The administration of prooxidants to whole animals leads to the inactivation of many enzymatic systems, including the hepatic, microsomal, ATP-dependent Ca++-pump. This inactivation involves the metabolism of the prooxidant by the cytochrome P-450 system to produce either a toxic metabolite or a reactive oxygen species. We have examined the effect of several prooxidants on the Ca++-pump and found that there are distinct biochemical differences in their mode of pump inactivation. Inactivation by CCl-4 was greatest under nitrogen whereas nitrogen blocks the inactivation of the pump by menadione, 2,3-dimethoxy-1,4-naphthoquinone and paraquat. Furthermore, the inactivation by these latter compounds was blocked by catalase and superoxide dismutase, which had no effect on the inactivation by CCl-4. Inactivation by none of these agents was affected by hydroxyl radical scavengers or metal chelators, except EDTA. The toxicity of none of these agents was due to lipid peroxidation. Of particular importance, the pump inactivation by CCl-4 was enzymatically both blocked and reversed by GSH, while that observed with menadione ani 2,3-dimethoxy-1-4-naphthoquinone was blocked but only partially reversed by GSH. The reversal of the inhibition of the Ca++-pump supports our hypothesis that the GSH protection of the pump from oxidant damage may be catalyzed by either the thiol:protein-disulfide oxidoreductases or the cytosolic glutathione peroxidases which reduces phospholipid hydroperoxides. Our overall goal is to determine which of these enzymes catalyzes the GSH protection of the Ca++-pump. To achieve this goal we propose four specific aims. First, we will determine whether the addition of either enzyme to intact microsomes enhances the GSH protection of the pump. Second, we will prepare antibodies to the Ca++-ATPase and Ca++-sequestration proteins and immunopurify these proteins from microsomes after incubation under a variety of conditions. These studies will indicate what effect oxidants have on the pump proteins and whether either of the GSH-dependent enzymes system we have purified could catalyze the GSH protection of the Ca++-pump. Third, we will incubate a partially reconstituted system consisting of purified cytochrome P-450 chain enzymes, Ca++-pump proteins and the GSH-dependent enzymes and determine what effect the prooxidants have on the pump proteins. Finally, we shall prepare monoclonal antibodies to the GSH-dependent enzymes to determine whether, in intact microsomes, the antibodies can block the protective effect of GSH observed during the metabolism of the various prooxidants.

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