EVIDENCE THAT HUMAN CLASS-THETA GLUTATHIONE-S-TRANSFERASE T1-1 CAN CATALYZE THE ACTIVATION OF DICHLOROMETHANE, A LIVER AND LUNG CARCINOGEN IN THE MOUSE - COMPARISON OF THE TISSUE DISTRIBUTION OF GST T1-1 WITH THAT OF CLASS-ALPHA-GST, CLASS-MU-GST AND CLASS-PI-GST IN HUMAN

The cDNA encoding human glutathione S-transferase (GST) T1 has been ex pressed as two recombinant forms in Escherichia coli that could be pur ified by affinity chromatography on either IgG-Sepharose or nickel-aga rose; one form of the transferase was synthesized from the pALP 1 expr ession 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 purif ied recombinant proteins from E. coli cultures were approx. 15 mg/l fo r the protein A fusion and 25 mg/l for the C-terminal polyhistidine-ta gged GST Tl-1. The purified recombinant proteins were catalytically ac tive, although the protein A fusion was typically only 5-30% as active as the histidine-tagged GST. Both recombinant forms could catalyse th e conjugation of glutathione with the model substrates 1,2-epoxy-3-(4' -nitrophenoxy)propane, 4-nitrobenzyl chloride and 4-nitrophenethyl bro mide 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 redu ction of cumene hydroperoxide. In addition, recombinant human GST Tl-1 was found to conjugate glutathione with dichloromethane, a pulmonary and hepatic carcinogen in the mouse. Immunoblotting with antibodies ra ised against different transferase isoenzymes showed that GST T1-1 is expressed in a large number of human organs in a tissue-specific fashi on that differs from the pattern of expression of classes Alpha, Mu an d 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.