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, α-subunit 40600, β-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 α-subunit of the decarboxylase. 5. The molar ratio of phosphate to α-subunits of the decarboxylase was estimated by radioamidination of amino groups of pyruvate dehydrogenase [32P]phosphate complex by using methyl [1-14C]acetimidate, followed by separation of α-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 α-chains (i.e. one phosphate group/α2β2 tetramer). Complete phosphorylation corresponded to three phosphate groups per α2β2 tetramer. 6. Subunit molar ratios in the complex were also estimated by the radioamidination technique. Results corresponded most closely to molar ratios of 4 α-subunits:4 β-subunits:2 dihydrolipoyl acetyltransferase subunits:1 dihydrolipoyl dehydrogenase subunit.
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Research Article|
August 01 1978
Regulation of pig heart pyruvate dehydrogenase by phosphorylation. Studies on the subunit and phosphorylation stoicheiometries
Peter H. Sugden;
Peter H. Sugden
1Nuffield Department of Clinical Biochemistry, Radcliffe Infirmary, Oxford OX2 6HE, U.K.
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Philip J. Randle
Philip J. Randle
1Nuffield Department of Clinical Biochemistry, Radcliffe Infirmary, Oxford OX2 6HE, U.K.
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Publisher: Portland Press Ltd
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 1978 London: The Biochemical Society
1978
Biochem J (1978) 173 (2): 659–668.
Citation
Peter H. Sugden, Philip J. Randle; Regulation of pig heart pyruvate dehydrogenase by phosphorylation. Studies on the subunit and phosphorylation stoicheiometries. Biochem J 1 August 1978; 173 (2): 659–668. doi: https://doi.org/10.1042/bj1730659
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