Pyruvate dehydrogenase was partially purified from Ehr lich ascites tumor cell mitochondria and its kinetic proper ties were determined. The apparent Kmvalues for pyruvate, nicotinamide adenine dinucleotide, and coenzyme A (CoA)

Pyruvate dehydrogenase was partially purified from Ehr lich ascites tumor cell mitochondria and its kinetic proper ties were determined. The apparent Kmvalues for pyruvate, nicotinamide adenine dinucleotide, and coenzyme A (CoA) were 46 j.@M,110 @M, and 36 p@M, respectively. Reduced nicotinamide adenine dinucleotide and acetyl-CoA inhibited enzyme activity competitively to nicotinamide adenine dinu cleotide (K = 22 /.@M) and C0A (K = 58 @tM), respectively. Copurified a-ketoglutarate dehydrogenase displayed appar ent Km values for a-ketoglutarate, nicotinamide adenine dinucleotide, and CoA of 1.25 mM, 67 @M, and 50 @M, respectively. Pyruvate dehydrogenase, but not a-ketoglutarate dehy drogenase, was inactivated specifically by adenosine tn phosphate with concomitant phosphorylation, and it was reactivated at 10 mM Mg2@by a protein fraction separated from the complex during purification. The rate of inactiva tion was decreased by pynuvate or pyrophosphate. The existence of active and inactive forms of pyruvate dehydrogenase in Ehrlich ascites tumor cells was demon strated. Active form and total activity were determined to be 74.0 ±1.5 and 93.6 ±4.9 munits/g packed cells (mean ± SE., n = 25), respectively.