of vol. 263, no. 5, pp. 10687-10697,1988 by the and ... · the journal of biological chemistrv 0...

THE JOURNAL OF BIOLOGICAL CHEMISTRV 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 263, No. 22, Issue of Auguat 5, pp. 10687-10697,1988 Printed in U. S.A.

Regulation of Malate Dehydrogenase Activity by Glutamate, Citrate, &-Ketoglutarate, and Multienzyme Interaction*

(Received for publication, July 7, 1987)

Leonard A. Fahien$, Edward H. Kmiotek, Michael J. MacDonaldg, Barbara Fibich, and Milka Mandic From the Departments of Pharmacology and $Pediatrics, University of Wisconsin Medical School, Madison, Wisconsin 53706

Binding experiments indicate that mitochondrial aspartate aminotransferase can associate with the a-ketoglutarate dehydrogenase complex and that mitochondrial malate dehydrogenase can associate with this binary complex to form a ternary complex. Formation of this ternary complex enables low levels of the a- ketoglutarate dehydrogenase complex, in the presence of the aminotransferase, to reverse inhibition of malate oxidation by glutamate. Thus, glutamate can react with the aminotransferase in this complex without glutamate inhibiting production of oxalacetate by the malate dehydrogenase in the complex. The conversion of glutamate to a-ketoglutarate could also be facilitated because in the trienzyme complex, oxalacetate might be directly transferred from malate dehydrogenase to the aminotransferase. In addition, association of malate dehydrogenase with these other two enzymes enhances malate dehydrogenase activity due to a marked decrease in the K, of malate.

The potential ability of the aminotransferase to transfer directly a-ketoglutarate to the a-ketoglutarate dehydrogenase complex in this multienzyme system plus the ability of succinyl-CoA, a product of this transfer, to inhibit citrate synthase could play a role in preventing a-ketoglutarate and citrate from accumulating in high levels. This would maintain the catalytic activity of the multienzyme system because a-ketoglutarate and citrate allosterically inhibit malate dehydrogenase and dissociate this enzyme from the multienzyme system. In addition, citrate also competitively inhibits fumarase. Consequently, when the levels of a- ketoglutarate and citrate are high and the multienzyme system is not required to convert glutamate to a-ketoglutarate, it is inactive. However, control by citrate would be expected to be absent in rapidly dividing tumors which characteristically have low mitochondrial levels of citrate.

Recent experiments (1) demonstrate the key role of malate metabolism in mitochondrial respiration and the role of malate as a mediator of hormone-induced enhancement of mitochondrial respiration. Conversion of malate to oxalacetate by

* This work was supported by National Institutes of Health Grants CA40445 and AM28348 and by a grant to the University of Wisconsin Medical School from the National Institutes of Health Division of Research Facilities and Resources. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accord- ance 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.

malate dehydrogenase (( S)-malate:NAD+ oxidoreductase, EC 1.1.1.37) is essential for transamination of glutamate by aspartate aminotransferase (L-aspartate:oxoglutarate aminotransferase, EC 2.6.1.1). These combined reactions are the mitochondrial part of the malate aspartate shuttle and are of importance in gluconeogenesis and ureogenesis (1,2) and for the release of insulin by pancreatic islets (3-4). In addition, since the mitochondria of rapidly dividing tumors have a low level of citrate (5), glutamate is the main metabolic fuel in these tumors because transamination of glutamate is required to provide the a-ketoglutarate dehydrogenase complex with substrate (6-8).

Aspartate aminotransferase and citrate synthase compete for oxalacetate generated by malate dehydrogenase. In order for either the aminotransferase or citrate synthase to receive oxalacetate from malate dehydrogenase, they apparently must be in close proximity to malate dehydrogenase (9, 10). In the case of malate dehydrogenase and citrate synthase, this can be accomplished because these two enzymes can form a complex (9) and also because both of these enzymes associate with the inner mitochondrial membrane (11, 12). However, we and others have found that malate dehydrogenase does not associate with the aminotransferase (13, 14) and that the aminotransferase markedly decreases binding of malate dehydrogenase to the membrane (15).

In this paper, we study kinetic consequences resulting from the binding of malate dehydrogenase to multienzyme struc- tures. In addition, we have investigated the effects of citrate and other potential modifiers of malate dehydrogenase on both of these multienzyme interactions and malate dehydrogenase activity. We have also studied the effects of citrate and other modifiers on fumarase, citrate synthase, and aspartate aminotransferase, which either provide malate to or receive oxalacetate from malate dehydrogenase.

MATERIALS AND METHODS

Enzyme and Reagents-Bovine and/or rat liver mitochondrial aspartate aminotransferase, malate dehydrogenase, citrate synthase, and the bovine heart cy-ketoglutarate and pyruvate dehydrogenase complexes were prepared by previously described methods (16-24). Mitochondrial aspartate aminotransferase and malate dehydrogenase were removed from preparations of liver citrate synthase by chro- matographing on DEAE-Sephadex as described previously for pre- paring mitochondrial fumarase (19). The eluted citrate synthase was then further purified as described previously (20). The bovine heart a-ketoglutarate and pyruvate dehydrogenase complexes were obtained from Sigma. Although a high molecular weight band beneath the decarboxylase (M, - 100,000) was found on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the commercial preparation of the cy-ketoglutarate dehydrogenase complex, there were no marked differences found between our preparation and commercial preparations in the kinetic and polyethylene glycol experiments described. Pig heart citrate synthase, mitochondrial malate dehydrogenase, and cytosolic aspartate aminotransferase were obtained from

10687

10688 Regulation of Malate Dehydrogenase Activity Boehringer Mannheim. Polyethylene glycol 6000 was obtained from Fischer and prepared as a 40% (w/v) solution. Other enzymes, coen- zymes, substrates, and reagents were obtained from Sigma. Stock solutions of all reagents used in assays were adjusted to the pH of the assays.

Before use in these experiments, the a-ketoglutarate and pyruvate dehydrogenase complexes were dialyzed extensively uersus 0.02 M potassium phosphate, 0.1 mM EDTA, 1 mM dithioerythritol, pH 7.0, at 4 "C. The enzymes were then precipitated by adding polyethylene glycol (14%, w/v). The precipitated enzyme was solubilized and dialyzed extensively uersus 1 mM dithioerythritol and 0.1 mM EDTA plus the Tris or phosphete buffer used in the specific experiment. The enzymes were then chromatographed on a Pierce Extracti-Gel Affinity Pak column equilibrated with buffer. The results described in this paper were similar if either Lubrol or Triton was used to solubilize the dehydrogenase complex from mitochondria.

Concentration of Enzymes-The concentrations of pure aspartate aminotransferase, malate dehydrogenase, citrate synthase, and succinate thiokinase were determined by measuring the absorbance at 280 nm and using previously measured values of A%'", (22-25). The concentrations of the a-ketoglutarate and pyruvate dehydrogenase complexes were determined with previously described methods using bovine serum albumin as a standard (26). The concentration of other enzymes was determined by measuring the absorbance at 280 nm and arbitrarily using a value of A%'nm = 1.0.

Enzyme Assays-Aspartate aminotransferase (16, 17, 22), succinate thiokinase (25, 27), malate dehydrogenase (28), citrate synthase (24), and the a-ketoglutarate and pyruvate dehydrogenase complexes (29, 30) were routinely assayed using principles described previously. Malate oxidation by mitochondrial malate dehydrogenase was assayed by measuring the rate of DPNH production at 340 nm in the presence of DPN (1.0 mM), malate (1.0 mM), and either 100 p M acetyl-coA plus an excess of citrate synthase or 10 mM glutamate plus an excess of aspartate aminotransferase.

Coprecipitatwn of Enzymes in Polyethylene Glycol-This was done essentially as described previously (9,15). Solutions of enzymes plus ligands were incubated in 1 ml of 14% (w/v) polyethylene glycol plus either 14 mM potassium phosphate, 0.1 mM EDTA, pH 7.0, or 14 mM Tris chloride, 0.1 mM EDTA, pH 7.0, at 25 "C. During the incubation, the turbidities of the solutions were read at 510 nm in an Aminco- Bowman spectrofluorometer. After 20-min incubations, the solutions were centrifuged at 25 "C for 10 min at 20,000 x g in a Sorvall RC 2B centrifuge. The supernatant solutions were then removed, and the pellets were resuspended in 1 ml of 0.02 M potassium phosphate, 0.1 mM EDTA, pH 7.0. The amount of protein and enzyme activity in the original samples, the supernatant solutions, and the dissolved pellets was determined with the standard assays of enzyme activity. In assays of enzyme activity, the supernatant and the solubilized precipitated fraction were diluted in the phosphate buffer, and 1% polyethylene glycol was added to the assays so that the level of polyethylene glycol would be the same in assays of the supernatant and the precipitated fraction. With the exception of experiments performed in Tris buffer with the a-ketoglutarate dehydrogenase complex alone, the sum of total enzyme units in the supernatant plus the total units in the precipitate essentially equaled the total units added. Thus, the amount of enzyme in the precipitate could be calculated by multiplying the fraction of enzyme units in the precipitate by the total amount of enzyme units incubated. Previous investigators studied multienzyme interactions with these dehydrogenase complexes in phosphate buffer in the presence of high levels of enzymes (29, 30). A disadvantage of this is that under these conditions, the dehydrogenase complexes are quite insoluble, even in the absence of a second enzyme. This was also found to be the case in experiments performed with lower (0.2 mg/ml) levels of enzyme in phosphate buffer. However, in Tris buffer plus 0.5 mM Mg2' and 0.1 mM EDTA, it was found, on the basis of assays of protein concentration, that in 14% polyethylene glycol, only about 10-15% of either dehydrogenase complex precipitated when incubated alone. However, a disadvantage of these conditions was that the a-ketoglutarate dehydrogenase complex, when incubated alone, was not stable (64% recovery of total enzyme activity with 15% of total activity in the precipitate). This problem could be overcome by incubating the a- ketoglutarate dehydrogenase complex with a second enzyme which, according to experiments performed in phosphate buffer, could associate with this complex. This resulted in coprecipitation of the dehydrogenase complex with the second enzyme and protection of the a-ketoglutarate dehydrogenase complex. In the absence of the dehydrogenase complex, the second enzyme did not precipitate. In addi-

tion, although the pyruvate dehydrogenase complex did not coprecipitate with the a-ketoglutarate dehydrogenase complex, it protected the a-ketoglutarate dehydrogenase complex. Therefore, experiments were also performed with the a-ketoglutarate dehydrogenase complex in Tris buffer in the presence of an excess of the pyruvate dehydrogenase complex. In these experiments, both dehydrogenase complexes were stable and did not precipitate unless a specific second complex- ing enzyme was added. The addition of 1 mM dithioerythritol to these incubations did not alter enzyme-enzyme interactions or enzyme stability.

Enzyme Cosedimentation-Enzymes were incubated for 30 min at 4 "C and then centrifuged at 4 "C for 120 min at 150,000 X g. The amount of enzyme activity in the noncentrifuged sample, the supernatant solution, and the solubilized pellet was then determined as described above for experiments performed in polyethylene glycol.

Enzyme Turbidity-These experiments were performed in an Aminco-Bowman spectrofluorometer equipped with an E polarizer. The instrument was adjusted prior to each experiment so that a 0.5 mg/ml solution of blue dextran would read 195.

Preparation of Mitochondrial Extracts-Mitochondrial extracts from pancreatic islets and Morris 7777 hepatomas were prepared as described previously (4).

Analysis of Kinetic Results-Inhibition of enzymes by ligands was evaluated with Equation 1:

where u is the initial velocity; V is the maximal velocity; K, is the Michaelis constant of the varied substrate; [SI and [I] are the concentration of substrate and inhibitor, respectively; Ki is the dissociation constant of the inhibitor from the enzyme; and K,' is the dissociation constant of the inhibitor from the enzyme-substrate complex. Data corresponding to Equation 1 were fitted to Equation 1 using the least-squares method as described previously (31). In the case of competitive inhibition, the value of Ki' is infinity.

RESULTS AND DISCUSSION

Inhibition of Fumarase and Citrate Synthase by Citrate- Citrate is competitive with fumarate in the fumarase reaction (Table I); and in agreement with previous results (32), we found that citrate is also competitive with oxalacetate in the citrate synthase reaction. With the exception of isocitrate, the other metabolites listed in the legend to Fig. 1 produced less than 20% inhibition of these enzymes under the same conditions, where the same concentration of citrate inhibited 40-50%. Isocitrate was about 2-fold less potent as an inhibitor than citrate of citrate synthase and fumarase. As shown in Table I, the Ki of citrate is in the range of the K, of fumarate in the fumarase reaction. Therefore, citrate might be a physiologically significant inhibitor of fumarase. In the case of

TABLE I Inhibition of mitochondrial enzymes by citrate

CoA (200 WM), dipyridyl disulfide (200 WM), citrate synthase (0.05 pg/ Assays of citrate synthase were performed in the presence of acetyl-

ml), and various concentrations of oxalacetate (10-100 /.&). Assays of malate dehydrogenase were performed as described in the legend to Fig. 2, with malate as the varied substrate. Assays of fumarase (0.3 rg/ml) were performed with fumarate (0.1-2 mM) as the varied substrate. Assays of aspartate aminotransferase (0.1 gg/ml) were performed with oxalacetate (10-100 pM) as the varied substrate in the presence of glutamate (10 mM), NHJl (20 mM), TPNH (100 @M), and glutamate dehydrogenase (0.1 mg/ml). Other experimental conditions were as described in the legend to Fig. 1. The kinetic constants Ki and K,' are defined in Equation 1.

Kinetic constant Enzyme K; K,' Km KdKm

~ ~~~

mM

Citrate synthase 0.40 CQ 0.007 57 Malate dehydrogenase 0.84 1.8 0.42 2.0 Fumarase 1.0 m 0.33 3.0 Aspartate aminotransferase High High High

Regulation of Malate Dehydrogenase Activity 10689

0 . 0 4 0 W

0.030

Y

0.020

0.010 1 - 1.0 2 .o

(Inhibitor or Mg2+) mM

FIG. 1. Plot of velocity (change in absorbance at 340 nm/ min) of malate dehydrogenase uersus concentration of inhibitor of M 3 + . These experiments were performed with pig heart mitochondrial malate dehydrogenase (0.08 pg/ml) in the presence of DPN (1.0 mM), malate (1.0 mM), acetyl-coA (100 pM), and citrate synthase (10 pg/ml) in 20 mM potassium phosphate, 0.1 mM EDTA, pH 7.0, at 25 "C. The additions were NaCl (curue A ) , MgClz (curue B ) , pyruvate (curue C ) , fumarate (curve D), aspartate isocitrate, or succinate (curue E ) , glutamate (curue F ) , a-ketoglutarate (curue G ) , and citrate (curve H). Plots of velocity in the presence of either 2 mM a-ketoglutarate or 2 mM citrate as a function of MgC12 concentration are shown in curves I and J, respectively.

citrate synthase, the Ki of citrate is much higher than the K , of oxalacetate. However, in many mitochondria, the level of citrate can be over 2 mM, and the ratio of citrate to oxalacetate is also quite high (33). Consequently, citrate could be a significant inhibitor of citrate synthase. According to Equa- tion 1 and the values shown in Table I, when fumarate and oxalacetate are present in levels equal to their K , in the fumarase and citrate synthase reactions, respectively, 2 mM citrate would produce 50% inhibition of fumarase and 70% inhibition of citrate synthase. Thus, 2 mM citrate could decrease conversion of fumarate to citrate. Furthermore, in assays of citrate synthase with 20 p~ oxalacetate and assays of fumarase with 0.5 mM malate, inhibition by 2.0 mM citrate was not significantly decreased (4.1-fold) by 0.5 mM MgC12. Therefore, since in normal mitochondria the level of citrate can exceed 2.0 mM, whereas the mitochondrial level of free M$+ is 0.5 mM, it is unlikely that M e would play a significant role in decreasing inhibition of these enzymes by citrate (33,34).

It is known that citrate synthase can be bound to the pyruvate dehydrogenase complex (30). Consequently, fumarase was assayed by coupling this reaction with malate dehydrogenase and citrate synthase in either the presence (17 fig/ ml) or absence of the pyruvate or a-ketoglutarate dehydrogenase complex. It was found that these dehydrogenase complexes did not alter inhibition of fumarase by citrate. Fur- thermore these dehydrogenase complexes did not alter inhibition of citrate synthase by citrate.

Inhibition of Malate Dehydrogenase-Because of the unfa- vorable equilibrium and product inhibition, malate oxidation by malate dehydrogenase is difficult to assay over a low, physiological pH range unless a second enzyme is added to react with DPNH or oxalacetate. As shown in Fig. 2 (curues

1

I 1 2 5 5.0

[GLUTAMATE] m M

FIG. 2. Plot of velocity of malate dehydrogenase activity versus glutamate concentration. These experiments were performed with pig heart mitochondrial malate dehydrogenase in the presence of 1.0 mM DPN, 1.0 mM malate, and 0.5 mM M&lz with either 2 pg/ml bovine liver aspartate aminotransferase (curues A and C) or 100 p~ acetyl-coA plus 3 pg/ml pig heart citrate synthase (curues B and D) in the presence (curues A and B ) or absence (curues C and D) of a 16 pg/ml concentration of the a-ketoglutarate dehydrogenase complex. Remaining experimental conditions are given in the legend to Fig. 1.

C and D), this can be accomplished by adding either acetyl- CoA plus citrate synthase or glutamate (2.0 mM or above) plus aspartate aminotransferase. Consequently, in these assays, the rate of DPNH production was linearly related to the concentration of malate dehydrogenase and was not altered by changing the level of citrate synthase or aminotransferase. This indicates that citrate synthase and aminotransferase are in excess. They are in excess because under the conditions of these assays, the kinetic properties of citrate synthase and aminotransferase would enable these enzymes (in the levels used in these assays, 2 pg/ml) to convert even a low (5 pM) steady-state level of oxalacetate into their respective products at an initial rate of between 40 and 80 nmol of product/ml/ min. This would be adequate to maintain oxalacetate at a level considerably lower than the K , or Ki of oxalacetate in the malate dehydrogenase reaction. This would prevent significant reversibility of the malate dehydrogenase reaction and product inhibition of this reaction by oxalacetate. A higher level of citrate synthase or aminotransferase might be required to monitor accurately the malate dehydrogenase reaction if this rate were measured by determining the rate of product formation from either the aminotransferase or citrate synthase in a coupled assay. However, in the assay employed, the product (DPNH) of the malate dehydrogenase reaction was measured, and the role of citrate synthase and aminotransferase was to maintain oxalacetate at a level lower than its kinetic constants in the malate dehydrogenase reaction. Thus, in these assays of malate oxidation, it is not necessary for citrate synthase and the aminotransferase to convert quantitatively and instantaneously oxalacetate into a product.

Although, under these conditions, both the aminotransferase and citrate synthase have a comparable capacity to react with oxalacetate, the rate of malate oxidation is considerably greater in the presence of acetyl-coA plus citrate synthase then it is in the presence of a high (5.0 mM) level of glutamate plus the aminotransferase (Fig. 2, curues C and D ) . This does not result from acetyl-coA activating malate dehydrogenase because increasing the level of acetyl-coA had no effect on the reaction in the presence of citrate synthase, and adding it (200 FM) had no effect on the reaction in the presence of the aminotransferase (data not shown). However,

10690 Regulation of Malate Dehydrogenase Activity

high levels of glutamate inhibit in the presence of both citrate synthase and the aminotransferase. Therefore, glutamate inhibits malate dehydrogenase; and consequently, when the level of glutamate exceeds 1 mM (Fig. 2, curues C and D), the rate of malate oxidation is comparable in both assays. Fur- thermore, in both assays, double-reciprocal plots of velocity uersus malate concentration in the presence of constant levels of glutamate intersect on the ordinate (Fig. 3), and secondary plots of these results in terms of the reciprocal of velocity uersus glutamate concentration (over a range O f 1.0-10 mM

I / [MALATE] mM”

I/[MALATE] mM“ FIG. 3. Double-reciprocal plot of velocity of malate dehy-

drogenase reaction uersuB malate concentration in presence and absence of glutamate and a-ketoglutarate or pyruvate dehydrogenase complex. These experiments were performed with pig heart mitochondrial malate dehydrogenase in the presence of 100 pM acetyl-coA, 1.0 mM DPN, and 0.5 mM MgC12. Upper, results obtained in the presence of bovine liver aspartate aminotransferase (2 pg/ml). The additions were 5.0 mM glutamate (curue A ) , 2.0 mM glutamate (curue B ) , and 1.0 mM glutamate (curue C). The results obtained in the presence of a 16 pg/ml concentration of either the pyruvate or a-ketoglutarate dehydrogenase complex plus 5,2 , and 1.0 mM glutamate are shown in curues D-P, respectively. These results were also plotted as the reciprocal of velocity uersus glutamate concentration (over a range from 1.0 to 5.0 mM) in the presence of constant levels of malate. The intercepts on the ordinate of these secondary plots were then used for double-reciprocal plots of velocity uersus malate concentration in the absence of glutamate and in the absence (curve A ’ ) or presence (curueB’) of a 16 pg/ml concentration of the dehydrogenase complexes. Lower, results obtained when malate dehydrogenase was assayed in the presence of citrate synthase (3 pg/ ml). The additions were 10 mM glutamate (curue A ) , 5 mM glutamate (curue B ) , 2 mM glutamate (curue C), and 1 mM glutamate (curue D). The results obtained in the absence of glutamate and in the absence or presence of a 16 pg/ml concentration of either the a-ketoglutarate or pyruvate dehydrogenase complex or in the presence of glutamate (5 or 10 mM) plus a 16 pg/ml concentration of either dehydrogenase complex are all shown in curue E. Remaining experimental conditions are given in the legend to Fig. 1.

glutamate; data shown) yield an intersection point which is also consistent with glutamate being competitive with malate with a Ki of 1.5 mM. In addition, the intercept on the ordinate of these secondary plots (data not shown) and double-reciprocal plots of velocity uersus malate concentration in the presence of citrate synthase (Fig. 3, lower, curue E ) yield a K, of malate of -0.3 mM. Thus, the K,,, of malate and the Ki of glutamate are essentially the same in both assays.

As shown in Fig. 4, citrate and a-ketoglutarate are both noncompetitive inhibitors of malate oxidation and are considerably more potent inhibitors then the other metabolites tested (Fig. 1). Although these results were obtained in the assay with citrate synthase, citrate was also found to be an inhibitor in the assay with glutamate plus the aminotransferase. Similar experiments were not performed with a-ketoglutarate plus the aminotransferase because a-ketoglutarate, unlike citrate, can inhibit the aminotransferase (15).

Although double-reciprocal plots of velocity uersus malate concentration are linear in these assays over a range of malate concentrations from 0.2 to 4.0 mM (Figs. 3 and 4), higher levels of malate produce substrate activation and abolish inhibition by citrate (data not shown). Thus, these, as the results of previous studies of these interactions performed at pH 8.0 (35), are consistent with binding of malate to both an active and a regulatory site (activator site) on malate dehydrogenase and competition between malate and citrate at the regulatory site, However, a striking difference between our results obtained at pH 7.0 and results obtained at pH 8.0 by

r - r - ” o o ~

I I / V 5 0

-2 5 0 2 .5 5 .O I / [DPN] m M”

-2.5 0 2.5 5 0

I/[MALATE] mM“

FIG. 4. Double-reciprocal plot of velocity versus substrate concentration. Upper, DPN was the varied substrate in the presence of 1.0 mM malate and 2 mM citrate (curue A ) , 2 mM a-ketoglutarate (curue B ) , and absence of inhibitor (curue C). Lower, malate was the varied substrate in the presence of 1.0 mM DPN plus 2 mM citrate (curue A ) , 2 mM a-ketoglutarate (curue B ) , and absence of inhibitor (curve C). Remaining experimental conditions are given in the legend to Fig. 1.


us and others (35) is that at pH 8.0, citrate activates malate oxidation in the presence of low levels of malate. This could be because at pH 8.0, citrate has little effect on the K,,, of malate (over a range of 0.2-4.0 mM) or the K, of DPN (35), whereas at pH 7.0, citrate increases the K,,, of both substrates (Fig. 4). This suggests that at pH 7.0, binding of citrate to the regulatory site decreases binding of both DPN and malate to their active sites. This is apparently related to the marked effects of pH on the conformation of malate dehydrogenase, which results in citrate having a 6-fold higher affinity for malate dehydrogenase at pH 7.0 than at pH 8.0 (35).

We also found differences between the effects of glutamate and a-ketoglutarate at pH 8.0 uersus 7.0. At pH 8.0 and in the presence of low (1.0 mM) levels of malate, glutamate (4.0 mM) produced 1.4-fold activation, and a-ketoglutarate (4.0 mM) was slightly inhibitory. Both of these effects were also reversed by increasing the level of malate to 10 mM (data not shown). Thus, these results indicate that a-ketoglutarate also associates with the regulatory site at pH 8.0; but instead of being converted from an inhibitor to an activator, as citrate, its inhibitory properties are markedly decreased. Glutamate could have a higher affinity for the active than the regulatory site at pH 7.0, but an opposite specificity at pH 8.0. This would explain why glutamate is an activator at pH 8.0 but a competitive inhibitor of malate at pH 7.0.

At pH 8.0, the equilibrium of malate oxidation is more favorable; and thus, we and others (35) assayed this reaction in the absence of citrate synthase and aminotransferase. As mentioned above, these enzymes are required at pH 7.0. Thus, it is possible that at pH 7.0, these modifiers actually alter citrate synthase or aminotransferase instead of malate dehydrogenase. However, this is quite unlikely because as mentioned above, glutamate and a-ketoglutarate, unlike citrate, do not inhibit citrate synthase; citrate does not inhibit the aminotransferase (15)) and glutamate is a substrate and at these levels is not a substrate inhibitor of the aminotransferase. Levels of glutamate considerably higher than 10 mM are required for substrate inhibition of the aminotransferase (16,17). Thus, the specificity of these modifiers makes it quite unlikely that they are inhibiting citrate synthase or the aminotransferase instead of malate dehydrogenase. For example, if they were inhibiting citrate synthase and the aminotransferase, it is quite unlikely that in the presence of citrate synthase, a-ketoglutarate and citrate would have similar effects on malate oxidation (Figs. 1 and 4). Also, citrate would not inhibit malate oxidation in the presence of the aminotransferase; high levels of malate would not reverse the effects of these inhibitors; and the K, of malate and the Ki of glutamate would not be the same in the presence of either citrate synthase or the aminotransferase (Fig. 3). In addition, increasing the level of citrate synthase or aminotransferase did not alter inhibition by these modifiers, which indicates that in these assays, citrate synthase and aminotransferase are not rate-limiting. Again, it would not be expected that these enzymes would be rate-limiting because they are in excess, and the validity of the assay is not dependent upon their ability to convert instantaneously and quantitatively oxalacetate into a measured product.

It is also possible that binding of citrate synthase or the aminotransferase to malate dehydrogenase alters malate dehydrogenase activity. However, we found that these enzymes had no significant effect on malate oxidation at pH 8.0. Furthermore, the aminotransferase does not readily associate with malate dehydrogenase (14); and although citrate synthase does associate with malate dehydrogenase, the level of citrate synthase required for these interactions is over an

order of magnitude higher than that used in these assays (36). Furthermore, at pH 7.0, citrate synthase (data not shown) and the aminotransferase (Fig. 2) had no effect on malate oxidation in the absence of acetyl-coA and glutamate, respectively.

The above results suggest that at pH 7.0, glutamate, citrate, and a-ketoglutarate could be physiologically significant inhibitors of malate dehydrogenase. For example, when NH: is supplied to liver mitochondria so that the reaction sequence is

DPNH + NH4 + a-ketoglutarate + DPN + glutamate

Glutamate + oxalacetate + a-ketoglutarate + aspartate

DPN + malate + DPNH + oxalacetate

Malate + NH, + aspartate

the level of malate is decreased 2-fold so that it is in the range of the K, of malate in the malate dehydrogenase reaction; the level of citrate and a-ketoglutarate is decreased to about 2 mM; and the level of glutamate is increased to 20 mM (33). Under these conditions, the amount of inhibition of malate dehydrogenase produced by citrate, a-ketoglutarate, and glutamate would be 64, 52, and 87%, respectively. Furthermore, the K, of glutamate in the mitochondrial aspartate aminotransferase reaction is also quite high (18 mM) (16) and considerably higher than the Ki of glutamate in the malate dehydrogenase reaction. Therefore, in mitochondria, where these two enzymes are present in comparable levels (16), rather than in these experiments, where aminotransferase is in excess over malate dehydrogenase, a high level of glutamate would be required for optimal coupling of aminotransferase with malate dehydrogenase. This high level of glutamate could inhibit malate dehydrogenase, which would in turn decrease both aspartate aminotransferase and citrate synthase activity.

Although Me alone had no marked effect on malate dehydrogenase (Fig. 1, curue B ) , it slightly decreased inhibition by citrate (curue J ) and slightly enhanced inhibition by a-ketoglutarate (curue I). However, levels of free M$+ higher than those expected to be found in mitochondria (34) are required for these effects.

Effect of Dehydrogenase Complexes-Although citrate synthase forms a complex with the pyruvate dehydrogenase complex (30), we found that 16 pg/ml levels of the pyruvate dehydrogenase complex did not alter malate oxidation or inhibition of this reaction by 2 mM citrate or a-ketoglutarate in the presence of citrate synthase. The a-ketoglutarate dehydrogenase complex also had little effect when added to these assays in either the presence or absence of citrate, and neither of these dehydrogenase complexes altered malate oxidation in the presence of aminotransferase in either the presence or absence of citrate. The a-ketoglutarate dehydrogenase complex did increase the rate of DPNH production when added to the malate dehydrogenase assay in the presence of citrate synthase and 2 mM a-ketoglutarate. However, this resulted from a-ketoglutarate and the a-ketoglutarate dehydrogenase complex reacting with the coenzyme A generated by citrate synthase.

Although these dehydrogenase complexes do not alter malate dehydrogenase activity in either the presence or absence of citrate or a-ketoglutarate, even quite low (0.05 pg/ml) levels of the dehydrogenase complexes markedly increase malate dehydrogenase activity in the presence of glutamate (Fig. 5). This suggests that malate dehydrogenase associates with these dehydrogenase complexes, and this decreases inhibition of malate dehydrogenase by glutamate. Both the a-ketoglutarate

"_""" -----


I I I ] 2 5 5 0 7 5

[ a-Ketoglutamte Dehydrogenase Complex] pg/ml

0.025 I I I 1 I ]

2 5 5 0 7 5 [a-Ketoglutarate Dehydrogenase Compiex] pg/ml

FIG. 5. Plot of malate dehydrogenase activity versus concentration of a-ketoglutarate dehydrogenase complex. In these experiments, mitochondrial malate dehydrogenase was assayed in the presence of 1.0 mM DPN, 1.0 mM malate, and 0.5 mM MgC12 with the indicated concentrations of the a-ketoglutarate dehydrogenase complex. Upper, results obtained in the presence of 10 mM glutamate plus 0.15 pg/ml bovine liver malate dehydrogenase plus 2 pg/ml bovine liver aspartate aminotransferase (curue A ) or 0.19 pg/ml pig heart malate dehydrogenase plus 5 pg/ml pig heart citrate synthase and 100 PM acetyl-coA (curue B ) . Lower, results obtained in the presence of 1.0 mM glutamate, 100 pM acetyl-coA with heart malate dehydrogenase plus either 2 pg/ml liver aspartate aminotransferase (curve A ) or 3 pg/ml heart citrate synthase (curve B ) . Remaining experimental conditions are given in the legend to Fig. 1.

and the pyruvate dehydrogenase complexes are similar in terms of their ability to reverse inhibition by glutamate (Fig. 3, upper, curves D-F; and lower, curve E). However, these dehydrogenase complexes have a different effect on malate dehydrogenase in the presence of citrate synthase than they do in the presence of the aminotransferase. That is, when the level of glutamate is too low (1 mM) to inhibit markedly malate dehydrogenase, the dehydrogenase complexes produce greater activation of malate dehydrogenase in the presence of the aminotransferase than they do in the presence of citrate synthase (Fig. 2, curves A and B; and Fig. 5, lower, curves A and B) . As shown in Fig. 3 (upper, curves A-C versus D-F), this is because in the presence of the aminotransferase, the dehydrogenase complex markedly decreases the K,,, of malate. The dehydrogenase complexes also apparently decrease the K,,, of malate in the presence of aminotransferase and in the absence of glutamate because the intercepts on the ordinate of secondary plots ( l / v versus glutamate) of these results plotted versus the reciprocal of malate concentration are also consistent with a marked decrease in the K,,, of malate (Fig. 3, upper, curves A ’ versus B’). Alternatively, when malate dehydrogenase is assayed in the presence of citrate synthase, the K,,, of malate is not altered by the dehydrogenase complexes in either the presence or absence of glutamate (Fig. 3,

lower, curve E). These results suggest that ternary complexes are formed among malate dehydrogenase, the dehydrogenase complex, and either citrate synthase or aminotransferase; and the aminotransferase-dehydrogenase complex has different effects than the citrate synthase-dehydrogenase complex on malate dehydrogenase activity. Thus, although the K,,, of malate and the Ki of glutamate are the same in the absence of the dehydrogenase complex and in the presence of either the aminotransferase or citrate synthase and, in both cases, the dehydrogenase complex prevents inhibition of malate dehydrogenase by glutamate, the dehydrogenase complex activates malate dehydrogenase in the presence of the aminotransferase by decreasing the K,,, of malate. It is conceivable that these dehydrogenase complexes are not bound to malate dehydrogenase, but are bound to citrate synthase and the aminotransferase, and this decreases inhibition of these enzymes by glutamate. This is possible because pyruvate dehydrogenase can activate citrate synthase (30). However, as mentioned above, glutamate is not a potent inhibitor of these enzymes; and in these assays, these enzymes are in excess. Furthermore, we found that neither dehydrogenase complex had a significant effect on aminotransferase activity.

The effects of the dehydrogenase complexes on malate dehydrogenase do not result from a malate dehydrogenase contaminant. These reactions are dependent upon added malate dehydrogenase. Thiamine pyrophosphate and pyruvate were not present in these assays. In assays coupled with aminotransferase, there is no source of coenzyme A, and in both coupled assays, the a-ketoglutarate level was absent or quite low, unless it was added. Pyruvate was not present in these assays. Therefore, the effects of the dehydrogenase complexes on malate dehydrogenase are not artifacts due to a reaction between DPN and the dehydrogenase complex. The effects of the dehydrogenase complexes are quite specific since quite low (0.05 pg/ml) levels produce marked activation (Fig. 5). Increasing the level of citrate synthase, unlike adding the dehydrogenase complexes, does not alter inhibition by glutamate (data not shown). The effects of the pyruvate and a- ketoglutarate dehydrogenase complexes on both liver and heart malate dehydrogenases are quite similar (Fig. 5). How- ever, if the cytosolic aspartate aminotransferase is substituted for the mitochondrial aminotransferase, then even high (17 pg/ml) levels of the dehydrogenase complexes do not reverse inhibition of malate dehydrogenase by glutamate (data not shown).

Physical Evidence of Enzyme-Enzyme Interaction-Accord- ing to the above results, low (0.05 pg/ml) levels of extensively dialyzed dehydrogenase complexes produce marked activation of malate dehydrogenase in the presence of 0.5 mM M F and 0.1 mM EDTA. This suggest that this activation results from heteroenzyme interaction. Therefore, additional experiments were performed to verify this conclusion. It was previously demonstrated that citrate synthase is bound to the pyruvate dehydrogenase complex (30). As shown in Tables 11-IV, citrate synthase and aspartate aminotransferase associate with both dehydrogenase complexes under a wide variety of conditions. Furthermore, although malate dehydrogenase is not bound markedly (26%) to the a-ketoglutarate dehydrogenase complex alone under the conditions of these assays with low (0.5 mM) but physiological levels of Mg+ (Table VI, adding aminotransferase or citrate synthase to malate dehydrogenase plus the &-ketoglutarate dehydrogenase complexes markedly increases binding of malate dehydrogenase (Table V). Espe- cially in the presence of the aminotransferase, this binding of malate dehydrogenase (100%) cannot be accounted for on the basis of the interaction between malate dehydrogenase and


TABLE I1 Enzyme-enzyme interaction in Tris buffer plus polyethylene glycol In these experiments, enzymes were incubated alone or with either

the a-ketoglutarate or pyruvate dehydrogenase complex in 1 mi of polyethylene glycol, 14 mM Tris chloride, 0.1 mM EDTA, and 0.5 mM MgC12, pH 7.0, at 25 “C. After 20 min, the enzymes were centrifuged, and the precipitate and supernatant were assayed for enzyme activity as described under “Materials and Methods.” In A, the concentrations of the pyruvate and a-ketoglutarate dehydrogenase complexes were 0.12 and 0.1 mg/ml, respectively. The concentration of the second enzyme was 0.1 mg/ml, and the concentration of polyethylene glycol was 14% (w/v). In B, the concentrations of the pyruvate dehydrogenase complex, second enzyme, and polyethylene glycol were 0.6 mg/ ml, 1.2 mg/ml, and 5% (w/v), respectively. Under these conditions, there was complete recovery of a-ketoglutarate dehydrogenase activity only in the presence of citrate synthase and mitochondrial aspartate aminotransferase (see “Materials and Methods”). PDH, pyruvate dehydrogenase complex; Asp-AT, mitochondrial aspartate aminotransferase; CS, citrate synthase; STK, succinate thiokinase; MDH, mitochondrial malate dehydrogenase; IDH, TPN-isocitrate dehydrogenase; C-Asp-AT, cytosolic aspartate aminotransferase; C-MDH, cytosolic malate dehydrogenase; KDH, a-ketoglutarate dehydrogenase complex.

Enzyme precipitated

Second KDH PDH Second enzyme enzyme

Alone +Second Alone +Second enzyme enzyme Alone +KDH +PDH

% A. None 15 10

Asp-AT 78 70 10 cs 90 83 11 STK 50 15 7 MDH 15 IDH

12 14 5 10 9

C-Asp-AT 15 10 2 C-MDH 15 10 4

12 10 PDH

40 55 53 71 25 18 12 14 8 10 2 2 2 4

12

B. None Asp-AT cs

2 95 2 19 2 2 7

TABLE I11 Enzyme-enzyme interaction in phosphate buffer

plus polyethylene glycol In these experiments, enzymes were incubated in 14% polyethylene

glycol, centrifuged, and assayed as described in the legend to Table 11, except that incubations were in 14 mM potassium phosphate, 2 mM MgC12, 0.1 mM EDTA, and the concentration of the dehydrogenase complexes was 0.1 mg/ml. Abbreviations are the same as those defined in the legend to Table 11. Under these conditions, over 80% of the dehydrogenase complex precipitated in the presence or absence of the second enzyme.

Second Precipitation of second enzyme enzyme Alone +KDH +PDH

% Asp-AT 2 71 85 cs 10 50 STK

50 6

MDH 28 14

5 47 85 IDH 9 16 15 C-Asp-AT 2 7 2 C-MDH 5 7 5

aminotransferase alone (12%), citrate synthase alone (35%), or dehydrogenase complex alone (26%). Therefore, these binding results indicate that malate dehydrogenase associates with the complex between either citrate synthase or the aminotransferase and the a-ketoglutarate dehydrogenase complex. Similar results are obtained with a 0.19 mg/ml concentration of the a-ketoglutarate dehydrogenase complex,

with the pyruvate dehydrogenase complex alone (data not shown), or with a 0.09 mg/ml concentration of the a-ketoglutarate dehydrogenase complex plus a 0.12 mg/ml concentration of the pyruvate dehydrogenase complex (Table V). In all cases, adding either aminotransferases or citrate synthase to malate dehydrogenase plus dehydrogenase complex enhances binding of malate dehydrogenase. Furthermore, in experiments performed with the mixture of the two dehydrogenase complexes, binding of the two dehydrogenase complexes is essentially the same. This indicates that the two dehydrogenase complexes have comparable affinities for these enzymes and agrees with the kinetic results described above, which demonstrate that both dehydrogenase complexes are comparable with respect to their abilities to reverse inhibition of malate dehydrogenase by glutamate. In addition, cytosolic aspartate aminotransferase is not bound to either dehydrogenase complex (Table 11), which is consistent with the fact that in the presence of the cytosolic enzyme, these dehydrogenase complexes do not reverse inhibition by glutamate.

As shown in Table VI, 2 mM citrate or 2 mM a-ketoglutarate markedly decreases binding of malate dehydrogenase and citrate synthase to the dehydrogenase complexes. However, 10 mM glutamate has considerably less of an effect. If aspartate aminotransferase is also added, citrate, but not glutamate, again essentially eliminates binding of malate dehydrogenase and citrate synthase, but does not eliminate binding of aspartate aminotransferase to the dehydrogenase complex. Thus, these results indicate that the dehydrogenase complexes reverse inhibition of malate dehydrogenase by glutamate, but not by citrate and a-ketoglutarate, because citrate and a- ketoglutarate dissociate malate dehydrogenase from these multienzyme complexes. The failure of high levels of glutamate and NaCl (Table VI) to dissociate these complexes indicates that citrate and a-ketoglutarate do not dissociate these complexes as a result of increasing ionic strength. These effects are also not the result of chelation of MgZ+ by citrate because these experiments were performed in the presence of EDTA, and citrate has a similar action in the absence of M F (Table VI). It has been previously shown that low (0.1 mM) levels of a-ketoglutarate dissociate the citrate synthase-malate dehydrogenase complex, but low levels of citrate did not have this effect (12). However, our results demonstrate that higher (2 mM) but physiological levels of citrate (33) decrease association of malate dehydrogenase and citrate synthase with these dehydrogenase complexes and apparently also with each other.

It seems probable that citrate and a-ketoglutarate prevent association of malate dehydrogenase with these dehydrogenase complexes because at pH 7.0, they are bound to an allosteric site on malate dehydrogenase, and this site is part of the binding site which associates with other enzymes. Alternatively, glutamate, which is a competitive inhibitor, might not dissociate malate dehydrogenase from the multienzyme system because it is bound to the active site.

The concentration of malate dehydrogenase used in these binding experiments is several orders of magnitude higher than that used in assays. Malate dehydrogenase can undergo a concentration-dependent dissociation (35). Therefore, although citrate is bound to high levels of malate dehydrogenase at pH 7.0 (35), it is possible that a-ketoglutarate is not bound, but dissociates malate dehydrogenase from the multienzyme system via some other mechanism. However, in fluorescence experiments similar to those performed previously (35) but under the conditions of these assays (pH 7.0), it was found that both 2 mM citrate and 2 mM a-ketoglutarate markedly decreased binding of TPNH to malate dehydrogenase (1 mg/

10694 Regulation of Malate Dehydrogenase Activity TABLE IV

Sedimentation of enzymes with dehydrogenase complexes In these experiments, enzymes were incubated either alone or with a dehydrogenase complex at 4°C for 30 min

and then centrifuged, and the pellet and supernatant were assayed as described under “Materials and Methods.” Conditions A were 0.2 mg/ml of each enzyme with or without a 0.7 mg/ml concentration of the a-ketoglutarate dehydrogenase complex in 20 mM potassium phosphate, 0.1 mM EDTA, 1 mM dithioerythritol, and 2 mM MgC12, pH 7.0. Conditions B were 1.2 mg of the second enzyme and a 0.6 mg/ml concentration of the pyruvate dehydrogenase complex in 20 mM Tris chloride, 0.5 mM MgC12, and 0.2 mM EDTA, pH 7.0. In all of these experiments, over 90% of the dehydrogenase complex precipitated with and without the second enzyme, and there was no loss in the activity of the second enzyme. See legend to Table I1 for definitions of enzyme abbreviations; DH, dehydrogenase complex.

Enzyme sedirnented

Enzyme(s) added Conditions Asp-AT MDH STK IDH cs +DH -DH +DH -DH +DH -DH +DH -DH +DH -DH

% MDH, Asp-AT, STK A 28 1 23 1 13 1 IDH A 2 1 C-MDH A 2 1 Asp-AT B 16 1 cs B 7 1

B C-MDH 2 1

TABLE V Binding of mitochondrial mnlnte dehydrogenase

In these experiments, 0.1 mg/ml concentrations of the enzymes were incubated either alone or with a 0.19 mg/ ml concentration of the a-ketoglutarate dehydrogenase complex or a 0.09 mg/ml concentration of the a-ketoglutarate dehydrogenase complex plus a 0.12 mg/ml concentration of the pyruvate dehydrogenase complex in 1 ml of 14% (w/v) polyethylene glycol plus 14 mM Tris chloride, 0.1 mM EDTA, and 0.5 mM MgC12, pH 7.0, at 25 “C for 20 min. The incubations were then centrifuged and assayed as described under “Materials and Methods.” Abbreviations are the same as those defined in the legend to Table 11.

Additions Enzyme precipitated

MDH ASPAT KDH PDH cs STK

MDH MDH, KDH MDH, Asp-AT MDH, KDH, Asp-AT ASP-AT, KDH MDH, KDH, PDH MDH, ASP-AT, KDH, PDH Asp-AT, KDH, PDH CS, MDH CS, KDH CS, KDH, MDH CS. KDH. PDH cs; KDH; PDH, MDH CS, MDH, Asp-AT CS, MDH, Asp-AT, KDH, PDH CS, MDH, Asp-AT, KDH, PDH, STK

14 26 12

100

27 100

35

90

90 57 78 78

20 88 87

91 91

22 100 100

ml), and 2 mM citrate also decreased binding of DPNH. Binding of DPNH could not be studied in the presence of a- ketoglutarate because under these conditions, it is a substrate of malate dehydrogenase.

The ability of citrate to dissociate also citrate synthase from these multienzyme systems could account for our inability to find an effect of these dehydrogenase complexes on inhibition of citrate synthase by citrate.

These heteroenzyme interactions are specific. As shown in Tables 11-V and previous results (9, 11, 14, 15, 30, 37-39), many other proteins are excluded from these interactions. Only glutamate dehydrogenase, carbamyl-phosphate synthase I, and, to a lesser extent, citrate synthase have been found to associate with malate dehydrogenase and the aminotransferase (9, 14, 15, 37, 38). Only malate dehydrogenase, the aminotransferase, and the pyruvate dehydrogenase complex have been found to interact with citrate synthase (9, 30, 38). Only citrate synthase and Complex I have been found to associate with the pyruvate dehydrogenase complex (11, 30).

%

25

75 75 20 25 60 75 69 73

30 82 90 75 70 72 83 100 77 70 60

45 57 68 100 57 68 100 20

Only succinate thiokinase, Complex I, and DPN-isocitrate dehydrogenase have been found to associate with the a- ketoglutarate dehydrogenase complex (11, 30, 39). Further- more, as shown in Table 11, there is no apparent interaction between the two dehydrogenase complexes. As discussed previously, all of these interactions have potential physiological significance in terms of facilitating a direct transfer of ligands from one enzyme to the other (9, 11, 14, 15, 30, 37, 38). Furthermore, although the complexes among the aminotransferase, citrate synthase, and the dehydrogenase complexes enhanced binding of malate dehydrogenase, they did not enhance binding of TPN-isocitrate dehydrogenase, cytosolic malate dehydrogenase, and cytosolic aspartate aminotransferase (data not shown). This is apparently because these enzymes have a low affinity for the dehydrogenase complexes, citrate synthase, and the aminotransferase. Even succinate thiokinase, which is bound to the a-ketoglutarate dehydrogenase complex (Tables 11-IV) (29) but is not bound to these other enzymes (data not shown), is rather excluded in these


TABLE VI Effect of citrate and a-ketoglutarate on enzyme-enzyme interaction

In these experiments, 0.1 mg of the enzymes were incubated with 0.09 mg of the a-ketoglutarate dehydrogenase complex plus 0.12 mg of the pyruvate dehydrogenase complex in 1 ml of 14% (w/v) polyethylene glycol, 14 mM Tris chloride, 0.1 mM EDTA, 0.5 mM MgCl,, pH 7.0, plus 2 mM sodium citrate, 2 mM a-ketoglutarate, 4 mM NaCl, or 10 mM glutamate at 25 'C. After 20 min, the enzymes were centrifuged and assayed as described under "Materials and Methods." Abbreviations are the same as those described in the legend to Table 11.

Enzyme precipitated

MDH Asp-AT KDH PDH cs %

Additions

KDH, PDH, CS, MDH 90 71 70 60 KDH, PDH, CS, MDH, citrate 22 41 34 18

CS, KDH, PDH, MDH, Asp-AT, citrate I 70 85 73 10 CS, KDH, PDH, MDH, Asp-AT, citrate - M$+ 7 70 85 80 12 CS, KDH, PDH, MDH, Asp-AT, NaCl 78 100 51 69 100 KDH, PDH, CS, MDH, a-ketoglutarate 29 55 60 41 KDH, PDH, CS, MDH, glutamate 65 86 93 58

KDH, PDH, MDH, Asp-AT, glutamate 93 86 75 61

CS, KDH, PDH, MDH, Asp-AT 78 100 57 68 100

KDH, PDH, MDH, ASP-AT 100 91 60 75 -

0.020

U 1 1 o.oloo

5 IO

[SUCCINATE THIOKINASE] pg/ml

FIG. 6. Plots of a-ketoglutarate dehydrogenase activity versus concentration of succinate thiokinase. These experiments were performed in the presence (curve A ) or absence (curue B ) of 2 PM GDP. Assay conditions were: 1 mM potassium phosphate, 2 mM a-ketoglutarate, 5 WM coenzyme A, 1 mM DPN, 2 mM MgC12, 200 p M thiamine pyrophosphate, 1 mM dithioerythritol, 50 mM Tris chloride, 0.1 mM EDTA, pH 7.4, at 25 "C. Velocity is in units of change in absorbance at 340 nm/min.

multienzyme systems (Table V). This is probably because it is displaced by the aminotransferase and/or citrate synthase. Thus, under a wide variety of conditions and in both the presence and absence of polyethylene glycol, there is considerably more binding of the aminotransferase than succinate thiokinase to the a-ketoglutarate dehydrogenase complex (Tables I1 and IV). In addition, in the presence of low (0.5 mM) but physiological levels of MP, there is more coprecipitation of the a-ketoglutarate dehydrogenase complex by the aminotransferase than by succinate thiokinase (Table 11). In experiments performed by previous investigators (29), quite high (3 mg/ml) levels of the a-ketoglutarate dehydrogenase complex were required for 25% binding of 0.5 mg/ml succinate thiokinase in the presence of high (1-5 mM) levels of M$+. This indicates that although succinate thiokinase is bound, it does not have a high affinity. Under these conditions, succinate thiokinase increased a-ketoglutarate dehydrogenase activity (29). However, this only occurred in the presence of low ((10 pM) levels of coenzyme A. As shown in Fig. 6, we found that this also requires GDP. Therefore, this activation might, in part, result from the succinate thiokinase reaction regen- erating coenzyme A.

DPN-isocitrate dehydrogenase also associates with the a- ketoglutarate dehydrogenase complex (39). However, the stability of this complex is in the range of that of the complex with succinate thiokinase.

Although we, as well as others (29), find greater binding of succinate thiokinase to the a-ketoglutarate than to the pyru-

vate dehydrogenase complex and significant binding of citrate synthase to the pyruvate dehydrogenase complex, we, unlike others (30), also find significant binding of citrate synthase to the a-ketoglutarate dehydrogenase complex under a wide variety of conditions (Tables I1 and 111). Furthermore, in polyethylene glycol and Tris buffer plus 0.5 mM M$+ and 0.1 mM EDTA, both the a-ketoglutarate and pyruvate dehydrogenase complexes coprecipitate with citrate synthase (Table 11), and both of these dehydrogenase complexes reverse inhibition of citrate synthase-malate dehydrogenase by glutamate in the absence of polyethylene glycol (Figs. 3 and 5). However, when quite high (3 mg/ml) levels of the dehydrogenase complex, citrate synthase (0.5 mg/ml), and M e (1-5 mM) are incubated in the absence of a chelator, there is greater binding (30 versus 10%) of citrate synthase to the pyruvate than to the a-ketoglutarate dehydrogenase complex (30). Since we and others (40) have found that omitting EDTA from our assays can produce a marked increase in a-ketoglutarate dehydrogenase activity in either the presence or absence of M%+, this greater specificity found in the absence of a chelator might be due to Ca2+ or some other divalent cation. Thus, our preparation of the a-ketoglutarate dehydrogenase complex, which was dialyzed uersw 0.1 mM EDTA and incubated in the presence of 0.1 mM EDTA, might be freer of Ca2+ and other divalent cations. There also can apparently be different binding specificity in the presence of high levels of enzyme. Thus, although low (0.1 mg/ml) levels of both citrate synthase and the aminotransferase have an almost equivalent affinity for the dehydrogenase complexes in the presence of 0.5 mM M$+ (Table 11), high (1.0 mg/ml) levels of aminotransferase in the absence of polyethylene glycol associate to a considerably greater extent than high levels of citrate synthase (16 versus 7%; Table IV) with the pyruvate dehydrogenase complex. Also, in the absence of polyethylene glycol, high levels of the aminotransferase produce a considerably greater increase in turbidity than high levels of citrate synthase when added to the dehydrogenase complexes (Table VII). This indicates that when the level of aminotransferase is high, it associates to a greater extent than citrate synthase with the dehydrogenase complexes, perhaps as a result of the aminotransferase inducing polymerization of the dehydrogenase complexes. Enzymes which are not bound to the dehydrogenase complexes under these conditions (0.5 mM M$'), such as cytosolic aspartate aminotransferase and mitochondrial malate dehydrogenase, do not produce an increase in turbidity


TABLE VI1 Turbidity of pyruvate and a-ketoglutarate dehydrogenase complexes

In these experiments, a 1.0 mg/ml concentration of either mitochondrial or cytosolic aspartate aminotransferase, citrate synthase, or malate dehydrogenase was added to a 0.05 mg/ml concentration of each of the dehydrogenase complexes as indicated. Turbidity was measured as described under “Materials and Methods.” Readings were stable for at least 5 min, and the results shown were those obtained after 5 min. These experiments were performed in 14 mM Tris chloride, 0.1 mM EDTA, and 0.5 mM MgCL, pH 7.0, at 25 “C. Abbreviations are the same as those described in the legend to Table 11.

Added Turbidity enzyme -KDH, PDH +KDH, PDH

arbitrary units None 5 181 Asp-AT 60 543 cs 60 310 MDH 50 289 C-Asp-AT 60 235

(Table VII). This is the case even though, under these conditions, mitochondrial malate dehydrogenase and aminotransferase have a comparable molecular weight and charge (16).

Variables in the methods employed to remove Triton from the complexes could conceivably also account for the great binding which we observed of citrate synthase to the a- ketoglutarate dehydrogenase complex. We markedly decreased the level of Triton by extensive dialysis and chromat- ographing over an Extracti-Gel cell column. Furthermore, we obtained similar results if the enzyme was solubilized from mitochondria with either Lubrol or Triton. Previous investigators (29, 30, 39) did not mention how the level of Triton was decreased in their preparations. We are presently making a systematic investigation of these variables, i.e. protein concentration, chelator, divalent cation level, method of solubi- lizing the complexes from the mitochondria, and method of removing Triton or Lubrol from the mitochondria. However, our results do clearly demonstrate that low levels of the rather Triton-free dehydrogenase complexes in the presence of physiological levels of Mg2+ and EDTA can associate with either citrate synthase or the aminotransferase, and binding of malate dehydrogenase to these binary complexes alters the kinetic properties of malate dehydrogenase.

Physiological Significance-According to previous results (1, 4, E), the apparently essential, direct transfer of oxalacetate from malate dehydrogenase to the aminotransferase could not readily take place within a complex between these two enzymes alone or on the mitochondrial membrane. This is because mitochondrial aspartate aminotransferase has a low affinity for mitochondrial malate dehydrogenase (14). Also, although both enzymes have a high affinity for the inner mitochondrial membrane, binding of the aminotransferase to the membrane facilitates dissociation of malate dehydrogenase from the membrane (15). However, our results indicate that aminotransferase can be bound to the a-ketoglutarate dehydrogenase complex, and malate dehydrogenase can have a higher affinity for this binary complex than for either of the two constituents alone. Thus, it is conceivable that this ternary complex could place malate dehydrogenase and the aminotransferase in close proximity, and this could facilitate a direct transfer of oxalacetate. This would not only provide the aminotransferase with substrate, but could also decrease product inhibition of malate dehydrogenase by oxalacetate. This complex could also enable high levels of glutamate to react with the aminotransferase without glutamate inhibiting

malate dehydrogenase. Furthermore, malate dehydrogenase is activated in this trienzyme system because the K,,, of malate is markedly decreased. In addition, in this trienzyme complex, a-ketoglutarate might be directly transferred from the aminotransferase to the a-ketoglutarate dehydrogenase complex. This would provide the dehydrogenase complex with substrate and prevent a-ketoglutarate from accumulating, inhibiting malate dehydrogenase, and dissociating malate dehydrogenase from the multienzyme complex. Dissociation of malate dehydrogenase from the multienzyme complex would further enhance its susceptibility to inhibition by glutamate and oxalacetate. The decreased oxalacetate production plus the inability of malate dehydrogenase to transfer directly oxalacetate to the aminotransferase would markedly decrease aminotransferase activity.

The catalytic activity of the trienzyme complex could also favor aminotransferase over citrate synthase activity by gen- erating succinyl-CoA, which inhibits citrate synthase (32). We found that succinyl-CoA does not inhibit the aminotransferase. Inhibition of citrate synthase or, more specifically, a decrease in the mitochondrial level of citrate would also favor the catalytic activity of the trienzyme system because citrate also inhibits malate dehydrogenase and dissociates it from the multienzyme system. Thus, when the levels of citrate and a- ketoglutarate are high and the a-ketoglutarate dehydrogenase complex can be adequately supplied with free a-ketoglutarate or a direct transfer of a-ketoglutarate from DPN-isocitrate dehydrogenase (39), the trienzyme system would not be required, and it would be dissociated and inhibited.

A complex can also be formed among citrate synthase, malate dehydrogenase, and the a-ketoglutarate dehydrogenase complex. However, the catalytic properties of this ternary complex are favored to a lesser extent than those of the analogous complex with the aminotransferase instead of citrate synthase. This is because in the complex with citrate synthase, the K,,, of malate is not decreased. Furthermore, citrate synthase, unlike the aminotransferase, can be inhibited and dissociated from the multienzyme complex by citrate (Table VI and Ref. 15).

These enzyme-enzyme interactions take place with levels of enzymes considerably lower than the levels found in mitochondria. Indeed, 0.2 pg/ml levels of the dehydrogenase complex can almost completely reverse inhibition of malate dehydrogenase by glutamate. Thus, it seems probable that these interactions take place in uiuo. Furthermore, in view of the high affinity of these enzymes for each other, it seems quite unlikely that these enzymes would be exclusively in the free form in mitochondria.

Direct inhibition by citrate of malate dehydrogenase, citrate synthase, and fumarase and direct inhibition of malate dehydrogenase by a-ketoglutarate could also be of physiological significance. This inhibition was found to be similar with the pure bovine liver, rat liver, and pig heart enzymes and in studies of these enzymes in mitochondrial extracts of pancreatic islets and Morris 7777 hepatomas. Thus, inhibition of this type is a rather common feature of these enzymes. Fur- thermore, recent results (33, 41) obtained with liver mitochondria indicate that malate dehydrogenase is not in equilib- ria, and there is not one well-mixed pool of mitochondrial oxalacetate. Thus, it seems possible that a-ketoglutarate and citrate could decrease malate dehydrogenase, both by their direct inhibitory effects and by dissociating malate dehydrogenase from these multienzyme complexes.

In the mitochondria of rapidly dividing tumors, glutamate- malate metabolism is highly compartmentalized (5,s). It has been proposed that this compartmentalization could, in part,


result from association of malate dehydrogenase with multienzyme complexes (8). This seems quite possible in view of the fact that the mitochondria of these tumors also apparently have a low level of citrate, which should enhance association of malate dehydrogenase with the multienzyme system. This might play a role in the observed preferential reactivity of added malate with malic enzyme uersus malate dehydrogenase in the mitochondria of these tumors (8). Thus, association of malate dehydrogenase with a multienzyme complex of matrix enzymes might decrease the ability of this enzyme to react with malate as it passes through the mitochondrial membrane. Furthermore, less inhibition of fumarase by citrate could enhance the ability of this enzyme to generate fumarate, which is an allosteric activator of the tumor mitochondrial malic enzyme (42). The low level of citrate in these mitochondria should also decrease inhibition of citrate synthase by citrate, and this could play a role in the observed preferential reactivity of oxalacetate generated by malate dehydrogenase with citrate synthase rather than with aspartate aminotransferase. Although the actual level of citrate in tumor mitochondria was not measured in these studies (5), it is apparently quite low. This is because although these mitochondria have ample aconitase and isocitrate dehydrogenase activity, the endogenous level of citrate is so low in these mitochondria that this activity is not detected unless citrate is added and citrate export is blocked.

These interactions could also play a role in the antagonism between glucagon and insulin in pancreatic islets. According to previous results (43,44), glucagon increases the mitochondria ratio of malate to a-ketoglutarate. This would decrease product inhibition of glutamate dehydrogenase by a-ketoglutarate (16); and according to our results, this would also enhance malate dehydrogenase and aspartate aminotransferase activity. An increase in the activity of these reactions can facilitate insulin release (3,4).

There are several ways in which malate dehydrogenase could associate with binary complexes between the aminotransferase and the a-ketoglutarate dehydrogenase complex. For example, malate dehydrogenase could be bound to different sites on the dehydrogenase complex than the aminotransferase; or one of the two polypeptide chains of the aminotransferase could associate with the dehydrogenase complex, and the other polypeptide chain could associate with malate dehydrogenase. This latter type of interaction could be similar to that found previously by Beeckmans and Kanarek (45). These investigators demonstrated that the aminotransferase can be bound to malate dehydrogenase immobilized on Seph- arose. Thus, it is conceivable that malate dehydrogenase could associate with aminotransferase when its mobility is decreased due to its association with the a-ketoglutarate dehydrogenase complex.

Physiological advantages of malate dehydrogenase forming a complex with either the a-ketoglutarate dehydrogenase- citrate synthase complex or the pyruvate dehydrogenase- aminotransferase complex are not readily apparent. However, there might be some mechanism in uiuo which destabilizes these complexes.

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