Characterization of methylation of rat liver cytosolic glutathione S-transferases by using reverse-phase h.p.l.c. and chromatofocusing.
Characterization of methylation of rat liver cytosolic glutathione S-transferases by using reverse-phase h.p.l.c. and chromatofocusing.
复制标题
使用反相 h.p.l.c 表征大鼠肝细胞质谷胱甘肽 S-转移酶的甲基化
DOI：
10.1042/bj2700483
复制
发表时间：
1990
期刊：
影响因子：
--
通讯作者：
Siegel,FL
中科院分区：
文献类型：
--
作者：
Johnson,JA;Neal,TL;Collins,JH;Siegel,FL
用反相高效液相色谱法分离大鼠肝胞液中的谷胱甘肽S-转移酶（GST）亚基;分离出五种主要蛋白质，并鉴定为亚基1、2、3、4和8。F.p.l.c.层析聚焦将亲和纯化的GST池解析成九种不同的同工酶。GST活性的5个基本（α类）二聚体峰为1-1、1-2a、1-2b、2-2a和2-2b。反相高压液相色谱分析显示亚基8也存在于指定为1-1、1-2a和1-2b的蛋白质峰中。4种中性（Mu类）同工酶为3-3、3-4、3-6和4-4。在反相h.p.l.c.之前，在体外甲基化GST池。或者f.p.l.c.色谱聚焦色谱聚焦表明Mu类亚型（3-3、3-4和4-4）是甲基化的主要GST，h.p.l.c.分析证实亚基3和4是主要的甲基接受GST亚基。钙调素的加入刺激GST同工酶3-3、3-4和4-4的体外甲基化分别增加了3.0、7.5和9.9倍。反相高压液相色谱也表明钙调素只刺激GST亚基3和4的甲基化。基本GST同工酶最低限度地甲基化和甲基化没有增强钙调蛋白。GST亚基3和4的甲基化的时间过程的调查表明，在孵育时间小于4小时的两个穆类亚基的甲基化刺激钙调蛋白，在这种条件下，亚基4是优选的基板。相反，在4或6小时的孵育时间基本上没有钙调素刺激的甲基化，亚基3的甲基化占主导地位。在2小时的孵育动力学参数进行了测定，在存在和不存在的钙调蛋白。加入钙调素使Vmax加倍。亚基3和4的甲基化，并降低亚基4的Km为S-腺苷-L-蛋氨酸的3.6倍。最后，甲基化是亚化学计量的，孵育6小时后，亚基4和3的甲基化范围分别为2.8 - 7.6%（摩尔比）。
Glutathione S-transferase (GST) subunits in rat liver cytosol were separated by reverse-phase h.p.l.c.; five major proteins were isolated and identified as subunits 1, 2, 3, 4 and 8. F.p.l.c. chromatofocusing resolved the affinity-purified GST pool into nine different isoenzymes. The five basic (Alpha class) dimeric peaks of GST activity were 1-1, 1-2a, 1-2b, 2-2a and 2-2b. Reverse-phase h.p.l.c. analysis revealed that subunit 8 was also present in the protein peaks designated 1-1, 1-2a and 1-2b. The four neutral (Mu class) isoenzymes were 3-3, 3-4, 3-6 and 4-4. The GST pool was methylated in vitro before reverse-phase h.p.l.c. or f.p.l.c. chromatofocusing. Chromatofocusing indicated that the Mu class isoforms (3-3, 3-4 and 4-4) were the primary GSTs methylated, and h.p.l.c. analysis confirmed that subunits 3 and 4 were the major methyl-accepting GST subunits. The addition of calmodulin stimulated the methylation in vitro of GST isoenzymes 3-3, 3-4 and 4-4 by 3.0-, 7.5- and 9.9-fold respectively. Reverse-phase h.p.l.c. also indicated that only the methylation of GST subunits 3 and 4 was stimulated by calmodulin. Basic GST isoenzymes were minimally methylated and the methylation was not enhanced by calmodulin. Investigation of the time course of methylation of GST subunits 3 and 4 indicated that at incubation times less than 4 h the methylation of both Mu class subunits was stimulated by calmodulin, and that under such conditions subunit 4 was the preferred substrate. In contrast, there was essentially no calmodulin-stimulated methylation at incubation times of 4 or 6 h, and the methylation of subunit 3 was predominant. Kinetic parameters at 2 h of incubation were determined in the presence and in the absence of calmodulin. The addition of calmodulin doubled the Vmax. for methylation of both subunits 3 and 4 and decreased the Km of subunit 4 for S-adenosyl-L-methionine 3.6-fold. Finally, methylation was substoichiometric and after 6 h of incubation ranged from 2.8 to 7.6% on a mole-to-mole basis for subunits 4 and 3 respectively.