Purification of rat kidney branched-chain oxo acid dehydrogenase complex with endogenous kinase activity

Abstract

A method was devised to purify branched-chain oxo acid dehydrogenase (BCOAD) from rat kidney which retains endogenous kinase activity. Incorporation of 32P into purified enzyme parallels the time course of enzyme inhibition by ATP. Phosphorylation occurs on a serine residue(s) of the 4600-MW subunit of the enzyme complex. Endogenous phosphatase activity is not present after purification, and added pyruvate dehydrogenase phosphate phosphatase does not re-activate BCOAD or liberate 32P from previously labeled enzyme. Apparently, BCOAD can be regulated by an endogenous protein kinase and the phosphorylation-cycle enzymes regulating BCOAD appear to be distinct from those associated with pyruvate dehydrogenase complex.