Studies by us have shown that glutathione-S-transferases (GSTs) constitute a family of proteins that bind diverse lipophilic substances such as steroid hormones, thyroid hormones, bilirubin and chemical carcinogens. GSTs also catalyze chemical transformations of certain compounds by conjugation with glutathione. Most GSTs have both high-affinity binding centers for non-substrate ligands and catalytic centers. In cells, binding by GSTs is usually high-capacity as well as high-affinity. The experiments in this proposal are designed to provide a structural basis for understanding binding of certain hormones, carcinogens and drugs to GSTs, and to explore physiological consequences of that binding. circular dichroism and other spectroscopic techniques, chemical probes, kinetic measurements and covalent affinity-labeling of binding sites, will be employed to determine binding specificities, relationships between binding of non-substrate ligands and catalytic activity, and subunit assembly mechanisms as influenced by structural variations. Recombinant DNA technology will be used to study the differential regulation of expression of individual members of this multigene family. Recently we showed that novel class-mu GSTs are expressed in brain testis but not in liver of both humans and rats, and we have characterized these forms by molecular cloning methods. Accordingly, brain GSTs will be used as a representative system to explore common transcriptional regulatory mechanisms in different species and to study tissue-specific gene expression. Little is known either about properties of extrahepatic GSTs or the nature of factors that regulate their expression. Binding characteristics, levels of GSTs, and regulation of expression of brain and testicular isoenzymes will therefore be analyzed. Regulation of GST gene expression will be studied in primary cultures of astrocytes, in glial cell lines, as well as neuronal cells which, in mammalian brains, ordinarily lack GSTs. The potential involvement of GSTs in functions of the blood-barriers of brain and testis will be explored. Our long-term goals are to define functions of GSTs in adaptive responses of cells and to determine whether these proteins can modulate the action of certain hormones, drugs, carcinogenic compounds, and cytotoxic agents; and whether cells can adapt to serve these functions by regulating the expression of specific GST genes.
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