Purification and molecular characteristics of mitochondrial phosphoenolpyruvate carboxykinase from bullfrog (Rana catesbeiana) liver.
Phosphoenolpyruvate carboxykinase from bullfrog liver mitochondria has been purified to electrophoretical and immunological homogeneity by an improved method using hydrophobic chromatography on Sepharose-hexane-GMP and affinity chromatography on phosphocellulose. The molecular weight was determined to be 70,000 by SDS-gel electrophoresis, 65,000 by Sephadex G-100 gel filtration and 72,000 by glycerol gradient centrifugation. The isoelectric point was determined to be 6.2, differing from that of the cytosol enzyme. The rabbit IgG fraction against the mitochondrial PEP carboxykinase precipitated not only the mitochondrial but also the cytosol enzyme. The dissociation constant of the nucleotide-enzyme complex was determined to be 3 microM for GTP, 8.5 microM for GDP, and 171 microM for GMP. The affinity of GTP for the enzyme was reduced in the presence of phosphoenolpyruvate or Mn2+, whereas that of GDP was not changed. GMP inhibited the enzyme competitively with GDP for the phosphoenolpyruvate carboxylation and competitively with GTP for the exchange reaction between [14C]HCO3- and oxaloacetate. The purified enzyme was found to have a cysteine residue which reacted with iodoacetamide to form inactive enzyme. Guanine nucleotides or IDP and Mn2+ at a lower concentration prevented the inactivation by iodoacetamide of the enzyme in a competitive manner. Binding of guanine nucleotide to the enzyme and the relation of the sulfhydryl group to the nucleotide binding are discussed.
D000818:Animals; D006150:Guanine Nucleotides / Q000378:metabolism; D007460:Iodoacetamide / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008930:Mitochondria, Liver / Q000201:enzymology*; D008970:Molecular Weight; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000302:isolation & purification* / Q000378:metabolism; D011892:Rana catesbeiana
D006150:Guanine Nucleotides; D008345:Manganese; D010729:Phosphoenolpyruvate Carboxykinase (GTP); D008274:Magnesium; D007460:Iodoacetamide