was supported by National Institutes of Health Grant CA 40445. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom reprint requests should be addressed Dept. of Phar- macology, University of Wisconsin Medical School, 1300 University Ave., Madison, WI 53706.

I Present address: Dept. of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, Great Britain.

protein-binding sites. Furthermore, citrate synthase can associate with and receive oxalacetate from malate dehydrogenase by a channeling mechanism (8-10). This could favor a transfer of oxalacetate from malate dehydrogenase to citrate synthase because malate dehydrogenase does not readily associate with or channel oxalacetate into the aminotransferase alone (11-14). Therefore, a transfer of oxalacetate from malate dehydrogenase to the aminotransferase is possibly facilitated by mitochondrial structures that place these two enzymes in close proximity to one another. Consequently, malate dehydrogenase may not be completely free in the matrix, but may, in part, be localized by these mitochondrial structures. This is consistent with previous results that demon- strated that when mitochondria are rendered permeable to molecules even larger than malate dehydrogenase, malate dehydrogenase and several other Krebs cycle enzymes are retained (15). Although malate dehydrogenase has a high affinity for the inner mitochondrial membrane and Complex I (4, 16-18) and the aminotransferase has a high affinity for a unique site on the membrane (4), the inner mitochondrial membrane may not be capable of placing these two enzymes in close proximity to one another. The aminotransferase, citrate, and high ionic strength displace malate dehydrogenase from the membrane (4,16). Furthermore, oxidation of malate by malate dehydrogenase may not readily take place on Com- plex I because NAD and malate displace malate dehydrogenase from Complex I (18). However, we have shown, using several different methods, that the aminotransferase forms a binary complex with the a-ketoglutaric dehydrogenase (EC 1.2.4.2) complex and that even in the presence of NAD, malate, and high ionic strength, malate dehydrogenase can associate with the binary complex (11, 12). Thus, in the ternary complex, malate dehydrogenase and the aminotransferase could be in close proximity to one another; and consequently, the ternary complex may be able to catalyze the combined malate dehydrogenase-aminotransferase reactions in liver mitochondria. Consistent with this, we found that adding the a-ketoglutarate dehydrogenase complex to the combined malate dehydrogenase-aminotransferase reaction markedly decreases the K,,, of malate (11). Another potential advantage of the ternary complex is that kinetic experiments (12) indicated that a-ketoglutarate can be directly transferred (19) from the aminotransferase to the a-ketoglutarate dehydrogenase complex or that binding of the a-ketoglutarate dehydrogenase complex to the aminotransferase can enhance dissociation of a-ketoglutarate from the aminotransferase so that a-ketoglutarate can rapidly react with the dehydrogenase complex without having to diffuse from the hetero-enzyme system.

Glutamate dehydrogenase (L-glutamate:NAD(P)+ oxidore- ductase (deaminating), EC 1.4.1.3) and carbamyl-phosphate synthase (ammonia) I (EC 6.3.4.16) are present in liver mi-

10411

10412 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 carbamyl- phosphate 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. Con- sequently, 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 carbamyl- phosphate synthase I can 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).

MATERIALS AND METHODS

Enzymes and Reagents-Bovine heart pyruvate dehydrogenase and a-ketoglutarate dehydrogenase complexes, bovine and rat liver mitochondrial glutamate dehydrogenases and aspartate aminotransferase, and rat liver carbamyl-phosphate synthase I and ornithine transcarbamylase were prepared as described previously (12, 20, 27- 31). Pig heart mitochondrial malate dehydrogenase, citrate synthase, and fumarase were obtained from Boehringer Mannheim. Other enzymes, coenzymes, substrates, and reagents were obtained from Sigma. Stock solutions of all reagents used in assays were adjusted to the pH of the assay.

Concentrations of Enzyme and Protein-The concentrations of pure pyruvate dehydrogenase and a-ketoglutarate dehydrogenase complexes were measured as described previously with bovine serum albumin as a standard (32). The concentrations of pure glutamate dehydrogenase, fumarase, carbamyl-phosphate synthase I, malate dehydrogenase, aspartate aminotransferase, succinate thiokinase, and citrate synthase were measured spectrophotometrically at 280 nm using previously determined values for extinction coefficients (29,

The concentration of protein in mitochondrial extracts was measured by the biuret reaction on trichloroacetate precipitates of the extracts (39).

Preparation of Mitochondria and Mitochondrial Extractions and Assay of Enzymes in Mitochondrial Extracts-Mitochondria were

33-38).

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 at 1100 X g) residues were repeatedly reho- mogenized 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 (2 min) 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.) The supernatant and fluffy layer were removed, and the mitochondria were suspended manually in H- medium (minus albumin and EGTA, plus 20 pg/ml leupeptin). Mi- tochondria 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 instead of 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 equal volume of 1.0% Triton X-100, 50 mM sodium Hepes (pH 7.5), 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.

For specific extraction of the pyruvate dehydrogenase complex, the extraction medium was an equal volume of 50 mM potassium MOPS (pH 6.9), 20 mM MgCl,, 20 mM calcium EGTA, 2 mM dichloroacetate, 2 mM azide, 40 pg/ml leupeptin, 1.0% Triton X-100, and 2 mM dithiothreitol.

the assay of Linn et al. (43), with the additions of inhibitors of NADH For the assay of the pyruvate dehydrogenase complex (based upon

oxidase and lactate dehydrogenase), a 10-50-pl aliquot of the extract was added to 1 ml of 0.5% Triton X-100, 50 mM sodium Hepes (pH 7.5), 0.1 mg/ml albumin, 0.1 mM EDTA, 0.1 mM dithiothreitol, 1 mM KCN, 5 p~ rotenone, 10 pg/ml leupeptin, 5 mM NAD, 0.2 mM CoA, 0.2 mM thiamine pyrophosphate, 1.25 mM MgC12, 1 mM pyruvate, and 10 mM oxalate. The reaction was started by the addition of pyruvate after the absorbance had reached a constant value. Oxalate (10 mM) reduced the activity of lactate dehydrogenase (measured with thiamine pyrophosphate and coenzyme A omitted and 40 p M NADH added to the pyruvate dehydrogenase complex assay to <2% of the pyruvate dehydrogenase activity) and did not affect the activity of the purified bovine heart pyruvate dehydrogenase complex. The

The abbreviations used are: Bes, N,N-bis(2-hydroxyethyl)-2-ami- noethanesulfonic acid; Hepes, N-2-hydroxyethylpiperazine-N'-2-eth- anesulfonic acid; HEPPS, N-2-hydroxyethylpiperazine-N'-3-pro- panesulfonic acid; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraace- tic acid; MOPS, 4-morpholinepropanesulfonic acid.

Glutamate-Malate Metabolism in Liver Mitochondria 10413

rate was linear with time only up to 20 p M NADH and, within this range, was also linear with respect to the concentration of extract (0.01-0.03 pmol/ml/min). When mitochondrial extract and pure bovine heart pyruvate dehydrogenase were combined, the measured rate equaled the sum of their activities. Adding pig heart lipoamide dehydrogenase or KCN did not change the rate, nor did citrate synthase plus oxalacetate. Bovine heart pyruvate dehydrogenase complex had a specific activity of 20 pmol/min/mg in this assay.

For specific extraction of pyruvate carboxylase, 5 volumes of 0.2% Triton X-100, 50 mM sodium Hepes (pH 7.5), 2 mM MgC12, 0.1 mM EDTA, 0.1 mM acetyl-coA, and 20 pg/ml leupeptin (at room temperature) were used as the extraction medium. Assays were performed spectrophotometrically at 412 nm by converting the generated oxalacetate into citrate (44). For the assay, a 5-pl aliquot of the extract was added to 1 ml of 50 mM sodium Hepes (pH 7.5), 50 mM KHC03, 10 mM sodium pyruvate, 2.5 mM ATP, 5.0 mM MgCl,, 0.1 mM acetyl- CoA, 0.2 mM 5,5-dithiobis(2-nitrobenzoic acid), and 0.1 mg/ml citrate synthase. The reaction was started with pyruvate after it reached a constant rate in its absence. The specific activity of purified rat liver pyruvate carboxylase under similar conditions is 34 pmol/min/mg (45).

For specific extraction of malate dehydrogenase, the extraction medium contained 400 volumes of 0.5% Triton X-100, 0.1 M potassium phosphate (pH 7.6), 1 mM EDTA, 1 mM dithiothreitol, and 1 mg/ml albumin. The assay was performed by adding 10-30-pl aliquots of the extract to 1 ml of the same solution (minus dithiothreitol and Triton X-100) plus 0.125 mM oxalacetate and 0.2 mM NADH. The reaction was started with oxalacetate, and the reaction in its absence was negligible. Pig heart mitochondrial malate dehydrogenase had a specific activity of 1200 pmol/min/mg in this assay.

We have found that the a-ketoglutarate dehydrogenase complex becomes gradually depleted of thiamine pyrophosphate during its isolation and that rebinding of the cofactor is a slow, temperature- dependent process (12). To ensure its saturation in the extract, the mitochondria were extracted by incubation for 15 min at 30 "C in 10 volumes of 0.5% Triton X-100,50 mM potassium phosphate (pH 7.0), 0.1 mM EDTA, 0.5 mM thiamine pyrophosphate, 2.0 mM MgCl,, 2.0 mM azide, 0.5 mM dithiothreitol, 10 pg/ml leupeptin, and 1.0 mg/ml albumin. About 25-50 p1 of the reconstituted diluted a-ketoglutarate dehydrogenase complex were then assayed in 1 ml containing 50 mM sodium Hepes (pH 7.5), 0.1 mg/ml albumin, 2.0 mM NAD, 5.0 mM a- ketoglutarate, 0.5 mM thiamine pyrophosphate, 2.0 mM MgC12, 5.0 mM calcium EGTA, 1.0 mM KCN, 0.2% (w/v) Triton X-100, 0.2 mM CoA, and 1.0 mM dithiothreitol. The specific activity of the pure bovine heart complex was 25 pmol/min/mg under these assay conditions.

For specific extraction of glutamate dehydrogenase, the extraction medium was 50 volumes of 0.2% Triton X-100, 50 mM potassium phosphate (pH 7.0), 0.1 mM EDTA, and 10 pg/ml leupeptin. For the assay, 10-20 pl of the extract were added to a 1-ml solution containing 50 mM potassium phosphate (pH 7.0), 0.1 mM EDTA, 2.5 mM leucine, 0.1 mM NADH, 1.0 mM a-ketoglutarate, 0.2% Triton X-100, 1 mM KCN, and 50 mM NHIC1. a-Ketoglutarate was added last after a constant rate (5% of rate with a-ketoglutarate) was achieved. Triton is inhibitory, but leucine both activates and overcomes this inhibition. The specific activity of pure rat liver glutamate dehydrogenase was 70 pmol/min/mg under these conditions.

For specific extraction of aspartate aminotransferase, the extraction medium was 50 volumes of 0.5% Triton X-100,O.l M potassium phosphate (pH 7.6), 1 mM EDTA, and 0.1 mg/ml albumin. A 10-20- pl aliquot of the diluted suspension was assayed in 1 ml of 50 mM sodium Hepes (pH 7.5), 0.1 mM EDTA, 0.1 mg/ml albumin, 10 mM aspartate, 10 mM a-ketoglutarate, 0.1 mM NADH, and 3 pg/ml mitochondrial malate dehydrogenase. The specific activity of the pure rat liver enzyme was 250 pmol/min/mg under these conditions.

NADP:isocitrate and NAD:isocitrate dehydrogenases were specifically extracted with an equal volume of 1.0% Triton X-100, 0.2 M potassium phosphate (pH 7.6), 2 mM ADP, 2 mM dithiothreitol, 0.2 mM EDTA, and 20 pg/ml leupeptin. For assays of NADP:isocitrate dehydrogenase, a 10-pl aliquot of the extract was added to 1 ml of a solution containing 25 mM sodium Hepes (pH 7.5), 0.2 mM NADP, 5 p M rotenone, 0.2% Triton x-100, 1.0 mM dithiothreitol, 1.0 mM MnSO,, 10 pg/ml leupeptin, and 5 mM DL-isocitrate. The specific activity of this enzyme from pig heart under comparable assay conditions is 30 pmol/min/mg (46).

For assays of NAD:isocitrate dehydrogenase, a 30-100-pl aliquot of the extract was added to 1 ml of a solution of 50 mM Bes/ triethanolamine (pH 7.2), 5 p~ rotenone, 10 pg/ml leupeptin, 0.5%

Triton X-100, 1.0 mM ADP, 1.0 mM dithiothreitol, 2.0 mM NAD, 1.0 mM MnC12,and 1.0 mM DL-isocitrate. The reaction was started with isocitrate after the reaction in its absence had reached a constant rate. The specific activity of pig heart NAD:isocitrate dehydrogenase is 26 pmol/min/mg with Mg2+ in this assay and would be -30% higher with Mn" (47, 48).

Alanine aminotransferase was specifically extracted by diluting the mitochondria (1:3) in 50 mM triethanolamine HCl (pH 7.8), 0.1 mM EDTA, 1 mM dithiothreitol, 20 pg/ml leupeptin, and 0.5% Triton X- 100. Although alanine aminotransferase has been reported to be unstable (49), there was no loss of its activity in this extract or the extract prepared with Method I (both containing leupeptin) over several hours a t 0 "C. In the assay adopted from the continuous assay of Swick and co-workers (50), 5-20 p1 of extract were added to a 1- ml solution containing 0.5% Triton X-100, 50 mM triethanolamine HCl (pH 7.8), 0.1 mM EDTA, 10 pg/ml leupeptin, 1.0 mM dithiothreitol, 5 pM rotenone, 50 mM alanine, 10 mM a-ketoglutarate, 0.2 mM NADH, and 10 units of lactate dehydrogenase. The reaction was started by addition of alanine after a constant absorbance was reached in its absence. The reaction was linear with time after a brief phase (1 nmol of NAD+ formed) when it was slightly faster. The specific activity of purified porcine kidney mitochondrial alanine aminotransferase under similar assay conditions is 213 pmol/min/mg (50).

For specific extraction of citrate synthase, the mitochondria were diluted 1:20 in 50 mM sodium Hepes (pH 7.5), 0.1 mM EDTA, 10 pg/ ml leupeptin, and 0.5% Triton X-100. For the assay, a 10-20-4 aliquot of the extract was added to 1 ml of essentially the same assay medium as described previously (51), except the medium contained 0.2 mM oxalacetate and 0.5% Trition X-100. Citrate synthase was then assayed spectrophotometrically at 412 nm as described previously (51, 52). The specific activity of pure rat liver citrate synthase under similar assay conditions is 124 pmol/min/mg (52).

Ornithine transcarbamylase was specifically extracted by diluting the mitochondria 1:20 in 0.05% albumin, 10 mM EDTA (pH 7.0), 1 mM dithiothreitol, and 0.5% Trition X-100 and then diluting 1:20 again in the same buffer minus dithiothreitol. For the assay, a 10-20- pl aliquot of diluted extract was added to a 1-ml solution of a previously described medium (53), except the medium contained 5 mM lithium carbamyl phosphate, 5 mM ornithine, and 0.05 M sodium HEPPS (pH 8.2) at 25 "C. The specific activity of pure rat liver ornithine transcarbamylase under similar assay conditions is 855 pmol/ml/min (54).

Citrate synthase and ornithine transcarbamylase activities assayed both in the original tissue homogenate and in the mitochondrial extract were measures of the yield of mitochondria. For assays of citrate synthase in the original liver homogenate, the homogenate was diluted 1:9 in the same medium described above for specific extraction of this enzyme from mitochondria. A 10-30-p1 aliquot was then added to the same assay medium described above. For assays of ornithine transcarbamylase in the original liver homogenate, the 1:9 dilution prepared for the assay of citrate synthase in the liver homogenate was diluted 1:19 in the same medium described for specific extraction of ornithine transcarbamylase (minus dithiothreitol), and a 10-20-pl aliquot was added to the assay medium described above.

Frozen mitochondria were also extracted with 0.1% cetyltrimeth- ylammonium bromide (Method 11) as described previously (3, 20, 27, 55), and some of the enzymes (malate dehydrogenase, fumarase, citrate synthase, glutamate dehydrogenase, and aspartate aminotransferase) in these extracts were assayed at pH 7.8 and 25 "C in 25 mM sodium arsenate, 0.1 mM EDTA as described previously (3, 20, 27, 52, 55). Slightly lower levels of aspartate aminotransferase and higher levels of glutamate dehydrogenase were found with this method.

Calculations of Mitochondrial Levels of Enzymes-The concentrations (C) of enzymes in milligrams of enzyme/milliliter of mitochondrial water were calculated by multiplying the ratio of the specific activity of the enzyme in the extract to the specific activity of the pure enzyme by the milligrams of mitochondrial protein/milliliter of mitochondrial water according to Equation 1:

C = units of enzyme

mg mitochondrial protein

mg pure enzyme units of enzyme (1)

mg mitochondrial protein ml mitochondrial water

10414 Glutamate-Malate Metabolism in Liver Mitochondria where 1 unit is micromoles of product generated by the enzyme/ minute in the assays described above. Since there can be 1 p1 of mitochondrial water/mg of mitochondrial protein (56), a value of 1000 was used for the milligrams of mitochondrial protein/milliliter of mitochondrial water. Molecular weight values used to convert enzyme concentrations into micromolar were as follows: malate dehydrogenase, 7 X lo4 (57); aspartate aminotransferase, 9 X 10' (58); NAD(P):isocitrate dehydrogenase, 6 X lo4 (59); glutamate dehydrogenase, 3.4 X lo6 (60); citrate synthase, 1 X lo6 (61); pyruvate carboxylase, 5 X lo6 (45); alanine aminotransferase, 8 X lo' (50); fumarase, 1.9 X 10' (62); NAD:isocitrate dehydrogenase, 3 X lo6 (47); a-ketoglutarate dehydrogenase complex, 2.7 X lo6 (63); pyruvate dehydrogenase complex, 7 X lo6 (64); carbamyl-phosphate synthase I, 1.6 X lo6 (65); succinate thiokinase, 7 X lo' (37); and ornithine transcarbamylase, 1.1 X lo6 (54). Mitochondrial concentrations of enzymes were not expressed in terms of concentration of active sites because in this paper we are mainly concerned with the concentration of sites on a given enzyme available to associate with a second enzyme. This may not be identical to the concentration of active sites.

Measurement of Stoichiometry and Dissociation Constants-These experiments were performed by incubating the enzymes in polyethylene glycol as described previously (8, 14). Adding polyethylene glycol results in conditions (in terms of solvent exclusion effects) that are more like the mitochondrial matrix than is an aqueous buffer (8). With the exception of the a-ketoglutarate dehydrogenase complex, which is quite insoluble in the absence of other enzymes, all of the other enzymes used were quite soluble when incubated alone but precipitated in the presence of specific additional enzymes. Conse- quently, the amount of enzyme bound could be estimated from the amount of enzyme precipitated with the added enzyme(s) (8, 11, 14). This method has been used previously in studies of the citrate synthase-pyruvate dehydrogenase complex system, where it was found that the dissociation constant and the stoichiometry are the same in either the presence or absence of polyethylene glycol (66). This can be the case in polyethylene glycol when an equilibrium level of hetero-enzyme complex is formed rapidly. The experiments described in this paper meet this requirement. In all cases, the turbidity of the multienzyme/polyethylene glycol suspension and the amount of enzyme coprecipitated were the same after either a 2- or 20-min incubation, indicating that equilibrium was reached rapidly.

Experiments with Divalent Cross-linker-These experiments were performed essentially as described previously (13, 67) with the divalent cross-linker dimethyl 3,3'-dithiobispropionimidate.

RESULTS

Stoichiometry and Dissociation Constants of Hetero-enzyme Complexes-We have shown with several different methods that the a-ketoglutarate dehydrogenase complex can associate with aspartate aminotransferase and malate dehydrogenase (11). In phosphate buffer, binding of the aminotransferase is not competitive with malate dehydrogenase; and in Tris buffer plus M P , aminotransferase enhances binding of malate dehydrogenase (11). The stoichiometry and dissociation constants of these hetero-enzyme interactions have not been determined. Fig. 1 shows the amount of aminotransferase (curve A ) and malate dehydrogenase (curve B ) bound when equal concentrations of both of these enzymes were added to a constant level of the a-ketoglutarate dehydrogenase complex in phosphate buffer. Curves E and F show the amount of aminotransferase and malate dehydrogenase precipitated, respectively, in the absence of the dehydrogenase complex. The points of curve C are the differences between the experimental points of curve A minus the experimental points of curve E, and the points of curve D are the differences between the experimental points of curve B minus the experimental points of curve F. Thus, curves C and D show the amount of aminotransferase and malate dehydrogenase, respectively, bound to the a-ketoglutarate dehydrogenase complex. The solid curves C and D are actually theoretical curves calculated for the case where the number (n) of aminotransferase- or malate dehydrogenase-binding sites on the a-ketoglutarate dehydrogenase complex equals 10 (for both) and the KO values of the

[ MDH] added nmol

lAsp AT] added nmol

FIG. 1. Plots of nanomoles of malate dehydrogenase and aspartate aminotransferase bound uersus nanomoles of malate dehydrogenase and aminotransferase added in presence or absence of a-ketoglutarate dehydrogenase complex. In these experiments, equal nanomoles of malate dehydrogenase (MDH) and aspartate aminotransferase (Asp AT) were incubated in either the presence (curves A and B ) or absence (curves E and F ) of 0.037 nmol of a-ketoglutarate dehydrogenase complex in a 1-ml volume of 14% (w/v) polyethylene glycol plus buffer as described in the legend to Table I. The nanomoles of aminotransferase bound are shown in curves A and E, and the nanomoles of malate dehydrogenase bound are shown in curves B and F. Curves C and D were calculated as described in the text by correcting for the nanomoles of aminotransferase (curve C) or malate dehydrogenase (curve D) bound in the absence of the a-ketoglutarate dehydrogenase complex, with the number of binding sites on the a-ketoglutarate dehydrogenase complex equal to 10 for both the aminotransferase and malate dehydrogenase and the dissociation constants equal to 0.4 and 1.1 p~ for the aminotransferase and malate dehydrogenase, respectively. Experi- mental conditions are described in the legend to Table I. Methods for determining the amount of enzyme bound are described under "Ma- terials and Methods."

aminotransferase (curve C) and malate dehydrogenase (curve D ) equal 0.43 and 1.1 PM, respectively. These theoretical curves were obtained by rearranging the equilibrium relationship (Equation 2) to Equation 3:

where [Dl and [a are, respectively, the total concentration and the concentration of bound a-ketoglutarate dehydrogenase complex in the incubation; and [E], [Et], and [nfl are, respectively, the concentration of total, free, and bound aminotransferase or malate dehydrogenase in the incubation. The values for KO and n were estimated from the best fit of the linear double-reciprocal plots of [nfl (obtained by converting the nanomoles of aminotransferase or malate dehydrogenase bound represented by the points on curves C and D, respectively, into nanomoles bound per milliliter of incubation or micromolar versm [Ef] (obtained by subtracting [nfl from [E]). Theoretical values for curves C and D were then obtained by reading from the double-reciprocal plots, the theoretical value of [ n a at a given [Ef] , and calculating [E] from the relationship [E] = [E/1 + [nXJ.

According to previous results, glutamate dehydrogenase has a higher affinity for the aminotransferase than for malate dehydrogenase, and both binary complexes containing gluta-


mate dehydrogenase are considerably more stable than malate dehydrogenase-aminotransferase (13, 14). Previous attempts to demonstrate a ternary complex were hampered by the fact that under the conditions employed (incubations at 10 "C), the affinity of glutamate dehydrogenase for malate dehydrogenase alone was sufficiently high so that it could not be readily determined whether malate dehydrogenase was bound to free glutamate dehydrogenase or to glutamate dehydrogenase-aminotransferase. However, when malate dehydrogenase (0.1 mg/ml or 1.4 p ~ ) was incubated with either the aminotransferase alone (0.1 mg/ml or 1.1 pM) or glutamate dehydrogenase alone (0.1 mg/ml or 0.3 pM) at a more physiological temperature (25 "C), there was little precipitation of any of the three enzymes, and the amount of precipitation was essentially the same as when each of the three enzymes was incubated alone (Table I, lines 2 and 4 uerszu 5-7). Therefore, at these low levels of enzyme and at 25 "C, binary complexes containing malate dehydrogenase were not formed. If they were formed, then more malate dehydrogenase would have been precipitated when incubated with each of the other two enzymes than when incubated alone. This is because adding each of the other two enzymes separately to malate dehydrogenase did not significantly decrease the level of free or soluble enzyme over that found when each of the enzymes was incubated alone. Therefore, in the presence of malate dehydrogenase and a second interacting enzyme, the amount of enzyme in the precipitate would equal the amount in the binary complex plus the same amount of enzyme precipitated when the enzyme was incubated alone.

In contrast to results obtained with malate dehydrogenase, when glutamate dehydrogenase (0.1 mg/ml) was incubated with the aminotransferase (0.1 mg/ml), 32% of the aminotransferase and 83% of the glutamate dehydrogenase were precipitated (Table I, line 1) uerszu 2% of the aminotransferase (line 6) and 7% of the glutamate dehydrogenase (line 5) when these two enzymes were incubated alone, indicating the formation of a glutamate dehydrogenase-aminotransferase complex. When malate dehydrogenase was added (line 3), it did not markedly alter precipitation of glutamate dehydrogenase or the aminotransferase, but 18% of the malate dehydrogenase was bound. Therefore, since malate dehydrogenase did not associate with each of the other two enzymes alone but was only bound in the presence of both of the other two enzymes, malate dehydrogenase associates with glutamate dehydrogenase-aminotransferase. Since glutamate dehydro-

TABLE I Effect of aspartate aminotransferase on binding of malate

dehydrogenase to glutamate dehydrogenase In these experiments, enzymes were incubated in 1 ml of polyeth-

ylene glycol (14%, w/v), 14 mM potassium phosphate, 0.1 mM EDTA (pH 7.0) at 25 "C. After 20 min, incubation mixtures were centrifuged at 25 "C and assayed as described under "Materials and Methods." 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; and malate dehydrogenase (MDH), 1.4 nmol.

Enzyme precipitated

Additions MDH AspAT GDH

nmol % nmol % nmol %

AspAT, GDH 0.35 32 0.25 83 MDH, GDH 0.09 6 MDH, GDH, AspAT 0.25 18 0.33 30 0.25 83

0.02 7

AspAT, MDH 0.06 4 0.05 4 GDH 0.02 7 AspAT 0.02 2 MDH 0.07 5

genase did not form a significant amount of a binary complex with malate dehydrogenase in the absence of aminotransferase, it is quite unlikely that glutamate dehydrogenase would form a significant amount of a binary complex with malate dehydrogenase in the presence of aminotransferase where 83% of the glutamate dehydrogenase is bound and only 0.05 nmol would be free. Similarly, although -69% or 0.76 nmol of the aminotransferase was soluble when incubated with glutamate dehydrogenase f malate dehydrogenase, this amount of soluble aminotransferase would not be expected to form a binary complex with malate dehydrogenase in view of the fact that 1.1 nmol of aminotransferase did not associate with malate dehydrogenase in the absence of glutamate dehydrogenase. To ascertain that this would be the case, we incubated malate dehydrogenase (1.4 nmol) with either 0.05 nmol of glutamate dehydrogenase or 0.8 nmol of aminotransferase and found no significant interaction (data not shown). Therefore, in the presence of all three enzymes, the only possible complexes would be those between glutamate dehydrogenase and the aminotransferase and the ternary complex among all three enzymes. Although the amount of malate dehydrogenase precipitated in the presence of all three enzymes (line 3) was only slightly higher than the sum of the amount of malate dehydrogenase precipitated when malate dehydrogenase was incubated alone (line 7), with aminotransferase alone (line 4), and with glutamate dehydrogenase alone (line 2), the blanks would not be additive. This is because since binary complexes containing malate dehydrogenase were not found, the only applicable blank for the amount of malate dehydrogenase precipitated in the presence of all three enzymes would be that of malate dehydrogenase when incubated alone (line 7).

The results obtained when the level of glutamate dehydrogenase in the incubation was increased from 0.3 to 1.2 p~ in the presence of the same concentration of aminotransferase (1.1 p ~ ) and malate dehydrogenase (1.4 p ~ ) used in the experiments shown in Table I are shown in Fig. 2. The solid curves (curues A and B ) are theoretical and were calculated (as described above for the a-ketoglutarate dehydrogenase complex) using a value of n = 1 for both malate dehydrogenase

I I

0 0.5 I 0 1.5

[Glutomote Dehydrogenase Addedlnmol

FIG. 2. Plots of nanomoles of enzyme bound versus nanomoles of glutamate dehydrogenase added. In these experiments, 1.4 nmol of malate dehydrogenase plus 1.1 nanomoles of aspartate aminotransferase were incubated with the indicated nanomoles of glutamate dehydrogenase in a volume of 1 ml under the conditions described in the legend to Table I. The nanomoles of aminotransferase (0) and glutamate dehydrogenase (A) bound are shown in curue A. The nanomoles of malate dehydrogenase bound are shown in curue B (0). The solid lines (curues A and B ) have been calculated for the case where one aminotransferase dimer and one malate dehydrogenase dimer are bound to one glutamate dehydrogenase hexamer with dissociation constants of 0.1 and 0.4 pM for the aminotransferase and malate dehydrogenase, respectively. Experimental conditions are described in the legend to Table I.


and the aminotransferase and KO values of 0.1 and 0.4 WM for the aminotransferase and malate dehydrogenase, respectively. According to these values of KD and n, glutamate dehydrogenase levels of 2.3 and 4.6 WM would be required for 90% saturation of the aminotransferase and malate dehydrogenase, respectively. These levels of glutamate dehydrogenase (0.8-1.6 mg/ml) are not practical to use in experiments of this type. However, the theoretical curues A and B agree reason- ably well with the experimental points, indicating that the K D

and n values used to calculate these curves are good approxi- mations of their true values.

The 1:l stoichiometry between glutamate dehydrogenase and the aminotransferase closely approximates (but does not rigorously characterize) the observed results. In all experiments (Table I and Fig. 2), the molar ratio of aminotransferase to glutamate dehydrogenase bound was slightly >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)2 complex. 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)n becomes 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 to form a quaternary complex (12). These interactions may 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 a- ketoglutarate 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 KO of 2.5 WM and -1.1 carbamyl- phosphate 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 WM and 2.6 carbamyl-phosphate synthase I monomers 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

0.05 0.10 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 ml under 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 by subtracting the amount of glutamate dehydrogenase bound (0.02 nmol) in the absence of the a-ketoglutarate dehydrogenase complex. Experimental conditions are given in the legend to Table I.

TABLE I1 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 % nmol % nmol % nmol %

MDH, CPS 0.46 33 0.51 27 AspAT, CPS 0.43 39 1.1 58 AspAT, CPS, MDH 0.70 50 0.43 39 1.3 68 AspAT, CPS, MDH, 0.85 61 0.59 54 1.7 89 0.3 100

AspAT, GDH, MDH 0.25 18 0.33 30 0.25 83 CPS, GDH 0.24 13 0.02 7 CPS 0.11 6

GDH

1:l stoichiometry (Table 11, line 1) observed with malate dehydrogenase indicates that essentially all of the bound malate dehydrogenase is in malate dehydrogenase-carbamyl- phosphate 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 synthase I-aminotransferase-(carbamyl-phosphate synthase I ) z complex and the remainder in an aminotransferase-(carbamyl-phosphate synthase I)z complex.

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 and carbamyl-phosphate synthase I bound was essentially the same in either the


presence or absence of malate dehydrogenase (line 2). These experiments were performed under the same conditions as for Table I, where as mentioned above, there was no evidence of a binary malate dehydrogenase-aminotransferase complex. Thus, the only explanation as to why the aminotransferase could enhance binding of malate dehydrogenase in the presence of carbamyl-phosphate synthase I would be that malate dehydrogenase associates with complexes between the aminotransferase and carbamyl-phosphate synthase I. The actual stoichiometry under these conditions (line 3) was 1.6 malate dehydrogenase dimers and 2.6 carbamyl-phosphate synthase I monomers bound per aminotransferase dimer. This stoichiometry was consistent with no binary complexes containing malate dehydrogenase and with 63% of the bound aminotransferase being in the form of malate dehydrogenase-carbamyl- phosphate synthase I-aminotransferase-(carbamyl-phosphate synthase I)*-malate dehydrogenase and the remainder in the form of aminotransferase-(carbamyl-phosphate synthase I)*- malate dehydrogenase.

When carbamyl-phosphate synthase I was incubated with glutamate dehydrogenase alone (Table 11, line 6), only 13% of the carbamyl-phosphate synthase I and 7% of the glutamate dehydrogenase were precipitated uersus 6% of the carbamyl- phosphate synthase I (Table 11, line 7) and 7% of the glutamate dehydrogenase (Table I, line 5 ) when each of these two enzymes was incubated alone. Thus, there is no significant interaction between these two enzymes. Even lesser amounts of a binary complex between these two enzymes would be expected when both malate dehydrogenase and the aminotransferase are present because these enzymes can associate with 83% of the glutamate dehydrogenase alone and 68% of the carbamyl-phosphate synthase I alone (Table I, line 3; same as Table 11, line 5; and Table 11, line 3). However, adding carbamyl-phosphate synthase I to the two dimers plus glutamate dehydrogenase (Table 11, line 4 uersus 5) enhanced binding of the two dimers, and there was more binding of the two high molecular weight enzymes than when they were incubated alone with the two dimers. Since there is no significant interaction between the two high molecular weight enzymes in the absence of the dimers and there should be less glutamate dehydrogenase-carbamyl-phosphate synthase I in the presence of the dimers, these results indicate that a complex can be formed among all four enzymes. The stoichiometry of the precipitate in the presence of all four enzymes was consistent with the formation of 0.3 nmol of carbamyl- phosphate synthase I-malate dehydrogenase-glutamate dehydrogenase-aminotransferase-carbamyl-phosphate synthase I or about the same amount of malate dehydrogenase-glutamate dehydrogenase-aminotransferase as formed (0.25 nmol) (Table 11, line 5) when carbamyl-phosphate synthase I was omitted. The amount of this ternary complex was not decreased by carbamyl-phosphate synthase I apparently because glutamate dehydrogenase has a higher affinity than carbamyl- phosphate synthase I for the dimers. Glutamate dehydrogenase did not decrease binding of carbamyl-phosphate synthase I apparently because carbamyl-phosphate synthase I associated with this ternary complex. In addition, since binding of glutamate dehydrogenase to the dimers does not markedly decrease the amount of free dimers (Table 11, line 5), the stoichiometry of the precipitate in the presence of all four enzymes was also consistent with the formation of the same amount (0.3 nmol) of malate dehydrogenase-carbamyl-phosphate synthase I-aminotransferase-(carbamyl-phosphate synthase Ibrnalate dehydrogenase as formed when glutamate dehydrogenase was omitted.

Incorporation of Fumarase-As shown in Table I11 (lines

TABLE I11 Zncorporation of fumarase into hetero-enzyme complexes

Experimental conditions are described in the legend to Table I. The amounts of enzymes in the 1-ml incubation mixture were as follows: fumarase (FUM), 0.26 nmol; glutamate dehydrogenase (GDH), 0.6 nmol; aspartate aminotransferase (AspAT), 1.1 nmol; malate dehydrogenase (MDH), 1.4 nmol; and a-ketoglutarate dehydrogenase complex (KDHC), 0.037 nmol. Methods for determining the amount of enzyme bound are described under “Materials and Methods.”

Enzyme precipitated

Additions FUM MDH AspAT GDH ~~~~

nmol % nmol % nmol % nmol %

FUM, MDH 0.016 6 0.14 10 FUM, AspAT 0.039 15 0.13 12 FUM, AspAT, MDH 0.039 15 0.14 10 0.13 12 FUM 0.041 16 FUM, GDH 0.010 4 0.12 2 FUM, GDH, MDH, 0.11 43 0.49 35 0.66 60 0.47 78

KDHC, FUM 0.042 16 KDHC, FUM, MDH, 0.13 50 0.24 17 0.28 25

AspAT

AspAT

1-4), there was no significant increase in precipitation of fumarase when incubated with malate dehydrogenase and/or aminotransferase over that found when fumarase was incubated alone. Similarly, precipitation of malate dehydrogenase and the aminotransferase was only slightly higher when these two enzymes were incubated with fumarase than when incubated separately or together in the absence of fumarase (Table 111, lines 1-3; uersus Table I, lines 4, 6, and 7). Thus, fumarase was a high molecular weight enzyme that did not readily support binding of malate dehydrogenase or the aminotransferase. Fumarase also did not associate with glutamate dehydrogenase alone (Table 111, line 5). However, when glutamate dehydrogenase was added to the other three enzymes (Table 111, line 6), malate dehydrogenase and the aminotransferase were again bound with a stoichiometry similar to that found in the absence of fumarase, and fumarase was also bound. Thus, since there was no evidence of binding of fumarase in the presence of dimers and absence of glutamate dehydrogenase or the presence of glutamate dehydrogenase and absence of dimers, fumarase apparently associates with the ternary complex.

When fumarase, aminotransferase, malate dehydrogenase, and glutamate dehydrogenase were incubated with the divalent cross-linker in the absence of polyethylene glycol and chromatographed on a Sephadex G-200 column (Fig. 4), a significant amount of the other three enzymes was eluted in the void volume (fraction 20 as measured with blue dextran) with glutamate dehydrogenase. The decrease in the elution volume of these enzymes was not due to inter- or intramolec- ular cross-linking of the aminotransferase, malate dehydrogenase, or fumarase. We have shown previously that under these experimental conditions, there is essentially no intra- molecular cross-linking of malate dehydrogenase or the aminotransferase alone or intermolecular cross-linking of malate dehydrogenase plus aminotransferase (Ref. 13; see also Ref. 67). Furthermore, when albumin was substituted for glutamate dehydrogenase, the other three enzymes were eluted as single peaks, and only a small amount of the other three enzymes was in the void volume (Fig. 4). These results cannot be utilized to estimate stoichiometry because the cross-linker decreases the specific activity of the enzyme (67). These results also do not prove that the aminotransferase was required for binding of malate dehydrogenase to glutamate


40 t A i 301 A / \ -I

I \ 1

FRACTION FIG. 4. Sephadex G-200 chromatography of mitochondrial

malate dehydrogenase, aspartate aminotransferase, fumarase, and glutamate dehydrogenase. In these experiments, 6.0 mg/ ml glutamate dehydrogenase (GDH) (0) or 6.0 mg/ml bovine serum albumin (0) was incubated with 2.0 mg/ml dimethyl 3,3’-dithiobispropionimidate, 1.0 mg/ml aspartate aminotransferase (Asp-AT), 1.0 mg/ml malate dehydrogenase ( M D H ) , and 1.0 mg/ml fumarase (FUM) for 2 h and chromatographed on a column of Sephadex G- 200 (2.5 X 27 cm). The void volume was determined by finding the blue dextran peak (fraction 20) at 625 nm as described previously (13, 67). Enzyme activities are in units of change in absorbance/ minute/milliliter of fraction from the column. Assay conditions are described under “Materials and Methods.” The elutions of glutamate dehydrogenase, fumarase, malate dehydrogenase, and aspartate aminotransferase are shown in the top, second, third, and bottom panels, respectively. The incubation was performed in 0.25 M sodium arsenate, 0.1 mM EDTA (pH 8.0) at 25 “C; and the column was equili- brated and eluted with the same buffer. The volume of fractions from the column was 1.9 ml.

dehydrogenase. At the high levels of enzymes used in these experiments (6.0 mg/ml glutamate dehydrogenase and 1.0 mg/ ml malate dehydrogenase), malate dehydrogenase is cross- linked to glutamate dehydrogenase in the absence of the aminotransferase (13). These results, however, are consistent with those obtained in polyethylene glycol in that they demonstrate that in the presence (unlike in the absence) of glutamate dehydrogenase, the three enzymes are sufficiently close to one another to permit cfoss-linking by a cross-linker with a bridge length of only 12 A (70).

Although the a-ketoglutarate dehydrogenase complex also did not associate with fumarase alone (Table 111, line 7), it could substitute for glutamate dehydrogenase in promoting interaction between the dimers and fumarase (line 8).

Effect of Ligands-We have previously shown that physiological levels of citrate enhance dissociation of the aminotransferase from glutamate dehydrogenase (71). As shown in Table IV (line 3), 1.0 mM levels of citrate decreased (but did not completely eliminate) binding of the aminotransferase, but decreased binding of fumarase and malate dehydrogenase essentially to the level observed in the absence of glutamate dehydrogenase (Table 111, line 3). These effects of citrate were specific in that adding a-ketoglutarate at 1.0 mM levels only slightly (1.1-1.3-fold) decreased the amount of each enzyme bound (Table 111, line 4), and adding fumarate at even 10 mM levels (Table IV, line 2) produced only a 1.2-1.4-fold decrease in binding of the enzymes to glutamate dehydrogenase. How-

TABLE IV 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 Meth- ods.”

Enzyme precipitated

Additions FUM MDH AspAT GDH ~ ~ _ _ _ _ ~ nmol % nmol % nmol % nmol %

None 0.13 50 0.77 55 0.90 82 0.82 68 Fumarate, 10 mM 0.09 36 0.62 44 0.61 56 0.84 70 Citrate, 1.0 mM 0.05 19 0.20 14 0.58 53 0.42 35 a-Ketoglutarate, 1.0 mM 0.11 41 0.70 50 0.78 71 0.61 51

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 bound in excess over malate dehydrogenase. The stoichiometry in the presence of added fumarate was consistent with equal (26% or 0.22 nmol) amounts of the bound glutamate dehydrogenase being in glutamate dehydrogenase-malate dehydrogenase and glutamate dehydrogenase-aminotransferase and the remainder or 0.39 nmol in a malate dehydrogenase- glutamate dehydrogenase-aminotransferase complex. This contrasts with a stoichiometry consistent with 94% or 0.77 nmol of the bound glutamate dehydrogenase being in a malate dehydrogenase-glutamate dehydrogenase-aminotransferase complex and the remainder or 0.05 nmol in a glutamate dehydrogenase-(aminotransferase)2 complex in the absence of fumarate (Table IV, line 1). Thus, high levels of fumarate and/or malate apparently decreased (but did not abolish) the ternary complex and equalized the affinity of both dimers for glutamate dehydrogenase.

Levels of Enzymes in Mitochondria-When Method I (see “Materials and Methods”) was employed, we recovered, based upon the ornithine transcarbamylase assay, 43 mg of mitochondrial protein/g of liver, wet weight, or 148 mg of mitochondrial protein/g of homogenate protein with a 66% yield of mitochondria. This gives a value of 65 mg of mitochondrial protein/g of liver or 224 mg of mitochondrial protein/g of homogenate protein. The yield was higher (81%) when it was based on the citrate synthase assay. However, -20-fold more protein was required for the citrate synthase assay compared with the ornithine transcarbamylase assay; and in the ornithine transcarbamylase assay, the control (no ornithine) rate was negligible.

When mitochondria and mitochondrial extracts were prepared with Method I, comparable (fl.2-fold) enzyme levels were found when the mitochondria were immediately extracted or were stored at -80 “C, thawed, and extracted. Comparable levels were also found for the indicated enzymes when Method I1 and the additional extraction procedures described under “Materials and Methods” were employed.

A major purpose of this paper was to determine if the apparent stoichiometries and dissociation constants of the hetero-enzyme complexes were compatible with their formation in liver mitochondria. Table V shows the levels of enzymes that can play a role in glutamate-malate metabolism calculated on the basis of the level of their activities in liver mitochondrial extracts as described under “Materials and Methods.”

As shown in Table V, the concentrations of aspartate

Glutamate-Malate Metabolism in Liver Mitochondria

TABLE V

10419

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 Specific activity Mitochondrial conc Enzyme Method

pH T Liver Pure enzyme mdml PM Substrate

“C pmol productlminlmg protein

MDH“ I1 7.8 25 Oxalacetate 7 700 10 140 I1 7.8 25 Malate 0.79 80 10 140 I 7.6 30 Oxalacetate 12.2 1200 10 140

AspAT I1 7.8 25 Aspartate 2.1 180 11.6 130 I1 7.8 25 Glutamate* 3.1 265 11.6 130 I 7.6 30 Aspartate 3.3 250 13.2 146

NADPIDH I 7.5 30 NADP 0.16 30 5.3 88 GDH I1 7.8 25 NADH 1.3 50 26 76

I 7.0 30 Leucine/NADH 1.7 70 24 71 OTC I 7.0 30 Ornithine 3.4 885 3.8 34 cs I1 7.8 25 Oxalacetate 0.41 165 2.5 25

I 8.1 30 Oxalacetate 0.31 124 2.5 25 PC I 7.5 30 Pyruvate 0.30 34 9 18 Fumarase I1 7.8 25 Malate 0.22 140 1.6 8

I1 7.8 25 Fumarate 0.12 74 1.6 8 AlaAT I 7.8 30 Alanine 0.16 213 0.70 8.6 NAD:IDH I 7.2 30 NAD 0.05 43 1.1 4 KDHC I 7.5 30 NAD 0.13 25 5.2 2 PDHC I 7.5 30 NAD 0.041 20 2.0 0.3

7.8 25 NADb 0.09 3.5

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.

aminotransferase, glutamate dehydrogenase, and a-ketoglutarate dehydrogenase complexes are 140, 70, and 2 pM, respectively. According to the data of others, the concentration of carbamyl-phosphate synthase I monomer would be 400 p~ (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 are considerably 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-ketoglutarate dehydrogenase 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 1.1 p ~ , 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-phosphate synthase 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 synthase also 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 a- ketoglutarate 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 and NAD:isocitrate dehydrogenase would not be expected to markedly displace the dimers from the a- ketoglutarate 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 that of citrate synthase (25 p ~ ) (Table V); and the levels of succinate thiokinase (60 p ~ ) (calculated from Ref. 37), citrate synthase, and NAD: isocitrate dehydrogenase (4 p ~ ) (Table V) are considerably lower than those of the dimers (140 p ~ ) (Table V). Further- 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


malate dehydrogenase, aminotransferase, citrate synthase, 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 can also 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 known hetero-enzyme interactions with the 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 comparatively lower levels of ornithine transcarbamylase (34 p ~ ) (Table V) and alanine aminotransferase (4 p ~ ) (Table V). Although NADP:isocitrate dehydrogenase is present a t high levels (88 p ~ ) (Table V) in liver mitochondria, it does not interact with the enzymes of the hetero-enzyme system in vitro (11,73,76) and apparently also does not channel a-ketoglutarate into the a-ketoglutarate dehydrogenase complex in intact mitochondria (77, 78).

DISCUSSION

According to previous results (11,12), the aminotransferase is not competitive with malate dehydrogenase for the a - ketoglutarate dehydrogenase complex, but can enhance binding of malate dehydrogenase. According to our results, similar types of interactions take place between these two dimers and carbamyl-phosphate synthase I and glutamate dehydrogenase. As discussed in the Introduction, malate dehydrogenase may be capable of supplying the aminotransferase with oxalacetate in mitochondria only in structures of this type, which would place these two enzymes in close proximity to one another.

I t is known that binding of the aminotransferase to glutamate dehydrogenase alters the fluorescence of a fluorescent probe on glutamate dehydrogenase (79) and results in an increase in the degree of polarization of a covalently attached group on the aminotransferase (80, 81). Consequently, the aminotransferase could facilitate binding of malate dehydrogenase in the presence of glutamate dehydrogenase because the conformational change induced in glutamate dehydrogen-

TABLE VI Exclusion of succinate thiokinase and citrate synthase from hetero-

enzyme interactions These experiments were performed as described in the legend to

Table I. With the exception of fumarase and carbamyl-phosphate synthase I (present at 0.05 and 0.3 mg, respectively), the amount of each enzyme in the 1-ml incubation mixture was 0.1 mg. In terms of nanomoles, the amounts of enzymes added were as follows: aspartate aminotransferase (AspAT), 1.1 nmol; malate dehydrogenase (MDH), 1.4 nmol; citrate synthase (CS), 1.0 nmol; fumarase (FUM), 0.26 nmol; succinate thiokinase (STK), 1.1 nmol; and, where indicated, glutamate dehydrogenase (GDH), 0.3 nmol; a-ketoglutarate dehydrogenase complex (KDHC), 0.037 nmol; and carbamyl-phosphate synthase I (CPS), 1.9 nmol.

Additions Enzyme precipitated

FUM MDH AspAT GDH CS STK CPS %

None 15 9 12 10 8 KDHC 50 20 30 15 8 GDH, KDHC 46 45 59 88 16 8 CPS, GDH 40 70 54 100 10 8 90

ase enhances its affinity for malate dehydrogenase. Alterna- tively, the restriction in the rotational mobility of the bound aminotransferase may enhance the affinity of the aminotransferase for malate dehydrogenase. It is known that malate dehydrogenase associates with immobilized fumarase, the aminotransferase associates with the malate dehydrogenase moiety of this complex, and the aminotransferase is 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-ketoglutarate dehydrogenase complex, binding of malate dehydrogenase and the aminotransferase to malate dehydrogenase or aminotransferase al- ready bound to the a-ketoglutarate dehydrogenase complex may, in part, account for the large number of dimer-binding sites found in this ternary complex.

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 and a significant fraction of the malate dehydrogenase being associated with these high molecular weight enzymes. Furthermore, higher degrees of organization of the hetero-enzyme system are possible because malate dehydrogenase-glutamate dehydrogenase-aminotransferase can associate with the other two high molecular weight enzymes.

In previous assays of the coupled malate dehydrogenase- aminotransferase reaction performed in the presence of NAD, malate, and glutamate (ll), levels of the a-ketoglutarate dehydrogenase complex as low as 0.003 p~ markedly decreased the K, of malate in the malate dehydrogenase reaction. According to the binding experiments described in this paper, the KD of malate dehydrogenase for aminotransferase- a-ketoglutarate dehydrogenase complex is -1 p ~ , and there are about 10 malate dehydrogenase-binding sites/a-ketoglutarate dehydrogenase complex. This would indicate that the level of ternary complex would be quite low in the previous assays. However, the KD of malate dehydrogenase for pyri- doxal-P-aminotransferase-a-ketoglutarate dehydrogenase complex is measured in direct binding experiments. This could be higher than the KO of NADH-malate dehydrogenase-oxalacetate for pyridoxamine-P-aminotransferase-a-ketoglutarate dehydrogenase complex that was estimated in previous 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 assays would prevent malate dehydrogenase from dissociating into monomers (82). The polyethylene glycol used in the direct binding experiments could result in a KO slightly higher than the actual value because in the case of the citrate synthase- malate dehydrogenase complex, polyethylene glycol failed to precipitate a small fraction of extremely high molecular weight complex (83). However, the KO of citrate synthase- pyruvate dehydrogenase complex was the same in either the presence or absence of polyethylene glycol (66).

Possible Role of Inner Mitochondrial Membrane-In liver mitochondria, the aminotransferase may be localized in the vicinity of the inner mitochondrial membrane because when aspartate is generated by the aminotransferase, it is trans- ported into the cytosol without mixing with the total pool of aspartate in the matrix (84, 85). Furthermore, the liver inner mitochondrial membrane has a specific aspartate aminotransferase-binding site (4). The a-ketoglutarate dehydrogenase complex can also associate with the membrane and Complex I (17, 18). In both cases, the number of enzyme-binding sites


on the membrane is sufficiently high and the dissociation constant sufficiently low so that a high fraction of the total aminotransferase and a-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 the hetero-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 would be quite slow in the viscous mitochondrial matrix (6), and not all of the hetero- enzymes 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-Ma- late dehydrogenase would be less rigorously localized than the aminotransferase in the membrane-hetero-enzyme system described above. As mentioned in the 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 the previously 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-pyruvate dehydrogenase 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 extracts were 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

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), and aspartate aminotransferase (data not 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 citrate synthase and ornithine transcarbamylase) 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-fold higher 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 the values 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 counter- parts. 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.

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