Bovine kidney pyruvate dehydrogenase complex. Isolation of the component enzymes after limited proteolysis with papain.
1. Bovine kidney pyruvate dehydrogenase multienzyme complex is inactivated rapidly by papain. However, none of the component activities of the complex is destroyed during inactivation of the overall reaction. 2. The core component, lipoate acetyltransferase, is cleaved by papain to give principal fragments with Mr 26,500 and 26,000 (as determined by dodecylsulfate gel electrophoresis). Much more slowly, the alpha chain of the pyruvate dehydrogenase component is attacked. 3. Fragmented lipoate acetyltransferase retains its complete enzymatic activity and remains of high molecular weight. It is unable, however, to bind the other component enzymes, pyruvate dehydrogenase and lipoamide dehydrogenase. Therefore, the multienzyme complex is disassembled when treated with papain. 4. A method is described which allows the rapid and convenient isolation of nicked lipoate acetyltransferase as well as unfragmented pyruvate dehydrogenase and lipoamide dehydrogenase from papain-treated complex under very mild conditions. The two uncleaved component enzymes have identical properties and similar specific activities as enzyme preparations obtained by other, more laborious procedures.
D000123:Acetyltransferases / Q000302:isolation & purification; D000818:Animals; D002417:Cattle; D008058:Dihydrolipoamide Dehydrogenase / Q000302:isolation & purification; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D004591:Electrophoresis, Polyacrylamide Gel; D007668:Kidney / Q000201:enzymology*; D008970:Molecular Weight; D010206:Papain / Q000494:pharmacology*; D011768:Pyruvate Dehydrogenase Complex / Q000302:isolation & purification*; D008063:Thioctic Acid / Q000031:analogs & derivatives / Q000302:isolation & purification
D011768:Pyruvate Dehydrogenase Complex; D008063:Thioctic Acid; D008058:Dihydrolipoamide Dehydrogenase; D000123:Acetyltransferases; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D010206:Papain