Chu-Lin, Xia; (1992) Glutathione transferase pi structure and gene regulation. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

In the early stage of this research, amino acid residues that are essential for the activity of human pi class glutathione transferase (GST π) were identified using chemical modification with group-specific reagents. Protection from inactivation by substrates, substrate analogues and inhibitors was used as the criterion for active site specificity. The results suggested the apparent involvement of one cysteine (Cys), one lysine (Lys), one arginine (Ang) one histidine (His) or tyrosine (Tyr) or both, one aspartate (Asp) or glutamate Glu) and tryptophan (Trp) residues in the glutathione binding site (G-site) of GSTπ. It was concluded that without the knowledge of the three-dimensional structure of GST 7T, further work in this area would not be profitable and therefore, attention was turned to the regulation of GST π gene expression. Recently, the three-dimensional structure of porcine GST pi was solved and interpretation of these results in terms of the structure of the porcine GST pi by computerized modelling revealed that Tyr7, Arg13, Trp38, Lys44 and Asp98 were associated with the G-site. The roles of Cys47 and His71 in the active site are not clear. Studies of the regulation of GST π gene involved the fusion of fragments of both the GST π gene and 13.5 kb of its 5' flanking region to the chloramphenicol acetyl transferase (CAT) reporter gene. Transfection into a number of human cell lines (Hela, HepG2, EJ and MCF7) demonstrated that the consensus AP1 binding site, located between nucleotides -58 to -65, vas essential for the basal level promoter activity of the gene. A positive cis-acting DNA element between nucleotides +8 to +72 was also identified which could be bound by protein(s) both in vivo and in vitro. This element is part of the promoter since it functions in an orientation and position dependent manner. No other regulatory activity was identified within the 17 kb sequence analyzed. Regulation by both retinoic acid and insulin was observed, but the mechanisms of the regulation remain to be elucidated.

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