Full activation of rat liver pyruvate dehydrogenase in vitro by ADP was prevented by palmitoyl-CoA at a concentration sufficiently low to preclude substrate effects secondary to its oxidation by mitochondria. Activation of pyruvate dehydrogenase by ADP in livers of fat-fed rats was less than in the control animal. The results are consistent with the experiments demonstrating an inhibition of adenine nucleotide translocase and on increased intramitochondrial ATP/ADP ratio by palmitoyl-CoA which could account for the effect on pyruvate dehydrogenase. Inactivation of brain pyruvate dehydrogenase by ATP was also diminished by palmitoyl-CoA indicating that the effect was at the level of the adenine nucleotides rather than at either the pyruvate dehydrogenase kinase or phosphatase enzymes.The interconversion of the inactive and active MATERIALS AND METHODS forms of pyruvate dehydrogenase occurs via a phosphorylationJdephosphorylation of the enzyme by a specific pyruvate dehydrogenase kinase and phosphatase which are independent of adenosine 3' : 5'-monophosphate (cyclic AMP) [l]. Observations based on a number of experiments in vitro [2-61 indicate that the interconversion is regulated by the intramitochondrial ratios of at least three important reactants : ATP/ADP, NADHiNAD The basal incubation medium for the mitochondria contained 90mM KCl, 30mM Tris-HCI, 2mM MgC12, 10 mM potassium phosphate buffer, and 1.0 mM EDTA, pH 7.2, with the addition of adenine nucleotides and palmitovl-CoA as specified. Following in--Enzymes. Pyruvate dehydrogenase (lipoate) (EC 1.2.4.1); arylcubation for 5 min at 25 "c, the reaction mixture was amine acetyltransferase (EC 2.3.1.5).cooled rapidly and mitochondria sedimented in a
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