Purification and characterization of cytosol-specific phosphoenolpyruvate carboxykinase from chicken liver.
Phosphoenolpyruvate carboxykinase of chicken liver cytosol was purified to homogeneity by procedures including affinity chromatography with GTP as a ligand. The purified enzyme showed a molecular weight of 68,000 on gel electrophoresis in the presence of dodecyl sulfate. Comparative studies on this enzyme and its isozyme purified from chicken liver mitochondria were performed. As regards amino acid composition, the cytosolic enzyme was quite different from the mitochondrial enzyme, but was rather similar to rat liver cytosolic phosphoenolpyruvate carboxykinase. Specific activities of the cytosolic enzyme were 30-100% higher than those of the mitochondrial enzyme for oxaloacetate-CO2 exchange, oxaloacetate decarboxylation, and phosphoenolpyruvate carboxylation reactions, though the relative rates of the activities were similar, decreasing in the order given. Apparent Michaelis constants for oxaloacetate in the oxaloacetate decarboxylation reaction were 11.6 and 17.9 microM for the cytosolic and the mitochondrial enzyme, respectively, but the values for GTP, GDP, phosphoenolpyruvate, and CO2 in the oxaloacetate decarboxylation and phosphoenolpyruvate carboxylation reactions were 1.3-2.2 times higher for the cytosolic enzyme than for the mitochondrial enzyme. Thus, the fundamental catalytic properties of the chicken liver phosphoenolpyruvate carboxykinase isozymes were rather similar, despite the marked difference in amino acid compositions.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002645:Chickens; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003600:Cytosol / Q000201:enzymology*; D003653:Decarboxylation; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D008099:Liver / Q000201:enzymology*; D008930:Mitochondria, Liver / Q000201:enzymology; D008970:Molecular Weight; D010071:Oxaloacetates / Q000378:metabolism; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*
D000596:Amino Acids; D010071:Oxaloacetates; D010729:Phosphoenolpyruvate Carboxykinase (GTP)