title: Evidence that human class Theta glutathione S-transferase T1-1 can catalyse the activation of dichloromethane, a liver and lung carcinogen in the mouse. Comparison of the tissue distribution of GST T1-1 with that of classes Alpha, Mu and Pi GST in human.
abstract: The cDNA encoding human glutathione S-transferase (GST) T1 has been expressed as two recombinant forms in Escherichia coli that could be purified by affinity chromatography on either IgG-Sepharose or nickel-agarose; one form of the transferase was synthesized from the pALP 1 expression vector as a Staphylococcus aureus protein A fusion, whereas the other form was synthesized from the pET-20b expression vector as a C-terminal polyhistidine-tagged recombinant. The yields of the two purified recombinant proteins from E. coli cultures were approx. 15 mg/l for the protein A fusion and 25 mg/l for the C-terminal polyhistidine-tagged GST T1-1. The purified recombinant proteins were catalytically active, although the protein A fusion was typically only 5-30% as active as the histidine-tagged GST. Both recombinant forms could catalyse the conjugation of glutathione with the model substrates 1,2-epoxy-3-(4'-nitrophenoxy)propane,4-nitrobenzyl chloride and 4-nitrophenethyl bromide but were inactive towards 1-chloro-2,4-dinitrobenzene, ethacrynic acid and 1-menaphthyl sulphate. Recombinant human GST T1-1 was found to exhibit glutathione peroxidase activity and could catalyse the reduction of cumene hydroperoxide. In addition, recombinant human GST T1-1 was found to conjugate glutathione with dichloromethane, a pulmonary and hepatic carcinogen in the mouse. Immunoblotting with antibodies raised against different transferase isoenzymes showed that GST T1-1 is expressed in a large number of human organs in a tissue-specific fashion that differs from the pattern of expression of classes Alpha, Mu and Pi GST. Most significantly, GST T1-1 was found in only low levels in human pulmonary soluble extract of cells, suggesting that in man the lung has little capacity to activate the volatile dichloromethane.
authors.affiliation: Biomedical Research Centre, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, Scotland, U.K.
authors.forename: P J
authors.initials: PJ
authors.lastname: Sherratt
authors.forename: D J
authors.initials: DJ
authors.lastname: Pulford
authors.forename: D J
authors.initials: DJ
authors.lastname: Harrison
authors.forename: T
authors.initials: T
authors.lastname: Green
authors.forename: J D
authors.initials: JD
authors.lastname: Hayes
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002273:Carcinogens / Q000378:metabolism / Q000494:pharmacology*; D003001:Cloning, Molecular; D005982:Glutathione Transferase / Q000378:metabolism*; D006801:Humans; D008099:Liver / Q000187:drug effects* / Q000473:pathology; D008168:Lung / Q000187:drug effects* / Q000473:pathology; D008752:Methylene Chloride / Q000378:metabolism / Q000494:pharmacology*; D051379:Mice; D008969:Molecular Sequence Data; D009928:Organ Specificity; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D002273:Carcinogens; D011994:Recombinant Proteins; D008752:Methylene Chloride; D005982:Glutathione Transferase