2-oxoglutarate dehydrogenase complex from bovine-adrenal-cortex mitochondria. Purification and partial characterization

Abstract

The 2-oxoglutarate dehydrogenase complex from bovine adrenal-cortex mitochondria has been purified by polyethylene glycol fractionation, ultracentrifugation through a layer of sucrose, isoelectric precipitation and gel filtration of Sepharose 4 B. The specific activity of the preparation obtained wa 9.9 U/mtg of protein; the sedimentation coefficient, S20, w, was 30 S. The results of sodium dodecyl sulphate polyacrylamide gel electrophoresis indicated decomposition of the 2-oxoglutarate dehydrogenase complex into 3 clear-cut protein fractions with mobilities corresponding to molecular weights of 51 000, 56 000 and about 110 000. Michaelis constants for the reactants of the 2-oxoglutarate dehydrogenase complex reactions were: 2-oxoglutarate = 200 micro M; CoA - 4,5 micro M; NAD - 25 micro M.