The visualization by affinity electrophoresis of a specific association between the consecutive citric acid cycle enzymes fumarase and malate dehydrogenase.
Evidence is growing that the citric acid cycle, like many other metabolic pathways, might exist in vivo as a more or less tightly organized multi-enzyme cluster. The term 'metabolon' [Robinson, J. B. & Srere, P. A. (1985) J. Biol. Chem. 260, 10800-10805] was recently introduced to describe such a complex of sequential metabolic enzymes. We adopted the technique of affinity electrophoresis for the study of interactions between the cycle enzymes fumarase and malate dehydrogenase. This approach offers several advantages over our previously described affinity chromatographic technique [Beeckmans, S. & Kanarek, L. (1981) Eur. J. Biochem. 117, 527-535], one of which is the fact that the interaction can be directly visualized. The observed association is specific since both metabolically unrelated proteins and the cytoplasmic isoenzyme of malate dehydrogenase do not interact with fumarase. Several metabolites (citrate, isocitrate, 2-oxoglutarate, succinate, fumarate, malate, oxaloacetate, Pi, AMP, ADP, NAD+, NADH) were found not to affect the association between fumarase and mitochondrial malate dehydrogenase. Both ATP, Mg2+ -ATP and GTP disrupt the association when they are present at 1 mM concentrations. Lower non-physiological ATP concentrations do not, however, disturb the interaction. The presence of 1 mM ADP was found to abolish the disrupting effect of 1 mM ATP. The latter findings are suggestive of an interruption of the citric acid cycle at the level of fumarase under conditions of high energy load (i.e. high ATP/ADP ratios).
Laboratorium voor Chemie der Proteinen, Vrije Universiteit Brussel, Belgium.
S
S
Beeckmans
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Van Driessche
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L
Kanarek
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002846:Chromatography, Affinity; D002952:Citric Acid Cycle; D004586:Electrophoresis; D004587:Electrophoresis, Agar Gel; D005649:Fumarate Hydratase / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D008291:Malate Dehydrogenase / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology*; D013552:Swine
D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D008291:Malate Dehydrogenase; D005649:Fumarate Hydratase; D008274:Magnesium