Purification and characterization of the individual glutathione S-transferases from sheep liver.
Purification and characterization of the individual glutathione S-transferases from sheep liver.
复制标题
绵羊肝脏中单个谷胱甘肽 S-转移酶的纯化和表征。
DOI：
10.1016/0003-9861(83)90192-3
复制
发表时间：
1983
影响因子：
3.9
通讯作者：
Tu,CP
中科院分区：
文献类型：
--
作者：
Reddy,CC;Burgess,JR;Gong,ZZ;Massaro,EJ;Tu,CP
通过采用Sephadex G-150 上的凝胶过滤和S-己基谷胱甘肽连接的Sepharose-6B 柱上的亲和层析相结合，将谷胱甘肽S-转移酶(EC 2.5.1.18) 从105,000g 绵羊肝匀浆上清液中纯化至电泳均质。通过这种纯化方法可以恢复大约70%的原始谷胱甘肽S-转移酶对1-氯-2,4-二硝基苯的活性和谷胱甘肽过氧化物酶对氢过氧化异丙苯的活性。在开发该方法时特别重要的是，亲和柱层析后获得的酶制剂代表了羊肝谷胱甘肽S-转移酶的所有同工酶。通过 CM-纤维素和 DEAE-纤维素柱层析进一步纯化，将谷胱甘肽 S-转移酶按照从离子交换柱洗脱的顺序分别分解为 7 种不同的阳离子同工酶（命名为 C-1、C-2、C-3、C-4、C-5、C-6 和 C-7）和 5 种重叠的阴离子转移酶（命名为 A-1、A-2、A-3、A-4 和 A-5）。亚基组成的十二烷基硫酸钠 SDS 凝胶电泳数据表明，阳离子酶由两个具有相同 Mr 为 24,000 的亚基组成，而在除 A-1 之外的所有阴离子同工酶峰中均观察到具有 Mr 为 26,000 的主要亚基。阳离子同工酶约占与谷胱甘肽S-转移酶相关的总过氧化物酶活性的98%，而阴离子同工酶中只有A-1表现出一些过氧化物酶活性。研究发现同工酶 C-4 是绵羊肝脏中最丰富的谷胱甘肽 S-转移酶。通过对多种选定底物、亚基组成和等电点的特异性对各个转移酶进行表征，显示出与人肝脏谷胱甘肽S-转移酶的模式有一些相似之处。
The glutathioneS-transferases (EC 2.5.1.18) have been purified to electrophoretic homogeneity from 105,000gsupernatant of sheep liver homogenate by employing a combination of gel filtration on Sephadex G-150 and affinity chromatography onS-hexylglutathione-linked Sepharose-6B columns. Approximately 70% of the original glutathioneS-transferase activity toward 1-chloro-2,4-dinitrobenzene and glutathione peroxidase activity toward cumene hydroperoxide could be recovered by this purification method. Of particular importance in developing this procedure was the fact that the enzyme preparation obtained after affinity column chromatography represented all the isozymes of sheep liver glutathioneS-transferases. Further purification by CM-cellulose and DEAE-cellulose column chromatography resolved the glutathioneS-transferases into seven distinct cationic isozymes designated C-1, C-2, C-3, C-4, C-5, C-6, and C-7 and five overlapping anionic transferases designated A-1, A-2, A-3, A-4, and A-5, respectively, in the order of their elution from the ion-exchange columns. The sodium dodecyl sulfate SDS-gel electrophoretic data on subunit composition revealed that cationic enzymes are composed of two subunits with an identicalMrof 24,000 whereas a predominant subunit withMrof 26,000 was observed in all anionic isozyme peaks except A-1. Cationic isozymes accounted for approximately 98% of the total peroxidase activity associated with the glutathioneS-transferase whereas only A-1 of the anionic isozymes displayed some peroxidase activity. Isozyme C-4 was found to be the most abundant glutathioneS-transferase in the sheep liver. Characterization of the individual transferases by their specificity toward a number of selected substrates, subunit composition, and isoelectric points showed some similarities to those patterns for human liver glutathioneS-transferases.