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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM031555-02
Application #
3279641
Study Section
Pharmacology A Study Section (PHRA)
Project Start
1983-12-01
Project End
1986-11-30
Budget Start
1984-12-01
Budget End
1985-11-30
Support Year
2
Fiscal Year
1985
Total Cost
Indirect Cost
Name
University of California San Francisco
Department
Type
Schools of Medicine
DUNS #
073133571
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Whalen, Richard; Liu, Xiangdang; Boyer, Thomas D (2006) Identification of a short form of ubiquitin-specific protease 3 that is a repressor of rat glutathione S-transferase gene expression. Biochem J 394:519-26
Ranganathan, Perungavar N; Whalen, Richard; Boyer, Thomas D (2005) Characterization of the molecular forms of glutathione S-transferase P1 in human gastric cancer cells (Kato III) and in normal human erythrocytes. Biochem J 386:525-33
Whalen, Richard; Voss, Susan H; Boyer, Thomas D (2004) Decreased expression levels of rat liver glutathione S-transferase A2 and albumin during the acute phase response are mediated by HNF1 (hepatic nuclear factor 1) and IL6DEX-NP. Biochem J 377:763-8
Voss, Susan H; Whalen, Richard; Boyer, Thomas D (2002) Mechanism of negative regulation of rat glutathione S-transferase A2 by the cytokine interleukin 6. Biochem J 365:229-37
Selim, N; Branum, G D; Liu, X et al. (2000) Differential lobular induction in rat liver of glutathione S-transferase A1/A2 by phenobarbital. Am J Physiol Gastrointest Liver Physiol 278:G542-50
Whalen, R; Rockey, D C; Friedman, S L et al. (1999) Activation of rat hepatic stellate cells leads to loss of glutathione S-transferases and their enzymatic activity against products of oxidative stress. Hepatology 30:927-33
Whalen, R; Boyer, T D (1998) Human glutathione S-transferases. Semin Liver Dis 18:345-58
Branum, G D; Selim, N; Liu, X et al. (1998) Ischaemia and reperfusion injury of rat liver increases expression of glutathione S-transferase A1/A2 in zone 3 of the hepatic lobule. Biochem J 330 ( Pt 1):73-9
Voss, S H; Park, Y; Kwon, S O et al. (1996) Role of interleukin 6 and corticosteroids in the regulation of expression of glutathione S-transferases in primary cultures of rat hepatocytes. Biochem J 317 ( Pt 2):627-32
Whalen, R; Kempner, E S; Boyer, T D (1996) Structural studies of a human pi class glutathione S-transferase. Photoaffinity labeling of the active site and target size analysis. Biochem Pharmacol 52:281-8

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