Identification of specific subunits of highly purified bovine liver branched-chain ketoacid dehydrogenase.
Branched-chain alpha-ketoacid dehydrogenase has been purified to homogeneity from bovine liver mitochondria. The isolated complex has a specific activity of 5-8 mumol of reduced nicotinamide adenine dinucleotide min-1 (mg of protein)-1 as isolated and does not require the addition of exogenous lipoamide dehydrogenase for activity. Addition of porcine heart lipoamide dehydrogenase stimulated complex activity by no more than 20%. Four subunits copurify with the complex with molecular weights by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 55 000, 52 000, 46 500, and 37 500. Here we show that the 52 000-dalton subunit is the lipoyl-containing transacylase component of the complex. Data are presented to support the hypothesis that the branched-chain ketoacid dehydrogenase complex is physically and catalytically similar to, but separate from, the pyruvate and alpha-ketoglutarate dehydrogenase complexes. The transacylase of the branched-chain ketoacid dehydrogenase complex has an exposed trypsin-sensitive region. Proteolytic action of trypsin separates a lipoyl-containing component from the remainder of the protein. Data from our laboratory presented here and elsewhere define a specific function for three of the four subunits.
D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D000818:Animals; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D007658:Ketone Oxidoreductases / Q000032:analysis*; D046911:Macromolecular Substances; D008930:Mitochondria, Liver / Q000201:enzymology*; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000032:analysis*; D014357:Trypsin / Q000378:metabolism
D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D014357:Trypsin