title: Purification and characterization of branched chain alpha-keto acid dehydrogenase complex of bovine kidney.
abstract: A branched chain alpha-keto acid dehydrogenase-dihydrolipoyl transacylase complex was purified to apparent homogeneity from bovine kidney mitochondria. As usually isolated, the complex (s(20,w) = 40 S) contained little, if any, dihydrolipoyl dehydrogenase. When saturated with the latter enzyme the complex had a specific activity of about 12 mumol of alpha-ketoisovalerate oxidized per min per mg of protein at 30 degrees with NAD(+) as electron acceptor. In addition to alpha-ketoisovalerate, the complex also oxidized alpha-ketoisocaproate, alpha-keto-beta-methylvalerate, alpha-ketobutyrate, and pyruvate. The ratios of the specific activities were 2.0:1.5:1.0:1.0:0.4, and the apparent K(m) values were 40, 50, 37, 56, and 1000 muM. The complex was separated into its component enzymes. The branched chain alpha-keto acid dehydrogenase (6 S) consists of two different subunits with estimated molecular weights of 46,000 and 35,000. The dihydrolipoyl transacylase (20 S) contains apparently identical subunits of molecular weight about 52,000. In the electron microscope, the transacylase has the appearance of a cube, and the molecules of branched chain alpha-keto acid dehydrogenase appear to be distributed on the surface of the cube. In contrast to the pyruvate dehydrogenase complex of bovine kidney, the branched chain alpha-keto acid dehydrogenase complex apparently is not regulated by phosphorylation-dephosphorylation. Its activity, however, is subject to modulation by end-product inhibition.
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel; D007651:Keto Acids; D007658:Ketone Oxidoreductases / Q000302:isolation & purification*; D007668:Kidney / Q000201:enzymology; D008854:Microscopy, Electron; D008928:Mitochondria / Q000201:enzymology; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000302:isolation & purification*
chemical_list: D007651:Keto Acids; D009097:Multienzyme Complexes; D007658:Ketone Oxidoreductases