Inactivation of the plasma membrane ATPase ofSchizosaccharomyces pombe by hydrogen peroxide and by the fenton reagent (Fe2+/H2O2): Nonradicalvs. Radical-induced oxidation |
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Authors: | K Sigler G Gille V Vacata N Stadler M Höfer |
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Institution: | (1) Institute of Microbiology, Academy of Sciences of the Czech Republic, 142 20 Prague 4, Czech Republic;(2) Institute of Medical Chemistry, University of Veterinary Medicine, A-1210 Vienna, Austria;(3) Institute of Botany, University of Bonn, 53115 Bonn, Germany |
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Abstract: | In the absence of added Fe2+, the ATPase activity of isolatedSchizosaccharomyces pombe plasma membranes (5–7 μmolP
i per mg protein per min) is moderately inhibited by H2O2 in a concentration-dependent manner. Sizable inactivation occurs only at 50–80 mmol/L H2O2. The process, probably a direct oxidative action of H2O2 on the enzyme, is not induced by the indigenous membrane-bound iron (19.3 nmol/mg membrane protein), is not affected by the
radical scavengers mannitol and Tris, and involves a decrease of both theK
m of the enzyme for ATP and theV of ATP splitting. On exposing the membranes to the Fenton reagent (50 μmol/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 Fe2+ is added before H2O2 and can thus bind to the membranes. The lack of effect of radical scavengers (mannitol, Tris) indicates that HO− radicals produced in the bulk phase play no role in inactivation. Blockage of the inactivation by the iron chelator deferrioxamine
implies that the process requires the presence of Fe2+ ions bound to binding sites on the enzyme molecules. Added catalase, which competes with Fe2+ for H2O2, slows down the inactivation but in some cases increases its total extent, probably due to the formation of the superoxide
radical that gives rise to delayed HO− production. |
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