INACTIVATION OF THE PLASMA-MEMBRANE ATPASE OF SCHIZOSACCHAROMYCES-POMBE BY HYDROGEN-PEROXIDE AND BY THE FENTON REAGENT (FE2+ H2O2) - NONRADICAL VS. RADICAL-INDUCED OXIDATION/

In the absence of added Fe2+, the ATPase activity of isolated Schizosa ccharomyces pombe plasma membranes (5-7 mu mol P-i per mg protein per min) is moderately inhibited by H2O2 in a concentration-dependent mann er. Sizable inactivation occurs only at 50-80 mmol/L H2O2 The process, probably a direct oxidative action of H2O2 on the enzyme, is not indu ced by the indigenous membrane-bound iron (19.3 nmol/mg membrane prote in), is not affected by the radical scavengers mannitol and Tris, and involves a decrease of both the K-m of the enzyme for ATP and the V of ATP splitting. On exposing the membranes to the Fenton reagent (50 mu mol/L Fe2+ + 20 mmol/L H2O2), which causes a fast production of HO-. radicals, the ATPase is 50-60 % inactivated and 90 % of added Fe2+ is oxidized to Fe3+ within 1 min. The inactivation occurs only when Fe2is added before H2O2 and can thus bind to the membranes. The lack of e ffect of radical scavengers (mannitol, Tris) indicates that HO. radica ls produced in the bulk phase play no role in inactivation. Blockage o f the inactivation by the iron chelator deferrioxamine implies that th e process requires the presence of Fe2+ ions bound to binding sites on the enzyme molecules. Added catalase, which competes with Fe2+ for H2 O2, slows down the inactivation but in some cases increases its total extent, probably due to the formation of the superoxide radical that g ives rise to delayed HO. production.