1.Studies with intact mitochondria and with soluble pyruvate dehydrogenase indicate that pyruvate and a-ketobutyrate are oxidized by the same enzyme (pyruvate dehydrogenase), while a-ketovalerate is oxidized by a different enzyme.2. Pyruvate and u-ketobutyrate have about the same affinity for the enzyme, but pyruvate is oxidized a t a much higher rate.3. Acetyl-CoA and propionyl-CoA both behave as competitive inhibitors to CoA. The K{ for both is slightly higher than the Km for CoA. Accordingly the enzyme is only moderately inhibited by a high acetyl-CoA/CoA ratio. 4.NADH behaves mainly as a competitive inhibitor to NAD. The Kt is significantly lower than the Km for NAD. Accordingly the enzyme is strongly inhibited by a high NADH/NAD ratio.5. The significance of these properties of the enzyme for the regulation of the activity of pyruvate dehydrogenase in vivo is discussed.The reaction mechanism of pyruvate dehydrogenase has been extensively investigated, but relatively few studies on the substrate specificity and on t,he regulation of the enzyme have been published.Pyruvate dehydrogenase from Streptococcus femlis is active with a-ketobutyrate as substrate [l, 21, but the efficiency of this substrate with the mammalian enzyme has not been studied.Acetyl-CoA is reported to inhibit pig heart pyruvate dehydrogenase by competing with CoA ( K i = 12.5 pM, K , for CoA = 6.7 pM) [3].Hansen and Henning [4] found NADH to inhibit pyruvate dehydrogenase from Escherichia coli by competing with NAD. The K m and the Ki for NAD and NADH respectively were not determined, but the enzyme was found to be far more sensitive to the NADH/NAD ratio than to the acetyl-CoA/CoA ratio.Schwartz, Old and Reed [B] have found that phosphoenolpyruvate lowers the K m for pyruvate with pyruvate dehydrogenase from E . coli, while acetyl-CoA was a competitive inhibitor to pyruvate with ferricyanide as the electron acceptor. The physiological importance of the latter observation remains doubtful however, as the K ( for acetyl-CoA increased about fifty-fold when NAD was the electron acceptor. In the present communication studies on the metabolism of a-keto acids by mitochondria from rat organs and by soluble pyruvate dehydrogenase from pig heart and kidney are reported. The effects of the reaction products have also been studied. Mitochondria from rat liver, rat kidney, rat heart, and rat brain were prepared by conventional centrifugation procedures after homogenization of the tissues in loo/, sucrose containing 2 mM EDTA. The isolated mitochondria1 fractions were washed once in homogenizing medium and finally suspended in 0.15 M KC1. EXPERIMENTAL PROCEDURE MaterialsPig heart pyruvate dehydrogenase was prepared according to Scriba and Holzer [9] except that the ethanol fractionation step was omitted. Pig kidney pyruvate dehydrogenase was prepared by a modification of the procedure of Ishikawa, Oliver and Reed [lo]. Following their procedure we were unable to extract the pyruvate dehydrogenase from the second protamine precipitate with phosphate buf...

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