Evidence for the regulation of the branched chain alpha-keto acid dehydrogenase multienzyme complex by a phosphorylation/dephosphorylation mechanism.
The regulation of the branched chain alpha-keto acid dehydrogenase complex by covalent modification was investigated in rat liver mitochondria. Depletion of intramitochondrial calcium and magnesium caused an inactivation of the branched chain alpha-keto acid dehydrogenase complex. Following inactivation of the branched chain complex, addition of calcium or magnesium ions separately to incubations of mitochondria only partially reactivated the enzyme complex. However, simultaneous addition of calcium and magnesium activated the branched chain enzyme complex rapidly and nearly completely. Mitochondrial incubations were performed in the presence of [32P]phosphate under conditions known to activate or to inactivate the branched chain alpha-keto acid dehydrogenase complex. Evidence demonstrating that [32P]-phosphate was incorporated into two major protein bands separated in sodium dodecyl sulfate-polyacrylamide gels of the mitochondrial incubations is presented. Migration of the labeled mitochondrial protein bands in the gel system corresponded exactly to the migration of the alpha subunit of the purified heart-derived pyruvate dehydrogenase (decarboxylase, E1) and the alpha subunit of the purified kidney-derived branched chain alpha-keto acid dehydrogenase (decarboxylase, E1). Furthermore, when the measured activity of the branched chain complex was minimized, the amount of [32P]phosphate incorporated into the alpha chain of the branched chain enzyme was maximal. Conversely, incubation conditions which activated maximally the enzyme complex minimized the [32P]phosphate incorporation into the alpha subunit of the branched chain dehydrogenase.
D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D007658:Ketone Oxidoreductases / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008930:Mitochondria, Liver / Q000201:enzymology*; D009097:Multienzyme Complexes / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D011919:Rats, Inbred Strains
D009097:Multienzyme Complexes; D010710:Phosphates; D000001:Calcimycin; D004533:Egtazic Acid; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D008274:Magnesium; D002118:Calcium