Component X. An immunologically distinct polypeptide associated with mammalian pyruvate dehydrogenase multi-enzyme complex.
The mammalian pyruvate dehydrogenase multi-enzyme complex contains a tightly-associated 50 000-Mr polypeptide of unknown function (component X) in addition to its three constituent enzymes, pyruvate dehydrogenase (E1), lipoate acetyltransferase (E2) and lipoamide dehydrogenase (E3) which are jointly responsible for production of CoASAc and NADH. The presence of component X is apparent on sodium dodecyl sulphate/polyacrylamide gel analysis of the complex, performed in Tris-glycine buffers although it co-migrates with the E3 subunit on standard phosphate gels run under denaturing conditions. Refined immunological techniques, employing subunit-specific antisera to individual components of the pyruvate dehydrogenase complex, have demonstrated that protein X is not a proteolytic fragment of E2 (or E3) as suggested previously. In addition, anti-X serum elicits no cross-reaction with either subunit of the intrinsic kinase of the pyruvate dehydrogenase complex. Immune-blotting analysis of SDS extracts of bovine, rat and pig cell lines and derived subcellular fractions have indicated that protein X is a normal cellular component with a specific mitochondrial location. It remains tightly-associated with the 'core' enzyme, E2, on dissociation of the complex at pH 9.5 or by treatment with 0.25 M MgCl2. This polypeptide is not released to any significant extent from E2 by p-hydroxymercuriphenyl sulphonate, a reagent which promotes dissociation of the specific kinase of the complex from the 'core' enzyme. Incubation of the complex with [2-14C]pyruvate in the absence of CoASH promotes the incorporation of radio-label, probably in the form of acetyl groups, into both E2 and component X.
D000818:Animals; D000918:Antibody Specificity; D002417:Cattle; D002478:Cells, Cultured; D002850:Chromatography, Gel / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D007668:Kidney / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D010455:Peptides / Q000276:immunology / Q000302:isolation & purification*; D011494:Protein Kinases / Q000276:immunology; D017346:Protein Serine-Threonine Kinases; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D011768:Pyruvate Dehydrogenase Complex / Q000276:immunology / Q000302:isolation & purification*; D051381:Rats; D011915:Rats, Inbred BUF; D013347:Subcellular Fractions / Q000201:enzymology; D013552:Swine
D010455:Peptides; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D011768:Pyruvate Dehydrogenase Complex; C489061:pyruvate dehydrogenase complex component X, Bos taurus; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases