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, a nd subjected to oxidative inactivation. The phosphatase activity, based on the hydrolysis of p-nitrophenyl phosphate and phosphocholine, decreased sli ghtly 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 effecti ve than the H2O2/Fe2+ system in inactivating brain phosphatase. In a furthe r study, it was observed that the hydroxy radical scavengers mannitol, etha nol, or benzoate failed to prevent against H2O2/Cu2+-induced inactivation o f the phosphatase, excluding the involvement of extraneous hydroxy radicals in metal-catalyzed oxidation, In addition, it was found that both substrat es, p-nitrophenyl phosphate and phosphocholine, and an inhibitor, phosphate ion, at their saturating concentrations exhibited a remarkable, although i ncomplete, protection against the inactivating action of H2O2/Cu2+. A Simil ar protection was also expressed by divalent metal ions such as Mg2+ or Mn2 +. Separately, it was found that H2O2/Fe2+-induced inactivation was prevent ed by p-nitrophenyl phosphate or Mg2+ but not phosphate ions. Thus, it is i mplied that phosphocholine-hydrolyzing alkaline phosphatase in brain membra ne might be one of enzymes susceptible to metal-catalyzed oxidation.