Isolation of rabbit liver branched chain alpha-ketoacid dehydrogenase and regulation by phosphorylation.

Abstract

Branched chain a-ketoacid dehydrogenase was purified 1,400-fold from a Triton X-100 extract of rabbit liver by polyethylene glycol fractionation, chromatography on hydroxyapatite and Sepharose CL-2B, and high speed centrifugation.The complex eluted from Sepharose CL-2B prior to blue dextran, indicating an apparent M, > 2 x 10'.Electrophoresis on sodium dodecyl sulfate-polyacrylamide gels indicated subunits of M, = 51,000,47,000,45,000, and 38,000.Specific activity (pmol/min/mg of protein at 30 "c) was 5.2 with 200 p M a-ketoisovalerate.Full expression of enzyme activity required exogenous dihydrolipoamide reductase.The apparent K,,, values of the complex for a-ketoisovalerate, a-ketoisocaproate, a-keto-/3-methylvalerate, and pyruvate were 28,15, 14, and 715 p ~, respectively.The high K,,, for pyruvate and the lack of a M, = 42,000 subunit suggest that the complex was free of pyruvate dehydrogenase.Incubation of the complex with Ng+ plus ATP resulted in a time-dependent loss of activity, whereas with only M g + or ATP there was no loss in activity.Incubation of the complex with [y3'P]ATP resulted in a time-dependent increase in 32P incorporation which was located in the M, = 47,000 subunit.The pH optimum for the kinase reaction was approximately 7.1.Mg+ concentration for maximum kinase activity was 1.5 m M and for half-maximum activity, approximately 25 PM.Ca" inhibited kinase activity at optimum MgZ' concentration.The apparent K,,, of the kinase for ATP was 25 p ~.was a competitive inhibitor with respect to ATP (apparent Ki of 130 p ~) .Dichloroacetate protected against phosphorylation and inactivation of the complex, but higher concentrations of dichloroacetate were required to inhibit branched chain a-ketoacid dehydrogenase kinase than pyruvate dehydrogenase kinase.a-Ketoisocaproate protected the complex against phosphorylation by ATP but caused a paradoxical time-dependent inactivation of the complex independent of ATP.Branched chain a-ketoacid dehydrogenase (EC 1.2.4.4) complex, located within the matrix space of the mitochondrion (l), catalyzes the oxidative decarboxylation of the transamination products of leucine, isoleucine, and valine, uiz.a-ketoisocaproate, a-keto-/3-methylvalerate, and a-ketoisovderate,