The glutathione S-transferases (GSH-tases) are a family of enzymes present in high concentrations within the hepatic cytosol. The GSH-tases are important for the detoxification of a large number of reactive molecules, including drugs, carcinogens, and organic peroxides. These enzymes also bind a variety of ligands that they do not metabolize, such as bilirubin and bile acids and are thought to act as intracellular transport proteins. The binding of these nonsubstrate ligands causes a significant reduction in enzymatic activity. It is uncerain how these proteins carry out these two competing functions, that is, intracellular transport of some ligands and simultaneous detoxification of others. This is an important because high intracellular concentrations of bilirubin or bile acids, as are seen in patients with liver disease, could reduce the activity of the GSH-tases to a level at which they no longer perform their enzymatic functions. This would leave patients susceptible to injury by toxins normally metabolized by the GSH-tases. The biochemical basis for inhibition of GSH-tases by nonsubstrate ligands will be studied, using equilibrium dialysis and standard kinetic analysis of the inhibition of enzyme activity by the nonsubstrate ligands. Physiological conditions that affect the extent of inhibition caused by nonsubstrate ligands will be indentified using pure enzyme systems and then applied to studies with isolated hepatocytes. In the latter studies, the effect of the intracellular accumulation of bilirubin and bile acids on the ability of the GSH-tases to metabolize drugs and toxins will be determined. The mechanisms and kinetics of transfer of nonpolar ligands between membranes and the GSH-tases will be studied. These studies are important because most of the molecules bound by the GSH-tases are nonpoplar and will partition extensively into cellular membranes. Defining the role of the GSH-tases in the intracellular transport of these nonpolar molecules requires that one understands how the GSH tases interact with membrane-associated molecules. The membrane-to-protein transfer of nonpolar molecules will be studied using a stopped-flow apparatus, pure proteins, and artificial membranes (liposomes). The movement of the ligands will be monitored by following the change in protein fluorescence as the ligand (quencher) associates/dissociates from the GSH-tase.
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