Purification and immunochemical studies of pyruvate dehydrogenase complex from rat heart, and cell-free synthesis of lipoamide dehydrogenase, a component of the complex.
Pyruvate dehydrogenase complex was purified from rat heart. The complex showed four polypeptide bands on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, corresponding to lipoate acetyltransferase (mol.wt. 68 000), lipoamide dehydrogenase (mol.wt. 56 000), alpha-subunit (mol.wt. 41 000) and beta-subunit (mol.wt. 35 000) of pyruvate dehydrogenase. Rat heart pyruvate dehydrogenase complex was dissociated into three component enzymes and the antibodies against each component enzyme were prepared. Anti-pyruvate dehydrogenase and anti-lipoate acetyltransferase antibodies effectively precipitated pyruvate dehydrogenase complex, but an anti-lipoamide dehydrogenase antibody released lipoamide dehydrogenase from the complex and effectively precipitated lipoamide dehydrogenase. Lipoamide dehydrogenase was synthesized in a cell-free reticulocyte lysate system with total RNA from rat liver. Its translation product was detected as a putative precursor which is 3000 Da larger than the mature subunit. In cell-free translation programmed with free and membrane-bound polysomes, activity of mRNA coding for the precursor of the enzyme was much higher in free polysomes than in membrane-bound polysomes.
D000818:Animals; D002474:Cell-Free System; D008058:Dihydrolipoamide Dehydrogenase / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D007106:Immune Sera; D005779:Immunodiffusion; D007700:Kinetics; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D011132:Polyribosomes / Q000378:metabolism; D014176:Protein Biosynthesis; D011768:Pyruvate Dehydrogenase Complex / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D011817:Rabbits; D051381:Rats; D012156:Reticulocytes / Q000378:metabolism
D007106:Immune Sera; D011768:Pyruvate Dehydrogenase Complex; D008058:Dihydrolipoamide Dehydrogenase