Tamai K, Satoh K, Tsuchida S, Hatayama I, Maki T, Sato K
Second Department of Biochemistry, Hirosaki University School of Medicine, Japan.
Biochem Biophys Res Commun. 1990 Feb 28;167(1):331-8. doi: 10.1016/0006-291x(90)91769-o.
Treatment of Class Pi glutathione S-transferases (GST) such as rat GST P (7-7), human GST pi and mouse GST MII with 0.05-0.1 mM N-ethylmaleimide (NEM) in 0.1 M Tris-HCl (pH 7.8) resulted in almost complete inactivation of these forms, whereas no or less inactivation occurred for GSTs in Class Alpha and Mu under the same conditions. Inactivated GST P lost its S-hexyl-GSH-Sepharose column affinity. About 0.8 mol of [14C]NEM was found to be covalently bound to 1 mol of GST P subunit when 80% of the activity was lost. Similar treatment with N-dimethyl-amino-3,5-dinitrophenyl maleimide, a colored analogue of NEM, followed by trypsin digestion, HPLC and amino acid sequence analysis revealed that one cysteine residue at the 47th position from the N-terminal of the GST P subunit was preferentially modified. Subunits of GST P and GST pi are known to have 4 cysteine residues at the same corresponding positions. The present results suggest that the 47th cysteine residue may be located in the vicinity of the active site of Class Pi GSTs.
在0.1 M Tris-HCl（pH 7.8）中用0.05 - 0.1 mM N-乙基马来酰亚胺（NEM）处理Pi类谷胱甘肽S-转移酶（GST），如大鼠GST P（7 - 7）、人GST pi和小鼠GST MII，会导致这些酶几乎完全失活，而在相同条件下，Alpha类和Mu类GST没有或仅有较少程度的失活。失活的GST P失去了其与S-己基-GSH-琼脂糖柱的亲和力。当80%的活性丧失时，发现约0.8摩尔的[14C]NEM与1摩尔的GST P亚基共价结合。用N-二甲基氨基-3,5-二硝基苯基马来酰亚胺（NEM的一种有色类似物）进行类似处理，随后进行胰蛋白酶消化、HPLC和氨基酸序列分析，结果显示GST P亚基N端第47位的一个半胱氨酸残基被优先修饰。已知GST P和GST pi的亚基在相同的对应位置有4个半胱氨酸残基。目前的结果表明，第47位半胱氨酸残基可能位于Pi类GST活性位点附近。
