Studies on the bovine brain pyruvate dehydrogenase complex using the antibodies against kidney enzyme complex.
Pyruvate dehydrogenase complex (PDHC) was purified from bovine kidney with a specific activity of 12-16 mumol of NADH or acetyl-CoA formed/min/mg protein. The four peptides comprising its three catalytic components were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Rabbit antibodies against this highly purified PDHC (anti-PDHC) exhibited similar binding affinity to the phospho-PDHC as it did to the PDHC antigen. To test whether there exist brain isozymes of PDHC differing from kidney enzyme, which has been extensively characterized, the PDHCs in bovine brain and kidney were compared using this anti-PDHC. The PDHC activities in the brain and kidney mitochondrial extracts were inhibited to the same degree by varying amounts of anti-PDHC. Brain PDHC was precipitated with the anti-PDHC and resolved by SDS-PAGE. The four brain PDHC peptides isolated immunochemically with anti-PDHC had the same sizes as the kidney PDHC peptides. These PDHC peptides from kidney and brain were further compared by their peptide fragment patterns, which were generated by partial proteolysis with Staphylococcus aureus V8 protease or by CNBr and resolved by SDS-PAGE. The peptide patterns generated with the former method indicated that the alpha and beta peptides of the pyruvate dehydrogenase (E1) component and the peptide of dihydrolipoyl transacetylase (E2) component of kidney PDHC were very similar to the corresponding peptides immunologically isolated from brain. The peptide patterns generated with CNBr further confirmed that the beta E1 and E2 peptides of kidney PDHC were similar to the corresponding peptides from brain.
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D000939:Epitopes / Q000276:immunology; D007163:Immunosorbent Techniques; D007668:Kidney / Q000201:enzymology*; D008928:Mitochondria / Q000201:enzymology; D010446:Peptide Fragments / Q000302:isolation & purification; D011768:Pyruvate Dehydrogenase Complex / Q000276:immunology / Q000378:metabolism*; D012697:Serine Endopeptidases
D000906:Antibodies; D000939:Epitopes; D010446:Peptide Fragments; D011768:Pyruvate Dehydrogenase Complex; D010450:Endopeptidases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D003488:Cyanogen Bromide