Phosphorylation of branched-chain 2-oxo acid dehydrogenase complex in isolated adipocytes. Effects of 2-oxo acids.
Isolated adipocytes from rat epididymal fat-pads were incubated with [32P]Pi, and intracellular phosphoproteins were then analysed by SDS/polyacrylamide-gel electrophoresis and autoradiography. A phosphorylated polypeptide of apparent Mr 46,000 was identified as the alpha-subunit of branched-chain 2-oxo acid dehydrogenase complex by immunoprecipitation using antiserum raised against the homogeneous E1 component of branched-chain 2-oxo acid dehydrogenase complex. Immunoprecipitation of this phosphoprotein is blocked in a competitive manner by purified branched-chain 2-oxo acid dehydrogenase complex. Peptide mapping of the isolated phosphoprotein indicates that two sites on the polypeptide are phosphorylated in the intact cells. Addition of branched-chain 2-oxo acids to the incubation medium causes diminution in the extent of labelling of both phosphorylation sites on the alpha-subunit, an effect presumably mediated via their known inhibitory action on branched-chain 2-oxo acid dehydrogenase kinase. These observations provide direct evidence for phosphorylation of branched-chain 2-oxo acid dehydrogenase complex in intact cells.
D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D000273:Adipose Tissue / Q000166:cytology / Q000201:enzymology*; D000818:Animals; D001665:Binding Sites; D011232:Chemical Precipitation; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D007651:Keto Acids / Q000494:pharmacology; D007658:Ketone Oxidoreductases / Q000378:metabolism*; D008297:Male; D009097:Multienzyme Complexes / Q000378:metabolism*; D010748:Phosphopeptides / Q000032:analysis; D010750:Phosphoproteins / Q000276:immunology; D010766:Phosphorylation; D051381:Rats; D011919:Rats, Inbred Strains
D007651:Keto Acids; D009097:Multienzyme Complexes; D010748:Phosphopeptides; D010750:Phosphoproteins; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)