Purification and Characterization of Hepatic Inorganic Pyrophosphatase Hydrolyzing Imidodiphosphate

Abstract 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 MgCl 2 , CoCl 2 , and MnCl 2 to hydrolyze PP i and was insensitive to 2 m m sodium fluoride. The purified hydrolase released 2.1 μmol P i from PP i per minute per milligram of protein in the presence of 1 m m MgCl 2 , and the K m for PP i was 0.14 m m . It also hydrolyzed imidodiphosphate to yield P i and ammonia even in the absence of a divalent cation. The purified hydrolase liberated 2.2 μmol P i from imidodiphosphate per minute per milligram of protein and the K m for imidodiphosphate was 0.12 m m . Addition of 80 μ m MgCl 2 , CoCl 2 , or MnCl 2 to 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 PP i ( K i = 0.13 m m ). Moreover, one-half of the maximal hydrolysis of imidodiphosphate was inhibited by 10 μ m trans -1,2-diaminocyclohexane N,N,N′,N′ -tetraacetic acid or 45 μ m p -chloromercuriphenyl sulfonate. When the hydrolase was treated with heat or SDS, two activities capable of hydrolyzing PP i and imidodiphosphate gave similar inactivation profiles, indicating that one hydrolase participated in the hydrolysis of both substrates.