Kinetic and Regulatory Properties of Pyruvate Dehydrogenase from Ehrlich Ascites Tumor Cells¹ Free

Pyruvate dehydrogenase was partially purified from Ehrlich ascites tumor cell mitochondria and its kinetic properties were determined. The apparent K_m values for pyruvate, nicotinamide adenine dinucleotide, and coenzyme A (CoA) were 46 μm, 110 μm, and 36 μm, respectively. Reduced nicotinamide adenine dinucleotide and acetyl-CoA inhibited enzyme activity competitively to nicotinamide adenine dinucleotide (K_i = 22 μm) and CoA (K_i = 58 μm), respectively. Copurified α-ketoglutarate dehydrogenase displayed apparent K_m values for α-ketoglutarate, nicotinamide adenine dinucleotide, and CoA of 1.25 mm, 67 μm, and 50 μm, respectively.

Pyruvate dehydrogenase, but not α-ketoglutarate dehydrogenase, was inactivated specifically by adenosine triphosphate with concomitant phosphorylation, and it was reactivated at 10 mm Mg²⁺ by a protein fraction separated from the complex during purification. The rate of inactivation was decreased by pyruvate or pyrophosphate.

The existence of active and inactive forms of pyruvate dehydrogenase in Ehrlich ascites tumor cells was demonstrated. Active form and total activity were determined to be 74.0 ± 1.5 and 93.6 ± 4.9 munits/g packed cells (mean ± S.E., n = 25), respectively.