In the presence of oxidants many enzymatic systems show reduced activity. The calcium pump of the endoplasmic reticulum is one such system. Further it has been found for this pump that the inhibitory effect of oxidants is partially reversed by reduced glutathione. In our preliminary studies, we have observed that the hepatic, microsomal ATP-dependent calcium pump activity is reduced through an oxidative process catalyzed by cytochrome P- 450. As in other oxidative systems, this inhibition is reversed by catalase and reduced glutathione. Substrates and ingibitors of the cytochrome P-450 dependent mixed funciton oxidases partially reverses this inhibition. Menadione, but not paraquat or nitrofurazone, increases the inhibition of the calcium pump by NADPH. Carbon tetrachloride inhibits the pump directly in the presence of NADP+ and this inhibition is markedly increased by the addition of NADPH. Finally in preliminary studies, we have found that the balance between the oxidative and reductive processes is modulated by catecholamines and glucagon. It is the purpose of the current proposal to determine whether the reversal of the oxidant damage by reduced glutathione is catalyzed by the enzyme thiol:protein-disulfide oxidoreductase. In these studies we propose to initially purify this enzyme from the rat liver cytosol and two isozymes from rat liver microsomes. These purifications will be performed by published procedures. The three oxidoreductases will be used to supplement the microsomes and determine whether this supplementation increases the protective effect of reduced glutathione in the presence of menadione. In parallel experiments, we will prepare monoclonal antibodies to the oxidoreductases and determine whether such antibodies inhibit the protective effect of reduced glutathione. From these studies we would hope to elucidate whether the thiol:protein-disulfide oxidoreductases may serve to protect the cell from oxidant damage by restoring the protein sulfhydryls to their reduced state.
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