Pig liver pyruvate carboxylase. Purification, properties and cation specificity

Abstract

Pyruvate carboxylase was purified to apparent homogeneity from pig liver mitochondria and shown to be free of all kinetically contaminating enzymes. The enzyme has a mol wt of 520,000 and is composed of four subunits, each with a mol wt of 130,000. The enzyme can exist as the active tetramer, dimer and monomer, although the tetramer appears to be the form in which the enzyme is normally assayed. For every 520,000g of the enzyme there are 4 mol of biotin, 3 mol of zinc and 1 mol of magnesium. No significant concentrations of manganese were detected. Analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis indicates three polypeptide chains per monomer unit, each with a mol wt of 47,000. The amino acid analysis, stoicheiometry of the reaction and the activity of the enzyme as a function of pH are also presented. The enzyme is activated by a variety of univalent cations but not by Tris+ or triethanolamine+. The activity of the enzyme is dependent on the presence of acetyl CoA; the low rate in the absence of added acetyl CoA is not due to an enzyme bound acyl CoA. The dissociation constant for enzyme bound acetyl CoA is a marked function of pH.