Inorganic pyrophosphate-phosphohydrolytic activity associated with rat osseous plate alkaline phosphatase.
Purified membrane-bound alkaline phosphatase from rat osseous plate hydrolyzed pyrophosphate in the presence of magnesium ions, with a specific activity of 92.7 U/mg. Optimal apparent pH for pyrophosphatase activity was 8.0 and it remained unchanged on increasing the pyrophosphate concentration. In the absence of magnesium ions the enzyme had a Km = 88 microM and V = 36.7 U/mg for pyrophosphate and no inhibition by excess substrate was observed. Pyrophosphatase activity was rapidly destroyed at temperatures above 40 degrees C, but magnesium ions apparently protected the enzyme against denaturation. Sodium metavanadate (Ki = 1.0 mM) was a competitive inhibitor of pyrophosphatase activity, while levamisole (Ki = 8.2 mM) and theophylline (Ki = 7.4 mM) were uncompetitive inhibitors. Magnesium ions (K0.5 = 1.7 microM) stimulated pyrophosphatase activity, while cobalt (Ki = 48.5 microM) and zinc (Ki = 22.0 microM) ions were non-competitive inhibitors. Manganese and calcium ions had no effect on pyrophosphatase activity. The Mw of the pyrophosphatase protein was 130 kDa by gel filtration, but a value of 65 kDa was obtained by dissociative gel electrophoresis, suggesting that it was a dimer of apparently identical subunits. These results suggested that pyrophosphatase activity stems from the membrane-bound osseous plate alkaline phosphatase and not from a different protein.
Rezende
L A
LA
Departamento de Química, Faculdade de Filosofia, Ciências e Letras, USP, Ribeirão Preto, SP Brasil.
Ciancaglini
P
P
Pizauro
J M
JM
Leone
F A
FA
D000469:Alkaline Phosphatase / Q000032:analysis*; D000814:Aniline Compounds / Q000378:metabolism; D000818:Animals; D001857:Bone Matrix / Q000637:transplantation*; D002118:Calcium / Q000494:pharmacology; D003035:Cobalt / Q000494:pharmacology; D018483:Diaphyses / Q000637:transplantation*; D019281:Dimerization; D011756:Diphosphates / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology; D004790:Enzyme Induction; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000201:enzymology*; D006132:Growth Plate / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D043564:Inorganic Pyrophosphatase; D007978:Levamisole / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D009943:Organophosphorus Compounds / Q000378:metabolism; D010012:Osteogenesis; D011489:Protein Denaturation; D011755:Pyrophosphatases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D051381:Rats; D017208:Rats, Wistar; D013696:Temperature; D013806:Theophylline / Q000494:pharmacology; D014638:Vanadates / Q000494:pharmacology; D015032:Zinc / Q000494:pharmacology
D000814:Aniline Compounds; D011756:Diphosphates; D004791:Enzyme Inhibitors; D009943:Organophosphorus Compounds; D007978:Levamisole; D003035:Cobalt; D014638:Vanadates; D008345:Manganese; C068798:4-aminophenylphosphate; D004492:Edetic Acid; D013806:Theophylline; D000469:Alkaline Phosphatase; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D008274:Magnesium; D015032:Zinc; D002118:Calcium