Phosphorylation of branched-chain 2-oxo acid dehydrogenase complex in isolated adipocytes. Effects of 2-oxo acids

Abstract

Isolated adipocytes from rat epididymal fat-pads were incubated with [32P]P(i), and intracellular phosphoproteins were then analysed by SDS/polyacrylamide-gel electrophoresis and autoradiography. A phosphorylated polypeptide of apparent M(r) 46,000 was identified as the α-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 and 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 α-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.