Oxidative inactivation of brain alkaline phosphatase responsible for hydrolysis of phosphocholine.
Alkaline phosphatase, one of the enzymes responsible for the conversion of phosphocholine into choline, was purified from bovine brain membrane, where the phosphatase is bound as glycosylphosphatidylinositol-linked protein, and subjected to oxidative inactivation. The phosphatase activity, based on the hydrolysis of p-nitrophenyl phosphate and phosphocholine, decreased slightly after the exposure to H2O2. Inclusion of Cu2+ in the incubation with 1 mM H2O2 led to a rapid decrease of activity in a time- and concentration-dependent manner. In comparison, the H2O2/Cu2+ system was much more effective than the H2O2/Fe2+ system in inactivating brain phosphatase. In a further study, it was observed that the hydroxy radical scavengers mannitol, ethanol, or benzoate failed to prevent against H2O2/Cu2+-induced inactivation of the phosphatase, excluding the involvement of extraneous hydroxy radicals in metal-catalyzed oxidation. In addition, it was found that both substrates, p-nitrophenyl phosphate and phosphocholine, and an inhibitor, phosphate ion, at their saturating concentrations exhibited a remarkable, although incomplete, protection against the inactivating action of H2O2/Cu2+. A similar protection was also expressed by divalent metal ions such as Mg2+ or Mn2+. Separately, it was found that H2O2/Fe2+-induced inactivation was prevented by p-nitrophenyl phosphate or Mg2+ but not phosphate ions. Thus, it is implied that phosphocholine-hydrolyzing alkaline phosphatase in brain membrane might be one of enzymes susceptible to metal-catalyzed oxidation.
Sok
D E
DE
College of Pharmacy, Chungnam National University, Taejon, Korea.
D000469:Alkaline Phosphatase / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002413:Cations, Divalent / Q000378:metabolism; D002417:Cattle; D003300:Copper / Q000378:metabolism / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D016166:Free Radical Scavengers / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006868:Hydrolysis; D007202:Indicators and Reagents; D007501:Iron / Q000378:metabolism / Q000494:pharmacology; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction; D010767:Phosphorylcholine / Q000378:metabolism*; D013379:Substrate Specificity
D002413:Cations, Divalent; D016166:Free Radical Scavengers; D007202:Indicators and Reagents; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D016877:Oxidants; D010767:Phosphorylcholine; C008644:nitrophenylphosphate; D003300:Copper; D006861:Hydrogen Peroxide; D007501:Iron; D000469:Alkaline Phosphatase