The glutathione S-transferases (GST) are a family of enzymes that are present in high concentration in hepatic cytosol. The GST are important enzymes for the detoxication of a large number of reactive molecules. They detoxify these reactive molecules by forming glutathione conjugates which are potent inhibitors of GST and some may themselves by toxic. Hepatocytes have a mechanism that allows for the efficient excretion into bile of products of GST catalyzed reactions. The GST are also thought to function as transport proteins for a variety of non-polar molecules (bilirubin, heme, bile acids) however this role has not been established with certainty. In this proposal are described experiments designed to purify and characterize (radiation inactivation, reconstitution, amino acid analysis) the transport system for conjugates of glutathione that is known to be present in canalicular membranes of hepatocytes. Additionally, in intact hepatocytes the effect of a number of important factors (inducibility by phenobarbital, role of membrane potential, effect of extracellular matrix) on expression of this transport system will be investigated. Recent work has shown that expression of a certain form of GST is associated with malignant transformation of cells and this form is also expressed in hepatocytes in culture. The expression of GST in cultured hepatocytes under different conditions of culture will be investigated to gain insight into the factors that influenced tissue expression of the GST. Heme must move from its site of synthesis in the mitochondria to the ER to form the active cytochromes form their respective apoproteins. GST may facilitate this movement, although exactly how they accomplish this is uncertain. This laboratory has a system that allows for measuring movement of heme out of mitochondria and it will utilize this system to investigate how the GST serve as heme transport proteins. The precise mechanism that allows the GST to increase the rate of reaction between glutathione and toxic electrophiles is unknown. The latter can be determined by localization of the active site in the primary amino acid sequence of GST and then using site directed mutagenesis to alter certain amino acids in the active site. Recent work has now provided the information needed to develop strategies for site directed mutagenesis of GST and experiments are described that will allow for the development of an expression system for mutants of GST using E. coli for subsequent site directed mutagenesis studies.
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