A NEW METHOD FOR THE ISOLATION OF RAT-LIVER ACETYL-COA CARBOXYLASE

Abstract

Rat liver acetyl-CoA carboxylase was purified to homogeneity by a new method involving polyethylene glycol precipitation and DEAE and Sepharose 4B chromatography. The final product displays a single band on SDS [sodium dodecylsulfate] polyacrylamide gel electrophoresis of estimated MW 240,000. This material contains 5.5 .+-. 0.3 mol of alkali-labile phosphate/subunit and has a specific activity of 1.2 .+-. 0.2 U/mg protein. As compared to previous purification procedures for the liver enzyme, this product has a higher phosphate content, lower specific activity, and an absence of major proteolysis. Trypsin digestion of 32P-labeled acetyl-CoA carboxylase from hepatocytes reveals that the 32P-labeled phosphorylation sites are extremely labile to proteolytic digestion. Potential modification of isolated liver acetyl-CoA carboxylase by proteolysis and/or dephosphorylation must be ascertained prior to in vitro enzymatic studies.