Relationshiop between phosphorylation and activity of pyruvate dehydrogenase in rat liver mitochondria and the absence of such a relationship for pyruvate carboxylase.

Linn et al. (Linn, T. C., Pettit, F. H., and Reed, L. J. (1969) Proc. Natl. Acad. Sci. U. S. A. 62, 234-241) have shown that purified preparations of pyruvate dehydrogenase complex can be inactivated by phosphorylation and reactivated by dephosphorylation. The present study shows that these processes also take place in rat liver mitochondria. In addition, we have compared such changes with possible parallel phosphorylation of pyruvate carboxylase. Mitochondria were incubated with 32Pi, the pyruvate dehydrogenase and pyruvate carboxylase were isolated by immunoprecipitation and sodium dodecyl sulfate-acrylamide gel electrophoresis, and the 3zP content was determined. Incubation of mitochondria with fluoride, ionophore A23187, and ethylene glycol bis(P-aminoethyl ether) N,N’-tetraacetic acid to inhibit pyruvate dehydrogenase phosphatase led to greater than 97% inactivation of the complex and incorporation of up to 2.0 mol of phosphate per mol of pyruvate dehydrogenase (M, = 154,000). When mitochondria were incubated with dichloroacetate to inhibit pyruvate dehydrogenase kinase, maximal activity of the complex was observed and less than 0.01 mol of phosphate was incorporated per mol of enzyme. Under the same conditions, neither treatment resulted in significant changes in the maximal catalytic activity of pyruvate carboxylase nor was there any phosphorylation of the enzyme. We conclude that phosphorylation plays a major role in the control of pyruvate dehydrogenase complex in liver mitochondria but that this mechanism is probably not a factor in pyruvate carboxylase regulation.