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 MnCl2 to hydrolyze PPi and was insensitive to 2 mM sodium fluoride. The purified hydrolase released 2.1 mumol Pi from PPi per minute per milligram of protein in the presence of 1 mM MgCl2, and the Km for PPi was 0.14 mM. It also hydrolyzed imidodiphosphate to yield Pi and ammonia even in the absence of a divalent cation. The purified hydrolase liberated 2.2 mumol Pi from imidodiphosphate per minute per milligram of protein and the Km for imidodiphosphate was 0.12 mM. Addition of 80 microM MgCl2, CoCl2, or MnCl2 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 PPi (Ki = 0.13 mM). Moreover, one-half of the maximal hydrolysis of imidodiphosphate was inhibited by 10 microM trans-1,2-diaminocyclohexane N,N,N',N'-tetraacetic acid or 45 microM p-chloromercuriphenyl sulfonate. When the hydrolase was treated with heat or SDS, two activities capable of hydrolyzing PPi and imidodiphosphate gave similar inactivation profiles, indicating that one hydrolase participated in the hydrolysis of both substrates.
Hiraishi
H
H
Department of Biochemistry, Saga Medical School, Japan.
Ohmagari
T
T
Otsuka
Y
Y
Yokoi
F
F
Kumon
A
A
D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D002417:Cattle; D004164:Diphosphonates / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D043564:Inorganic Pyrophosphatase; D008099:Liver / Q000201:enzymology*; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D011489:Protein Denaturation; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013696:Temperature; D014461:Ultracentrifugation
D002413:Cations, Divalent; D004164:Diphosphonates; D008670:Metals; D013439:Sulfhydryl Reagents; C016514:imidodiphosphonic acid; D012967:Sodium Dodecyl Sulfate; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase