David J. Pulford

TL;DR: The biochemical functions of GST are described to show how individual isoenzymes contribute to resistance to carcinogens, antitumor drugs, environmental pollutants, and products of oxidative stress, and to allow identification of factors that may modulate resistance to specific noxious chemicals.

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.

TL;DR: Molecular cloning and heterologous expression of rat GSTA5-5 has led to the demonstration that it exhibits substantially greater activity for AFB1-8,9-epoxide than other rat transferases, and a novel aflatoxin-aldehyde reductase (AFAR) that is similarly induced by ethoxyquin is identified.

TL;DR: Computer-assisted analysis of the upstream sequence has indicated the presence of an antioxidant-responsive element (ARE), and several elements thought to be required for tissue-specific expression of the enzyme, in both upstream and intronic sequences.

TL;DR: A novel gene fusion approach which may be of more general use has been developed for investigating the function of calmodulin in the budding yeast Saccharomyces cerevisiae by fusing a portion of the Staphylococcus aureus spa gene (encoding protein A) to CMD1, which generates a yeast cal modulin with an affinity tag able to bind immunoglobulins.