Studies on the apparent instability of bovine kidney α-ketoglutarate dehydrogenase complex

Abstract

The α-ketoglutarate dehydrogenase complex in extracts of bovine kidney and liver mitochondria is inactivated rapidly at 25 °C. This inactivation is not accompanied by loss of activity of the three component enzymes of the complex. This inactivation can be prevented by extensive washing of the mitochondria with dilute phosphate buffer prior to rupturing the mitochondria by freezing and thawing. Evidence is presented that the washings contain a protease which cleaves a peptide bond or bonds in the dihydrolipoyl transsuccinylase component of the α-ketoglutarate dehydrogenase complex, and this limited proteolysis results in dissociation of α-ketoglutarate dehydrogenase and dihydrolipoyl dehydrogenase from the transsuccinylase. The protease appears to be specific for the transsuccinylase component of the mammalian α-ketoglutarate dehydrogenase complex. It does not affect the activity of the mammalian pyruvate dehydrogenase complex or the Escherichia coli α-ketoglutarate dehydrogenase complex. The protease has been purified about 100-fold from extracts of unwashed mitochondria from bovine kidney. It requires a thiol for activity and it is not affected by treatment with diisopropyl phosphorofluoridate or phenylmethyl sulfonylfluoride. A component has been detected in highly purified preparations of the bovine kidney α-ketoglutarate dehydrogenase complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which is present in trace amounts, if at all, in purified preparations of the bovine heart α-ketoglutarate dehydrogenase complex. This component is tightly bound to the transsuccinylase.