Improved purification of the pyruvate dehydrogenase complex from Ascaris suum body wall muscle and characterization of PDHa kinase activity.
An improved purification scheme for the isolation of the Ascaris suum pyruvate dehydrogenase complex directly from body wall muscle has been developed which yields a fully activated pyruvate dehydrogenase complex with substantial PDHa kinase activity. The apparent Km for coenzyme A (CoA) is much lower than previously reported and can only be accurately measured in the presence of a CoA-regenerating system. The alpha-pyruvate dehydrogenase subunit of the ascarid complex is unique and its migration on sodium dodecylsulfate polyacrylamide gels is altered after phosphorylation. PDHa kinase activity is inhibited by ADP, thiamine pyrophosphate, and physiological levels of pyruvate and propionate. In contrast, PDHa kinase activity is stimulated by elevated NADH/NAD+ and acetyl CoA/CoA ratios, although it appears that the NADH/NAD+ ratios required for half-maximal stimulation are more than an order of magnitude greater than those reported for mammalian pyruvate dehydrogenase complexes.
D000105:Acetyl Coenzyme A / Q000494:pharmacology; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D001200:Ascaris / Q000201:enzymology*; D003065:Coenzyme A / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D009243:NAD / Q000494:pharmacology; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D011768:Pyruvate Dehydrogenase Complex / Q000302:isolation & purification* / Q000378:metabolism
D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D011768:Pyruvate Dehydrogenase Complex; D009243:NAD; D000244:Adenosine Diphosphate; D000105:Acetyl Coenzyme A; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D003065:Coenzyme A