1887

Abstract

The pyruvate dehydrogenase complex of PAO was purified by affinity chromatography on ethanol-Sepharose 2B followed by sucrose density gradient centrifugation. The overall purification was 130-fold based on enzyme activity. The purified complex contained three major and one minor polypeptide components when analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. These were identified by heat treatment, limited proteolysis and peptide mapping as pyruvate dehydrogenase (E1; M 92500), acetyltransferases (E2; major component, M 76000, and minor component, M 77 800), and lipoamide dehydrogenase (E3; M 58000). The purified complex had a sedimentation coefficient of 48S and the specific activity for the overall reaction of the complex was 6·5 substrate transformed (mg protein) min at the optimum pH (7·8) and 25 °C.

The lesions in four mutants lacking overall pyruvate dehydrogenase complex activity were identified after partial purification of the corresponding cell-free extracts. Three strains, designated mutants, lacked pyruvate dehydrogenase activity (E1 component) and one strain, an mutant, lacked the activity of the acetyltransferase (E2 component).

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1980-10-01
2021-10-21
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