ACTIVATION OF ACETYL-COA CARBOXYLASE - PURIFICATION AND PROPERTIES OF A MN-2+-DEPENDENT PHOSPHATASE

Abstract

Acetyl-CoA carboxylase was isolated from rat liver by polyethylene glycol precipitation and avidin affinity chromatography. Sodium dodecyl sulfate electrophoresis of the enzyme gives 1 protein band (MW 250,000). Phosphate analysis of the carboxylase showed the presence of 8.3 mol of phosphate/mol of subunit (MW 250,000). The purified carboxylase has low activity in the absence of citrate (specific activity = 0.3 U/mg). Addition of 10 mM citrate activates the carboxylase 10-fold, with half-maximal activation observed at 2 mM citrate, well above the physiological citrate level. Using this carboxylase as a substrate, a protein that activates the enzyme .apprx. 10-fold was isolated from rat liver. This protein was purified to near homogeneity (MW 90,000). Incubation of this protein with 32P-labeled acetyl-CoA carboxylase results in a time-dependent activation of carboxylase with concomitant release of 32Pi, indicating that this protein is a phosphoprotein phosphatase. Both activation and dephosphorylation are dependent on Mn2+, but not citrate. This phosphatase doss not hydrolyze p-nitrophenyl phosphate but does show high affinity for acetyl-CoA carboxylase (Km = 0.2 .mu.M) as compared to its action on phosphorylase a (Km = 5.5 .mu.M) and phosphohistone (Km = 20 .mu.M). Activated acetyl-CoA carboxylase was isolated after dephosphorylation by the phosphatase. Such preparations contain .apprx. 5 mol of phosphate/mol of subunit and have specific activities of 2.6-3.0 U/mg in the absence of citrate. These activities are comparable to those of the phosphorylated carboxylase in the presence of 10 mM citrate. Thus, dephosphorylation by the Mn2+-dependent phosphatse renders the carboxylase citrate-independent, as compared to the phosphorylated form, which is citrate-dependent. This is the 1st report of a preparation of animal acetyl-CoA carboxylase that has substantial catalytic activity independent of citrate.