Organic anion-binding by human hepatic GSH S-transferases.
Organic anion-binding by human hepatic GSH S-transferases.
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人肝 GSH S-转移酶与有机阴离子结合。
DOI：
10.1016/0006-2952(86)90540-x
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发表时间：
1986
影响因子：
5.8
通讯作者：
Kaplowitz,N
中科院分区：
文献类型：
--
作者：
Takikawa,H;Sugiyama,Y;Stolz,A;Sugimoto,M;Kaplowitz,N
方法各种GSH S-转移酶的纯化。根据Jensson和Mannervik [8]纯化大鼠肝酶的方法，进行人肝GSH转移酶的纯化。使用了两个明显正常的手术肝脏样本。用SephadexG-75超微细柱层析，S-辛基谷胱甘肽与环氧活化的Sepharose6B亲和层析，在碱性和酸性pH范围内分离层析。四种形式的谷胱甘肽S-转移酶，确定在chromatofocusing：两个阳离子形式被发现在两个肝脏（洗脱在pH 9.0和8.7），而一个肝脏含有中性（pH 6.8）和其他阴离子形式（pH 5.4）。按照从色谱聚焦洗脱的顺序，阳离子形式被称为Cl和C2。根据Laemmli [9]的方法，通过十二烷基硫酸钠（SDS）聚丙烯酰胺凝胶电泳确认均一性。用这种技术，碱性和酸性形式表现为同源二聚体（亚基M = 25，000），中性形式是较大亚基（M = 25，000）的同源二聚体。26，~）。- Anjl-no-8.石脑油-烯磺酸盐（ANS）-缓蚀技术[10 - 131]。加入含有2 ml 0.01 M磷酸钠缓冲液的比色皿中（pH值7.4）（标准缓冲液）和纯化蛋白（0.1至0.3@VI），加入不同量的ANS（从5mM储备溶液起最多30天），在室温（25 - 27 ℃）下用Hitachi MPF-4-pec荧光计测定荧光（激发400nm和发射480nm）。为了阐明抑制的类型，ANS结合用阳离子荧光法测定。
MethodsPurification of various GSH S-trunsferases. Purification of human hepatic GSH-transferases was performed according to the approach of Jensson and Mannervik [8] for purification of rat liver enzymes. Two operative liver samples which were apparently normal were used. Briefly, the p~ fication procedure consists of Sephadex G-75 superfine chromatography, affinity chromatography on S-octylglutathione bound to epoxy-activated Sepharose 6B, and separate chromatofocusing both in the basic and acidic pH range. Four forms of GSH S-transferases were identified in chromatofocusing: two cationic forms were found on both livers (eluting at pH 9.0 and 8.7) whereas one liver contained a neutral (pH 6.8) and the other an anionic form (pH 5.4). The cationic forms are referred to as Cl and C2 in order of elution from chromatofocusing. Homogeneity was confirmed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis according to the method of Laemmli [9]. With this technique the basic and acidic forms appeared as homodimers (subunit M,= 25,000) and the neutral form was a homodimer of a larger subunit (M?= 26,~). l-Anjl~ no-8. naphtha~ enesuIfonate(ANS)~ uoresce~ ce inhibition technique [10-131. To cuvettes containing 2 ml of0.01 M sodium phosphate buffer (pH 7.4)(standard buffer) and purified protein (0.1 to 0.3@ VI), various amounts of ANS [up to 30 d from a 5 mM stock solution) were added, and fluorescence was determined (excitation 4OOnm and emission 480 nm) at room temperature (25-27’) with an Hitachi MPF-4~ pec~ ofluorom~ ter. To eiucidate the type of inhibition, ANS binding was determined with cationic