Purification and properties of inorganic pyrophosphatase of rat liver and hepatoma 3924A.
Inorganic pyrophosphatase (EC 3.6.1.1) has been purified to electrophoretic homogeneity from the soluble portion of the cytoplasm of rat Hepatoma 3924A and rat liver. It has a specific activity of 600 to 700 mumol inorganic orthophosphate liberated per min per mg protein at 25 degrees, a value in the same range as the highly purified enzymes from yeast and Escherichia coli. By all criteria applied, the hepatoma inorganic pyrophosphatase is identical with the liver enzyme. It is a dimer with subunits with molecular weights of approximately 30,000 to 33,000 and has a pH optimum of 7.4, a Km for pyrophosphate of 5 microM, and a Ka for Mg2+ of 0.3 mM with a pyrophosphate concentration of 0.2 mM. It is not inhibited by high Mg2+ concentrations up to 20 mM. Other metal ions such as Zn2+ and Ca2+ do not activate. Mn2+ activates to less than 10% that of Mg2+ at 0.6 mM and has no effect at 1 mM or higher. In the presence of optimal (4 mM) Mg2+ concentration, Ca2+, Mn2+, Hg2+, and F- at 0.2 mM inhibited strongly, but Zn2+ at 1 mM was not inhibitory. The enzyme had no phosphatase activity toward any of the purine or pyrimidine nucleoside mono-, di-, and triphosphates or toward p-nitrophenyl phosphate, beta-glycerophosphate, glucose 6-phosphate, or glucose 1-phosphate. Bromo- or iodoacetate at high concentration had no inhibitory effect, but p-chloromercuribenzoate and p-chloromercuriphenylsulfonate inhibited strongly at low concentration. The purified enzyme was very unstable but was protected markedly at or above the pH optimum of 7.4 by cysteine, dithiothreitol, and glutathione.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D011756:Diphosphates / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D005978:Glutathione / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D043564:Inorganic Pyrophosphatase; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008114:Liver Neoplasms, Experimental / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D011755:Pyrophosphatases / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity; D013696:Temperature
D011756:Diphosphates; D009596:Nitrophenols; D009943:Organophosphorus Compounds; C008644:nitrophenylphosphate; D000255:Adenosine Triphosphate; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D005978:Glutathione; D008274:Magnesium; D004229:Dithiothreitol