THE PURIFICATION AND PROPERTIES OF ACETYL-COENZYME A CARBOXYLASE KINASE

BRUCE ALAN LENT, Purdue University

Abstract

Acetyl-CoA carboxylase kinase has been purified to apparent homogeneity from rat liver. The kinase was found to exist in two forms: bound to carboxylase in a complex or in a free form that is in different stages of aggregation over a wide range of molecular weights. The molecular weight of the kinase subunit was 170,000 as determined by SDS gel electrophoresis. The incorporation of 1 mole of phosphate per mole of carboxylase subunit caused complete inactivation of the carboxylase. Acetyl-CoA carboxylase, inactivated by the action of the kinase, can be dephosphorylated and reactivated when incubated with phosphorylase phosphatase. The K(,m) of the kinase for acetyl-CoA carboxylase and ATP are 93 nM and 20 (mu)M, respectively. The kinase was found to be cAMP-independent, but activated by CoA. The protein kinase can phosphorylate acetyl-CoA carboxylase protamine, and histones, but could not act on HMG-CoA reductase or phosphorylase b. Phosphorylation and inactivation of acetyl-CoA carboxylase by acetyl-CoA carboxylase kinase requires coenzyme A. Coenzyme A did not enhance the phosphorylation of alternative substrates of the carboxylase kinase such as protamine or histones. The presence of Coenzyme A did not alter the K(,m) of the carboxylase kinase for its substrates, ATP and acetyl-CoA carboxylase. Fluorescence binding studies showed that CoA binds to carboxylase but not to the kinase. The K(,D) of CoA binding to carboxylase is 27 (mu)M. These results indicate that Coenzyme A, acting on acetyl-CoA carboxylase, may play an important role in the regulation of the covalent modification mechanism for acetyl-CoA carboxylase. The addition of the catalytic subunit of cyclic AMP-dependent protein kinase stimulates the inactivation of acetyl-CoA carboxylase by activating acetyl-CoA carboxylase kinase. Activation of the carboxylase kinase is accompanied by the incorporation of 0.6 moles of phosphate per mole of carboxylase kinase. The addition of the regulatory subunit of cyclic-AMP dependent protein kinase prevents the stimulation of carboxylase inactivation. The inactivation of carboxylase by the carboxylase kinase requires the presence of Coenzyme A even when using the activated carboxylase kinase. The enhanced inactivation of carboxylase is due to its increase phosphorylation and not to inhibition by the phosphorylated carboxylase kinase.

Degree

Ph.D.

Subject Area

Biology

COinS