Pig liver pyruvate carboxylase. Purification, properties and cation specificity.
1. Pyruvate carboxylase was purified to apparent homogeneity from pig liver mitochondria and shown to be free of all kinetically contaminating enzymes. 2. The enzyme has a mol. wt. of 520000 and is composed of four subunits, each with a mol. wt. of 130000. 3. 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. 4. For every 520000g of the enzyme there are 4mol of biotin, 3mol of zinc and 1mol of magnesium. No significant concentrations of manganese were detected. 5. Analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis indicates three polypeptide chains per monomer unit, each with a mol. wt. of 47000. 6. The amino acid analysis, stoicheiometry of the reaction and the activity of the enzyme as a function of pH are also presented. 7. The enzyme is activated by a variety of univalent cations but not by Tris(+) or triethanolamine(+). 8. 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.
D000105:Acetyl Coenzyme A; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001710:Biotin / Q000032:analysis; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000032:analysis; D008345:Manganese; D008930:Mitochondria, Liver / Q000201:enzymology*; D008970:Molecular Weight; D011766:Pyruvate Carboxylase / Q000032:analysis / Q000302:isolation & purification; D012967:Sodium Dodecyl Sulfate; D013053:Spectrophotometry; D013552:Swine; D015032:Zinc / Q000032:analysis
D000596:Amino Acids; D046911:Macromolecular Substances; D012967:Sodium Dodecyl Sulfate; D008345:Manganese; D001710:Biotin; D000105:Acetyl Coenzyme A; D011766:Pyruvate Carboxylase; D008274:Magnesium; D015032:Zinc