Purification and physical characterization of glutathione S-transferase K. Differential use of S-hexylglutathione and glutathione affinity matrices to isolate a novel glutathione S-transferase from rat liver.
Purification and physical characterization of glutathione S-transferase K. Differential use of S-hexylglutathione and glutathione affinity matrices to isolate a novel glutathione S-transferase from rat liver.
复制标题
谷胱甘肽 S-转移酶 K 的纯化和物理表征。差异使用 S-己基谷胱甘肽和谷胱甘肽亲和基质从大鼠肝脏中分离出一种新型谷胱甘肽 S-转移酶。
作者：
J. Hayes
描述了一种新的肝酶，谷胱甘肽S-转移酶K，与以前表征的转移酶不同，其对S-己基谷胱甘肽-琼脂糖6 B几乎没有亲和力，但可以分离，因为它结合到谷胱甘肽亲和基质。设计了一种纯化该新酶的方案，使用DEAE-纤维素，S-己基谷胱甘肽-Sepharose 6 B，谷胱甘肽-Sepharose 6 B和羟基磷灰石层析。最终的羟基磷灰石步骤导致三种色谱上可互变形式K1、K2和K3的洗脱。纯化的蛋白质具有6.1的等电点，并且包括被指定为Yk（Mr 25，000）的亚基;在十二烷基硫酸钠/聚丙烯酰胺凝胶电泳期间，其迁移略快于Ya亚基，但慢于肺Yf单体（Mr 24，500）。转移酶K显示出与其他肝转移酶不同的催化、免疫化学和物理性质。胰蛋白酶肽图表明，转移酶K是一个同源二聚体，或包括密切同源的亚基。胰蛋白酶指纹图谱还表明，尽管转移酶K在结构上与先前描述的肝形式分离，但Yk、Ya和Yc多肽之间存在有限的序列同源性。这些结构数据与随附论文[Hayes & Mantle（1986）Biochem. J. 233，779-788]中给出的免疫化学结果一致雅阁。
A novel hepatic enzyme, glutathione S-transferase K, is described that, unlike previously characterized transferases, possesses little affinity for S-hexylglutathione-Sepharose 6B but can be isolated because it binds to a glutathione affinity matrix. A purification scheme for this new enzyme was devised, with the use of DEAE-cellulose, S-hexylglutathione-Sepharose 6B, glutathione-Sepharose 6B and hydroxyapatite chromatography. The final hydroxyapatite step results in the elution of three chromatographically interconvertible forms, K1, K2 and K3. The purified protein has an isoelectric point of 6.1 and comprises subunits that are designated Yk (Mr 25,000); during sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, it migrates marginally faster than the Ya subunit but slower than the pulmonary Yf monomer (Mr 24,500). Transferase K displays catalytic, immunochemical and physical properties that are distinct from those of other liver transferases. Tryptic peptide maps suggest that transferase K is a homodimer, or comprises closely homologous subunits. The tryptic fingerprints also demonstrate that, although transferase K is structurally separate from previously described hepatic forms, a limited sequence homology exists between the Yk, Ya and Yc polypeptides. These structural data are in accord with the immunochemical results presented in the accompanying paper [Hayes & Mantle (1986) Biochem. J. 233, 779-788].