Purification and properties of a protein activator of phosphorylated branched-chain 2-oxo acid dehydrogenase complex.
The protein activator of phosphorylated branched-chain 2-oxo acid dehydrogenase complex was purified greater than 1000-fold from extracts of rat liver mitochondria; the specific activity was greater than 1000 units/mg of protein (1 unit gives half-maximum re-activation of 10 munits of phosphorylated complex). Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis gave two bands (Mr 47700 and 35300) indistinguishable from the alpha- and beta-subunits of the branched-chain dehydrogenase component of the complex. On gel filtration (Sephacryl S-300), apparent Mr was 190000. This and other evidence suggests that activator protein is free branched-chain dehydrogenase; this conclusion is provisional until identical amino acid composition of the subunits has been demonstrated. Activator protein (i.e. free branched-chain dehydrogenase) was inhibited (up to 30%) by NaF, whereas branched-chain complex was not inhibited. There was no convincing evidence for interconvertible active and inactive forms of activator protein in rat liver mitochondria. Activator protein was detected in mitochondria from liver (ox, rabbit and rat) and kidney (ox and rat), but not in rat heart or skeletal-muscle mitochondria. In rat liver mitochondrial extracts, branched-chain complex sedimented with the mitochondrial membranes, whereas activator protein remained in the supernatant. Activator protein re-activated phosphorylated (inactive) particulate complex from rat liver mitochondria, but it did not activate dephosphorylated complex. Liver and kidney, but not muscle, mitochondria apparently contain surplus free branched-chain dehydrogenase, which is bound by the complex with lower affinity than is the branched-chain dehydrogenase intrinsic to the complex. It is suggested that this functions as a buffering mechanism to maintain branched-chain complex activity in liver and kidney mitochondria.
D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D000818:Animals; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004032:Diet; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D066298:In Vitro Techniques; D007658:Ketone Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008930:Mitochondria, Liver / Q000201:enzymology; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010766:Phosphorylation; D011506:Proteins / Q000494:pharmacology*; D051381:Rats; D012969:Sodium Fluoride / Q000494:pharmacology; D014018:Tissue Distribution
D009097:Multienzyme Complexes; D011506:Proteins; D012969:Sodium Fluoride; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)