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
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
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DOI：
10.1042/bj3260837
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发表时间：
1997-09-15
影响因子：
4.1
通讯作者：
Hayes, JD
中科院分区：
文献类型：
--
作者：
Sherratt, PJ;Pulford, DJ;Hayes, JD
编码人谷胱甘肽S-转移酶（GST）T1的cDNA在大肠杆菌中以两种重组形式表达，可通过IgG-琼脂糖或镍-琼脂糖亲和层析纯化;一种形式的转移酶从pALP 1表达载体合成为金黄色葡萄球菌蛋白A融合物，而另一种形式是从pET-20 b表达载体合成的C-末端多组氨酸标记的重组体。从E.大肠杆菌培养物约为。蛋白A融合物为15 mg/l，C-末端多组氨酸标记的GST T1 -1为25 mg/l。纯化的重组蛋白具有催化活性，尽管蛋白A融合物的活性通常仅为组氨酸标记的GST的5-30%。这两种重组形式都能催化谷胱甘肽与模型底物1，2-环氧-3-（4 '-硝基苯氧基）丙烷、4-硝基苄基氯和4-硝基苯乙基溴的结合，但对1-氯-2，4-二硝基苯、依他尼酸和1-甲基萘硫酸盐无活性。重组人GST T1-1具有谷胱甘肽过氧化物酶活性，并能催化氢过氧化枯烯的还原。此外，重组人GST T1 -1被发现结合谷胱甘肽与二氯甲烷，肺和肝致癌物在小鼠中。用针对不同转移酶同工酶的抗体进行的免疫印迹显示，GST T1-1以组织特异性的方式在大量人体器官中表达，其不同于α类、Mu类和Pi类GST的表达模式。最重要的是，GST T1-1在人肺可溶性细胞提取物中仅以低水平存在，这表明人肺几乎没有能力激活挥发性二氯甲烷。
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 Tl-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 Tl-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.