Chang L H, Fan J Y, Liu L F, Tsai S P, Tam M F
Institute of Molecular Biology, Academia Sinica, Taipei.
Biochem J. 1992 Jan 15;281 ( Pt 2)(Pt 2):545-51. doi: 10.1042/bj2810545.
Glutathione S-transferase CL 3 subunits purified from 1-day-old-chick livers were digested with Achromobacter proteinase I and the resulting fragments were isolated for amino acid sequence analysis. An oligonucleotide probe was constructed accordingly for cDNA library screening. A cDNA clone of 1342 bases, pGCL301, encoding a protein of 26209 Da was isolated and sequenced. Including conservative substitutions, this protein has 75-79% sequence similarity to other Alpha family glutathione S-transferases. The coding sequence of pGCL301 was inserted into a baculovirus vector for infection of Spodoptera frugiperda (SF9) cells. The expressed protein has a high relative activity with ethacrynic acid (47% of the specific activity with 1-chloro-2,4-dinitrobenzene). The enzyme has a subunit molecular mass of 25.2 +/- 1.2 kDa (by SDS/PAGE), a pI of 9.45 and an absorption coefficient A1%1cm of 13.0 +/- 0.5 at 280 nm.
从1日龄雏鸡肝脏中纯化的谷胱甘肽S-转移酶CL 3亚基用无色杆菌蛋白酶I进行消化，所得片段经分离后用于氨基酸序列分析。据此构建了一个寡核苷酸探针用于筛选cDNA文库。分离并测序了一个1342个碱基的cDNA克隆pGCL301，其编码一种26209 Da的蛋白质。包括保守性替换在内，该蛋白质与其他α家族谷胱甘肽S-转移酶的序列相似性为75 - 79%。将pGCL301的编码序列插入杆状病毒载体，用于感染草地贪夜蛾（SF9）细胞。表达的蛋白质对依他尼酸具有较高的相对活性（相对于对1-氯-2,4-二硝基苯的比活性为47%）。该酶的亚基分子量为25.2±1.2 kDa（通过SDS/PAGE测定），pI为9.45，在280 nm处的吸收系数A1%1cm为13.0±0.5。
