Maruyama H, Niitsu Y, Takahashi Y, Hirata Y, Homma H, Urushizaki I
Nihon Naibunpi Gakkai Zasshi. 1985 Sep 20;61(9):893-911. doi: 10.1507/endocrine1927.61.9_893.
A new isozyme of Glutathione-S-transferase (GST) with more acidic pI (6.7) than other forms of GST hitherto reported was isolated from rat liver cytosol by consecutive chromatographies on a DEAE cellulose column, lysyl-GSH affinity column and Sephadex G-100 column. This anionic form of GST represented approximately one third of total GST activity in rat liver cytosol. Amino acid composition, immunological reactivity, enzymatic properties, and secondary structure as measured by circular dichroism of this form were distinct from those of cationic isozymes (GST-AA, GST-B, GST-X), presently investigated. The stoichiometric ratio of high affinity site for bilirubin to GST molecule differs amongst isozymes. The anionic form of GST bound two bilirubin per molecule whereas cationic GSTs bound only one bilirubin per two subunits. The most distinguished property of anionic GST was its strong affinity for glucocorticoid. The dissociation constant of anionic GST-corticosterone complex was as low as 2.0 X 10(-8) M. Corticosterone inhibited the enzyme activity of anionic GST in a noncompetitive fashion with an apparent Ki value of 8.6 X 10(-5) M and 1.1 X 10(-6) M for 1-chloro-2.4-dinitrobenzene and GST respectively. The anionic GST-corticosterone complex bound to DNA coupled Sepharose at 25 degrees C and passed through the column at 4 degrees C. Conversely, the complex bound to a DEAE cellulose column at 4 degrees C but passed through at 25 degrees C. These properties of anionic GST are quite similar to those of glucocorticoid receptor of rat liver cytosol reported previously.
通过在DEAE纤维素柱、赖氨酰谷胱甘肽亲和柱和Sephadex G - 100柱上连续进行色谱分离，从大鼠肝脏胞液中分离出一种谷胱甘肽 - S - 转移酶（GST）的新同工酶，其比迄今报道的其他形式的GST具有更酸性的pI（6.7）。这种阴离子形式的GST在大鼠肝脏胞液中约占总GST活性的三分之一。通过圆二色性测量的这种形式的氨基酸组成、免疫反应性、酶学性质和二级结构与目前研究的阳离子同工酶（GST - AA、GST - B、GST - X）不同。胆红素高亲和力位点与GST分子的化学计量比在同工酶之间有所不同。阴离子形式的GST每个分子结合两个胆红素，而阳离子GST每两个亚基仅结合一个胆红素。阴离子GST最显著的特性是其对糖皮质激素的强亲和力。阴离子GST - 皮质酮复合物的解离常数低至2.0×10⁻⁸ M。皮质酮以非竞争性方式抑制阴离子GST的酶活性，对于1 - 氯 - 2,4 - 二硝基苯和GST，其表观Ki值分别为8.6×10⁻⁵ M和1.1×10⁻⁶ M。阴离子GST - 皮质酮复合物在25℃下与DNA偶联的琼脂糖结合，并在4℃下通过柱子。相反，该复合物在4℃下与DEAE纤维素柱结合，但在25℃下通过柱子。阴离子GST的这些特性与先前报道的大鼠肝脏胞液糖皮质激素受体的特性非常相似。
