Oxidative Inactivation of Brain Alkaline Phosphatase Responsible for Hydrolysis of Phosphocholine

다국어 초록 (Multilingual Abstract)

Alkaline phosphatase, on of the enzymes responsible for the conversion of phosphocholine into choline, was purified from bovine brain membrane, where the phosphatase is bound as glycosylphosphatidylinositollinked protein, and subjected to oxidative inactivation. The phosphate activity, based on the hydrolysis of p-nitrophenyl phosphate and phosphocholine, decreased slightly after the exposure to H_2O_2. Inclusion of Cu^2+ in the incubation with 1 mM H_2O_2 led to a rapid decrease of activity in a time-and concentration-dependent manner. In comparison, the H_2O_2/Cu^2+ system was much more effective than the H_2O_2/Fe^2+ 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 H_2O_2/Cu^2+-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-nitrophenylphosphate and phosphocholine, and an inhibitor, phosphate ion, at their saturating concentrations exhibited a remarkable, although incomplete, protection against the inactivating action of H_2O_2/Cu^2+. A similar protection was also expressed by divalent metal ions such as Mg^2+ or Mn^2+. Separately, it was found that H_2O_2/Fe^2+ -induced inactivation was prevented by p-nitrophenyl phosphate or Mg^2+ 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.