and specific aims): The glutathione-S-transferases (GSTs) comprise a multi-class family of enzymes found in the cytosol of most cells. GSTs are enzymes of detoxification and protect cells from injury caused by a variety of endogenous and exogenous toxins and carcinogens. GSTs detoxify these reactive molecules by forming glutathione conjugates. The GSTs are dimeric proteins with independent catalytic sites and it is unclear how the dimeric structure is necessary for function. The GSTs also function as intracellular transport and binding proteins for water insoluble molecules (bilirubin, heme), but the relationship between the catalytic site and nonsubstrate ligand binding site remains undefined. The GST genes contain a number of regulatory elements that are thought to mediate increases in the expression of GSTs in response to drugs, but the physiologic factors that mediate expression are undefined. This proposal describes experiments designed to define how subunit interactions affect the catalytic activity of the GSTs. These studies will utilize site- directed mutagenesis to define the importance of specific amino acids in subunit interactions. The effect of these mutations on catalytic activity and results of radiation inactivation will be determined. The investigators will determine the location of the nonsubstrate ligand binding site in the GSTs with a radiolabeled affinity ligand that binds specifically to this site. The role of specific amino acids in nonsubstrate binding by site-directed mutagenesis will be determined. The investigators have found that GST expression decreases in cultured hepatocytes exposed to the cytokine interleukin 6 and dexamethasone. Using a chloramphenicol acetyltransferase (CAT) construct that contains the 5'-flanking sequence of a GST gene they will determine how the cytokines regulate GST expression. They have also observed that ischemia-reperfusion injury increases expression by hepatocytes of a form of GST that metabolizes products of oxidant injury. The molecular mechanism of the increase in expression will be determined. Lipocytes are nonparenchymal cells in the liver that upon activation become myofibroblasts and are thought to account for the production of collagen in the dirrhotic liver. The investigators have found that activation of these cells is associated with a decrease in the expression of a number of GST genes. Using the above CAT construct they will determine if the decrease in expression is mediated by the 5'- flanking sequence of the GST gene. These latter studies are important as products of lipid peroxidation are GST substrates and increase collagen production. Therefore, the loss of GST may accelerate fibrosis.
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