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Purification and Properties of Inorganic Pyrophosphatase of Rat Liver and Hepatoma 3924A¹

Fels Research Institute, Temple University School of Medicine, Philadelphia, Pennsylvania 19140

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 µmol inorganic orthophosphate liberated per min per mg protein at 25°, 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 K_m for pyrophosphate of 5 µM, and a K_a 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, ß-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.

¹ This work was aided by Grants BC-74 from the American Cancer Society and Grants CA-10916 and CA-12227 from the National Cancer Institute, Department of Health and Human Services. We also acknowledge with thanks generous support from the General Research support grant to Temple University School of Medicine.

² To whom requests for reprints should be addressed.

Received 3/15/82. Accepted 5/28/82.

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