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Title: Isolation and characterization of two murine P1-class glutathione S-transferases
Author: Bammler, Theodor K.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1996
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A 15-kilobase (kb) DNA fragment has been isolated from a mouse genomic library in λEMBL3 that contains two actively transcribed pi-class GST genes termed Gst p-1 and Gst p-2. Each of the two genes is approximately 3 kb in size and composed of seven exons interrupted by six introns. In addition to a TATA box and a sequence motif matching the phorbol-ester-responsive element, the promoters of Gst p-1 and Gst p-2 exhibit one and two G+C boxes (5'-GGGCGG-3') respectively. The hepatic expression of both pi genes was found to be significantly higher in males compared to females, and Gst p-1 mRNA levels were more abundant in both sexes than Gst p-2. The cDNAs corresponding to these genes were isolated from total liver RNA employing RT-PCR. The proteins encoded by the two murine pi-class genes, GSTP1-1 and GSTP2-2, which share 97% identity and differ in six amino acids only were expressed in Escherichia coli. Surprisingly, the catalytic activities of these recombinant enzymes toward a panel of electrophilic substrates were remarkably different, such that GSTP2-2 displayed 1-3 orders of magnitude lower activity than GSTP1-1. Site directed mutagenesis and kinetic analysis revealed that the amino acid differences at positions 10 (Val/Ser), 11 (Arg/Pro), and 104 (Val/Gly) were responsible for the dramatically reduced catalytic activity of GSTP2-2, affecting both binding of the electrophilic substrate and glutathione to the enzyme.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available