Purification and Characterization of Hepatic Inorganic Pyrophosphatase Hydrolyzing Imidodiphosphate

A 56-kDa inorganic pyrophosphatase consisting of 33-kDa subunits was purified from bovine liver almost to homogeneity. This hydrolase required divalent cations such as MgCl2, CoCl2, and MnCl2to hydrolyze PPiand was insensitive to 2 mmsodium fluoride. The purified hydrolase released 2.1 μmol Pifrom PPiper minute per milligram of protein in the presence of 1 mmMgCl2, and theKmfor PPiwas 0.14 mm. It also hydrolyzed imidodiphosphate to yield Piand ammonia even in the absence of a divalent cation. The purified hydrolase liberated 2.2 μmol Pifrom imidodiphosphate per minute per milligram of protein and theKmfor imidodiphosphate was 0.12 mm. Addition of 80 μmMgCl2, CoCl2, or MnCl2to the reaction mixture increased the hydrolysis rate of imidodiphosphate by 1.5-, 2.0-, and 3.4-fold, respectively. In the absence of divalent cations, the enzymatic hydrolysis of imidodiphosphate was inhibited competitively by PPi(Ki= 0.13 mm). Moreover, one-half of the maximal hydrolysis of imidodiphosphate was inhibited by 10 μmtrans-1,2-diaminocyclohexaneN,N,N′,N′-tetraacetic acid or 45 μmp-chloromercuriphenyl sulfonate. When the hydrolase was treated with heat or SDS, two activities capable of hydrolyzing PPiand imidodiphosphate gave similar inactivation profiles, indicating that one hydrolase participated in the hydrolysis of both substrates.