Regulation of pig heart pyruvate dehydrogenase by phosphorylation. Studies on the subunit and phosphorylation stoicheiometries.
1. The molecular weights of the subunits of purified pig heart pyruvate dehydrogenase complex were determined by sodium dodecyl sulphate/polyacrylamide-disc-gel electrophoresis and were: pyruvate decarboxylase, alpha-subunit 40600, beta-subunit 35100; dihydrolipoyl acetyltransferase 76100; dihydrolipoyl dehydrogenase 58200. 2. Inactivation of the pyruvate dehydrogenase complex by its integral kinase corresponded to the incorporation of 0.46nmol of P/unit of complex activity inactivated. 3. Further incorporation of phosphate into the complex occurred to a limit of 1.27nmol of P/unit of complex inactivated (approx. 3 times that required for inactivation). 4. Phosphate was incorporated only into the alpha-subunit of the decarboxylase. 5. The molar ratio of phosphate to alpha-subunits of the decarboxylase was estimated by radioamidination of amino groups of pyruvate dehydrogenase [(32)P]phosphate complex by using methyl [1-(14)C]acetimidate, followed by separation of alpha-subunits by sodium dodecyl sulphate/polyacrylamide-disc-gel electrophoresis. Inactivation of the complex (0.46nmol of P/unit of complex inactivated) corresponded to a molar ratio of one phosphate group per two alpha-chains (i.e. one phosphate group/alpha(2)beta(2) tetramer). Complete phosphorylation corresponded to three phosphate groups per alpha(2)beta(2) tetramer. 6. Subunit molar ratios in the complex were also estimated by the radioamidination technique. Results corresponded most closely to molar ratios of 4 alpha-subunits:4 beta-subunits:2 dihydrolipoyl acetyltransferase subunits:1 dihydrolipoyl dehydrogenase subunit.
D055598:Chemical Phenomena; D002621:Chemistry; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D008956:Models, Chemical; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism*
D010710:Phosphates; D011768:Pyruvate Dehydrogenase Complex