The goal of this work is to understand in biochemical and molecular terms the role which glutathione S-transferase (GST)-catalyzed conjugation reactions naturally play, or can be made to play by augmentation, in reducing the incidence of toxic alkylation events in cells. Initial studies using a bacterially-produced GST pi isozyme will combine site-directed mutagenesis of amino acid residues, epitope mapping of a panel of inhibitory monoclonal antibodies, and a collaboration which involves x-ray crystal analysis, to identify residues which are responsible for binding GSH and xenobiotic substrates to the GST pi enzyme. Random oligonucleotide mutagenesis of all amino acids within the GST pi and GST Yc isozymes will be done, and novel, increased-efficiency mutant enzymes will be identified by selecting GST- expressing E. coli cells in anti-BPDE (pi) or melphalan (Yc). The ability of naturally-occurring GST isozymes as well as kinetically- efficient mutant derivatives to confer resistance to electrophiles will be examined by introducing recombinant expression vectors into several mammalian systems. These will include: i) a transgenic line of FVB mice expressing a tandem-design human GST pi construct in dermal fibroblasts to suppress benzo(a)pyrene-induced fibrosarcoma formation, ii) expression of human mu-class GST constructs in lymphoblasts from human subjects carrying an inherited GST1 null-phenotype, and iii) cultured rodent cells (10T1/2, MatB mammary carcinoma) which are expressing recombinant GST mutants to determine the maximal degree of resistance to electrophiles that can be achieved. The final aspect of this work involves the use of novel cloning strategies to isolate cDNAs from a mouse liver cDNA-expression library that encode i) a plasma- membrane pump which exports GSH-conjugates from mammalian cells, and ii) a protein which mediates the induction of GST by antioxidant inducer molecules such as butylated hydroxyanisole.
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