184, 529-534 (1977)
Interaction between Citrate Synthase and Mitochondrial Malate Dehydrogenase in the Presence of Polyethylene Glycoll LAURA A. HALPER AND PAUL A. SRERE’ Pre-Clinical Science Unit, Veterans Administration The University and Department of Biochemistry, Received
4500 S. Lancaster
of Texas Health Science Center, Dallas,
June 15, 1977; revised
Texas 75216 Texas 75235
Pig heart citrate synthase and mitochondrial malate dehydrogenase interact in polyethylene glycol solutions as indicated by increased solution turbidity. A large percentage of both enzymes sediments when mixtures of the two in polyethylene glycol are centrifuged, whereas little if any of either enzyme sediments in the absence of the other. The observed interaction is highly specific in that neither cytosolic malate dehydrogenase nor nine other proteins showed evidence of specific interaction with either pig heart citrate synthase or mitochondrial malate dehydrogenase. Escherichia coli citrate synthase did not interact with pig heart citrate synthase, but did show evidence of interaction with pig heart mitochondrial malate dehydrogenase. The relation between enzyme behavior in polyethylene glycol solution and in the mitochondrion and the significance of possible in uiuo interactions between citrate synthase and mitochondrial malate dehydrogenase are discussed.
The observed rate of the Krebs cycle is much higher than the rate of the citrate synthase reaction calculated using the apparent intramitochondrial concentrations of acetyl coenzyme A, oxaloacetate, and enzyme. This conclusion is based upon the following data and reasoning. Krebs and Veech (1) have shown that the NAD+/ NADH ratio in rat liver mitochondria is 7 and that the malate concentration is 0.3 mM. Since the malate dehydrogenase (MDH)3 reaction has a K,,, at pH 7.0 of 2 x lo-“, the free oxaloacetate concentration can be calculated to be about 4 x lo-” M. Since the K, for oxaloacetate of rat liver citrate synthase (CS) is about 4 x lo-” M, if no other factors are involved citrate ’ This work was supported by the Veterans Administration and by a grant from the United States Public Health Service. a To whom all correspondence should be sent at the Pre-Clinical Science Unit, Veterans Administration Hospital. 3 Abbreviations used: MDH, malate dehydrogenase; CS, citrate synthase; m-MDH, mitochondrial MDH; PEG, polyethylene glycol; BSA, bovine serum albumin; c-MDH, cytosolic MDH; DTNB, 5,5’-dithiobis(2-nitrobenzoic acid); OD, optical density.
synthase could express about 1% of its maximum activity, a figure much too low to account for observed respiration rates of rat liver mitochondria. To reconcile this apparent discrepancy, a complex of citrate synthase and malate dehydrogenase has been proposed to exist near the inner mitochondrial membrane (2). If an enzyme complex containing CS and MDH does exist, then high concentrations of oxaloacetate might exist in the vicinity of the active site of citrate synthase to account for the observed rate of Krebs cycle activity. Complex formation between MDH and CS in solution has not been reported previously. However, it has been shown (3) that immobilization of pig heart MDH and CS on Sepharose or within polyacrylamide gels resulted in a two- to fourfold increase in the rate of conversion of malate to citrate as compared to the system of free enzymes when malate concentration was limiting. Nonionic polymer media for proteins have been used in an attempt to obtain an environment similar to intracellular conditions; such media may also be more
Copyright 0 1977by Academic Press, Inc. All rights of reproduction in any form reserved.
favorable than water for protein complex formation. Thus, physical interaction between aspartate aminotransferase and malate dehydrogenase from pig heart was recently reported to occur in a biphasic water, dextran, and carboxymethylpolyethylene glycol system (41, whereas no physical evidence of the existence of a complex could be found in buffer systems without added polymers (5). This paper describes apparent specific interactions of pig heart mitochondrial MDH (m-MDH) and CS in polyethylene glycol (PEG) solutions, as detected by changes in the turbidity of protein mixtures and changes in the precipitation behavior of the proteins when mixed together in PEG solutions. MATERIALS
The pig heart enzymes mitochondrial MDH (EC 18.104.22.168), CS (EC 22.214.171.124), NADP-dependent isocitrate dehydrogenase (EC 126.96.36.199), aspartate aminotransferase (glutamate-oxaloacetate transaminase) (EC 188.8.131.52), succinyl-Cob synthase (EC 184.108.40.206), and diaphorase (EC 220.127.116.11) and rabbit muscle aldolase (EC 18.104.22.168) and horse liver alcohol dehydrogenase (EC 22.214.171.124) were purchased from Boehringer-Mannheim. Bovine serum albumin (BSA) (Pentex) and pig heart cytosolic MDH (c-MDH) (EC 126.96.36.199) were purchased from Miles Laboratories. Cytosolic MDH was also purified in this laboratory and used as indicated in the text. Yeast alcohol dehydrogenase (EC 188.8.131.52) was purchased from Sigma Chemical Co. Citrate lyase (EC 184.108.40.206) from Streptococcus diacetilactis and citrate synthase (EC 220.127.116.11) from Escherichia coli were purified in this laboratory. The enzymes, usually obtained as ammonium sulfate suspensions, were dialyzed against 5 mM potassium phosphate buffer, pH 7.0, until they were free of ammonium sulfate. Oxaloacetic acid, 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB), malic acid, and NAD were purchased from Sigma Chemical Co. Coenzyme A was purchased from P-L Biochemicals, Inc. Acetyl-CoA was made from coenzyme A and acetic anhydride (6). Polyethylene glycol (M, = 6000-7500) was purchased from MC/B Manufacturing Chemists. PEG was used as received without further purification, since in preliminary studies no difference was observed in the behavior of the protein interactions when either purified [by precipitation from acetone with ether (7)] or unpurified PEG was used. Solutions of PEG were made up in 5 mM potassium phosphate buffer, pH 7.0, as stock 40% (w/v) solutions.
Citrate synthase activity was determined by monitoring the reaction with DTNB at 412 nm (8). The assay mixture contained 0.1 M Tris-Cl, pH 8.1, 0.1 mM DTNB, 0.3 mM oxaloacetate, and 0.2 mM acetyl-CoA. Malate dehydrogenase activity in the direction of formation of oxaloacetate was determined by following the increase in absorbance at 340 nm in an assay mixture which contained 0.1 M Tris-Cl, pH 8.1, 1 mM NAD, and 25 mM L-malate. Acetyl-CoA (0.2 mM) and CS (0.5-1.0 unit) were added to remove oxaloacetate by means of the citrate synthase reaction. All assays were performed in a total volume of 1.0 ml at 25°C. Protein concentrations were determined by absorbance measurements at 280 nm, using extinction coefficients (E:TJ of 17.8 for CS (9), 9.3 for cytosolic MDH (lo), 2.8 for mitochondrial MDH (ll), and 6.6 for BSA (12). For other proteins, and as noted in the text, protein concentrations were determined by reaction with fluorescamine (13). Enzyme samples and mixtures were incubated for 1 h at 10°C in 0.4 ml of 5 mM KPOI, pH 7.0, containing PEG. The optical density (OD) at 650 nm was determined as a measurement of turbidity. The diminution of transmitted light at 650 nm was attributed to light scattering, since the solutions examined do not absorb light at this wavelength. Optical densities were obtained using lo-mm-pathlength microcuvettes (Pyrocell) in a Gilford 2000 recording spectrophotometer employing a microaperture, with the temperature controlled at 10°C. To analyze protein precipitation, samples were placed into 6 x 52-mm polyethylene tubes after incubation with PEG and centrifuged at 10°C for 10 min at 20,OOOgusing Sorvall408 adaptors, a Sorvall SS-34 rotor, and a Sorvall RC2-B centrifuge. The supernatant solutions were then removed with a Pasteur pipet, and the pellets were resuspended in the original sample volume of 14% PEG. The resuspended pellets had low turbidities, since the protein concentrations were lower here than in the original samples. The completeness of precipitation was verified by remeasuring the optical density at 650 nm for the supernatant solution after centrifugation. Enzyme activities of the original samples, the supernatant solutions, and the resuspended pellets were determined. RESULTS
Increasing the PEG concentration increased the turbidity (OD, 650 nm> of a 1:l (w/w) mixture of CS and MDH (Table I). Increasing the ionic strength caused a decrease in the turbidity of an enzyme mixture (1:l) in 14% PEG (Table I). These results are in agreement with those reported by other workers (14, 15), in that
THE EFFECT OF POLYETHYLENE GLYCOL AND IONIC STRENGTH ON THE TURBIDITY OF MIXTURES OF CITRATE SYNTHASE AND MITOCHONDRIAL MALATE DEHYDROGENASE Ionic strength OD (650 nm) Percentage (w/ (x 10R) v) PEG
10” 12” 14” 16” 140 146 14b 14h 140
0.040 0.135 0.315 0.360 0.320 0.315 0.310 0.285 0.200
5.4 5.4 5.4 5.4 3.8 5.4 11.3 18.8 33.8
a Samples contained 0.2 mg/ml each of CS and of m-MDH, 2.0 pmol of potassium phosphate buffer, pH 7.0, and PEG at the concentrations indicated, in a total volume of 0.4 ml. Samples were incubated at 10°C for 1 h before OD readings were taken. b Samples contained 0.2 mg/ml each of CS and of m-MDH, 14% PEG (w/v), 0.2 pmol of potassium phosphate buffer, pH 7.0, and potassium chloride to the ionic strength indicated, in a total volume of 0.4 ml. Samples were incubated at 10°C for 1 h before OD readings were taken.
for a given protein composition, either increasing the concentration of PEG or decreasing the ionic strength of the solution decreases protein solubility as evidenced by the increase in turbidity. The specificity of the apparent interaction between mitochondrial MDH and CS was tested initially using either cytosolic MDH or BSA in place of mitochondrial MDH. Increasing the concentration of mMDH, c-MDH, or BSA in solutions containing a constant concentration of CS in 14% PEG caused an increase in turbidity only in the solution of CS with m-MDH (Fig. 1). Although the solution of CS alone was slightly turbid in 14% PEG, the addition of increasing amounts of m-MDH increased the turbidity much beyond that of a solution of CS at the same total protein concentration. Solutions of mitochondrial or cytosolic MDH in 14% PEG had very low optical densities at 650 nm (Fig. 2). When the experiment was performed with increasing amounts of CS and constant amounts of the other proteins, then again an in-
crease in the turbidity of solutions containing CS with m-MDH was observed, but there was little effect on the turbidity of solutions of CS with either c-MDH or BSA (Fig. 2). After samples containing CS, m-MDH, or c-MDH alone and in combination were centrifuged, the supernatant solutions and the resuspended pellets were assayed for enzyme activities (Table II). The presence of CS with m-MDH resulted in increased precipitation of both enzymes, whereas mMDH did not precipitate from solutions containing only that enzyme. In agreement with the turbidimetric studies, the presence of CS with c-MDH did not result in increased precipitation of either protein. Several other proteins from various sources were tested for possible interactions with either pig heart m-MDH or pig heart CS in 14% PEG solution (Table III).
01 0.2 0.3 0.4 CONCENTRATION OF CITRATE SYNTHASE (mg/ml)
FIG. 1. The effect on turbidity of adding malate dehydrogenase or bovine serum albumin to citrate synthase solutions. Samples contained 2.0 pmol of potassium phosphate buffer, pH 7.0, and 14% PEG (w/v) in a total volume of 0.4 ml. Optical density measurements were taken after samples had been incubated at 10°C for 1 h. 0, sample contained 0.2 mglml of citrate synthase plus m-MDH at the concentrations indicated; A, sample contained 0.2 mg/ ml of citrate synthase plus BSA at the concentrations indicated; 0, sample contained 0.2 mgiml of citrate synthase plus c-MDH at the concentrations indicated.
Of the pig heart mitochondrial enzymes creased the turbidity of BSA solutions to examined, only diaphorase affected the about the same extent. Proteins from turbidity of solutions containing either CS other sources either did not affect turbidity or m-MDH. However, diaphorase in- or increased the turbidity of solutions containing either CS, m-MDH, or BSA to TABLE II PRECIPITATION OF ENZYMES FROM PEG SOLUTIONS Proteind cs
FIG. 2. The effect on turbidity of adding citrate synthase or bovine serum albumin to malate dehydrogenase solutions. Samples contained 2.0 pmol of potassium phosphate buffer, pH 7.0, and 14% PEG (w/v) in a total volume of 0.4 ml. Optical density measurements were taken after samples had been incubated at 10°C for 1 h. 0, sample contained 0.2 mg/ml of m-MDH plus CS at the concentrations indicated; A, sample contained 0.2 mg/ml of mMDH plus BSA at the concentrations indicated; cl, sample contained 0.2 mg/ml of c-MDH plus CS at the concentrations indicated. TABLE OPTICAL DENSITY (650 nm) Protein”
MmDH 40 40 40
3’ 26 -
4e 13 2”
0 Samples were incubated in 0.4 ml of 5 mM potassium phosphate buffer containing 14% PEG for 1 h at 10°C and then centrifuged at 10°C for 10 min at 20,OOOg. b Protein concentrations were determined by fluorescamine assay. c Cytosolic MDH for these samples was isolated in this laboratory. d Amount of precipitated protein determined by enzymatic assays before and after centrifugation. Total recoveries (activity in pellet plus activity in supernatant solution) approximated 100%. e This number (2-4% of the total protein) may represent some contamination of the pellet with supernatant solution. III
PROTEIN SOLUTIONS IN 14% PEG AFTER 1 h AT 10°C Source
OD,,,b in the presence of PigknHt
cs CS and m-MDH Aspartate aminotransferase Succinyl-CoA synthase Isocitrate dehydrogenased Diaphorased Alcohol dehydrogenase Alcohol dehydrogenased Aldolased Citrate lyase cs
Pig heart Pig heart Pig heart Pig Heart Pig heart Pig heart Yeast Horse liver Rabbit muscle S. diacetilactis E. coli
0.365 0.035 0.005 0.070 0.115 0.000 0.235 0.165 0.040 0.350
Pig heart cs
0.000 0.000 0.070 0.175 0.045 0.350 0.350 0.000 0.015
0.360c 0.160 0.375 0.375 0.000
a Concentration of each protein = 0.2 mg/ml as determined by fluorescamine assay. b Optical density = OD of mixture - (OD of individual proteins at 0.2 mg/ml). c The optical density of this sample increased from 0.315 after 60 min of incubation to 0.360 after 90 min of incubation. d These proteins gave the following high turbidities when incubated at a concentration of 0.2 mg/ml in 14% PEG solutions: isocitrate dehydrogenase (OD = 0.090); diaphorase (OD = 0.195); horse liver alcohol dehydrogenase (OD = 0.330); rabbit aldolase (OD = 0.320).
about the same extent. The same final turbidity was observed for solutions of either CS and m-MDH without BSA or CS and m-MDH in the presence of BSA. Interaction was also observed for E. coli CS, which gave turbid solutions with pig heart m-MDH, but not with pig heart CS or with BSA. DISCUSSION
Ogston (16) suggested some years ago that the nonideal behavior exhibited upon mixing of protein solutions might be important in systems of high protein concentration such as cell cytoplasm. The use of nonionic polymer solutions may be a model for the mitochondrial matrix which contains up to 50% protein (17) and in which exclusion effects may operate to enrich both intermediates and enzymes (18) and perhaps even induce phase separation (19). Specific interaction between pig heart mitochondrial aspartate aminotransferase and mitochondrial malate dehydrogenase has been observed in a biphasic water, dextran, carboxymethylpolyethylene glycol system (4). No physical evidence of the existence of a complex was observed in buffer systems without added polymers even though kinetic evidence for the interaction was presented (5). The effect of nonionic polymers on protein solutions is apparently one of exclusion of the protein from the solvent by the polymer (20, 21). It has been reported (18) that pig heart mitochondrial MDH and CS which had been cross-linked with glutaraldehyde exhibited a small activation (lo-15%) of the overall rate of conversion of malate to citrate when PEG was present in the suspension medium. These results were interpreted as an exclusion effect of the polymer leading to a relative enhancement of the intermediates oxaloacetate and NADH and an apparently higher concentration of the enzymes such that the efficiency of the CS reaction was increased beyond the efficiency found when the two enzymes were mixed in solution. The solubility of individual proteins in PEG solution has been reported to be a function of the Stokes’ radius of the protein (14, 20). Although precipitation from pro-
tein mixtures cannot yet be accurately predicted (14), evidence presented here indicates that an interaction between mitochondrial MDH and CS occurs in PEG solutions. Both mitochondrial MDH and CS exhibited altered precipitation behavior when incubated together in the presence of PEG. Since cytosolic MDH, BSA, and other proteins could not substitute for mitochondrial MDH, some type of specific interaction in PEG solutions seems to be indicated. It is doubtful that the mitochondrial proteins were merely exerting a further exclusion effect when mixed in PEG solutions, since relatively low concentrations of protein were used. In addition, if only exclusion effects were operating, then BSA (M, = 70,000) (22), cytosolic MDH (M, = 74,000) (lo), and mitochondrial MDH (M, = 70,000) (23) might each be expected to behave similarly in PEG solutions containing CS due to the similarity in the molecular weights (and presumably the Stokes’ radii) of these proteins. However, Figs. 1 and 2 show that only CS and m-MDH increase turbidity as would be expected for complex formation. The turbidimetric data in Table II indicate that, of the enzymes examined, only pig heart CS or E. coli CS specifically interacts with pig heart m-MDH. If in uiuo complex formation occurs between m-MDH and CS, then other proteins must not interfere with this interaction. Table III shows that the same final turbidity is exhibited by solutions of either CS and m-MDH without BSA or CS and m-MDH in the presence of BSA. The present data do not indicate that an interaction between CS and m-MDH exists in mitochondria. However, the specificity of the interaction reported here, and the fact that the presence of additional protein (e.g., BSA) does not prevent this interaction, show that these enzymes can associate under the appropriate conditions. Since these interactions require high enzyme concentrations, studies of the kinetics of the reactions of the enzymes in these aggregates would be difficult. However, studies of other systems in which the enzymes are in putative interaction are in progress.
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