Characterization of an inhibitor on glutathione-S-tranferase by analyzing the changes of kinetic parameters

ZHANG Ling; YANG Xiao-lan; BAI Jing; LIAO Juan; LIU Hong-bo; LIAO Fei

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Characterization of an inhibitor on glutathione-S-tranferase by analyzing the changes of kinetic parameters

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- Characterization of an inhibitor on glutathione-S-tranferase by analyzing the changes of kinetic parameters
- Journal of Chongqing Medical University Vol. 36, Issue 10, Pages: 1218-1221(2011)
- 作者机构：
  
  重庆医科大学检验医学院
- 作者简介：
- 基金信息：
- DOI：
  CLC： R912
- Published：2011，
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[1]张灵,杨晓兰,白婧,廖娟,刘红博,廖飞.测定谷胱甘肽-S-转硫酶动力学参数的变化表征其抑制剂[J].重庆医科大学学报,2011,36(10):1218-1221.

ZHANG Ling, YANG Xiao-lan, BAI Jing, et al. Characterization of an inhibitor on glutathione-S-tranferase by analyzing the changes of kinetic parameters. [J]. Journal of Chongqing Medical University 36(10):1218-1221(2011)
[1]张灵,杨晓兰,白婧,廖娟,刘红博,廖飞.测定谷胱甘肽-S-转硫酶动力学参数的变化表征其抑制剂[J].重庆医科大学学报,2011,36(10):1218-1221. DOI：

ZHANG Ling, YANG Xiao-lan, BAI Jing, et al. Characterization of an inhibitor on glutathione-S-tranferase by analyzing the changes of kinetic parameters. [J]. Journal of Chongqing Medical University 36(10):1218-1221(2011) DOI：

摘要

目的:测定谷胱甘肽-S-转移酶（Glutathione-S-transferases

GST）动力学参数变化

建立表征其抑制剂的方法。方法:从猪肝经阴离子交换层析和亲和层析制备GST酸性同工酶

用还原型谷胱甘肽（Glutathione

GSH）和1-氯-2

4-二硝基苯（1

-chloro-2

4-dinitrobenzene

CDNB）合成S-（2

4-二硝基苯基）-谷胱甘肽（GS-DNB）为候选抑制剂

以GSH与CDNB为底物测定GST在GS-DNB作用下的米氏常数（Km）和最大反应速度（Vm）

从而确定GS-DNB对GST的抑制常数（Ki）。结果:此GST被纯化146倍以上

活性总收率近30%。该GST对GSH和CDNB的Km分别为42μmol/L和0.86 mmol/L

属于随机双底物动力学模型。GS-DNB对CDNB竞争性Ki为（21±1）μmol/L（n=2）;对GSH竞争性Ki为（17±1）μmol/L（n=2）。结论:产物GS-DNB是GST的高亲和力竞争性抑制剂;测定GST动力学参数随抑制剂浓度的变化可筛选其抑制剂。

Abstract

Objective:To establish a method based on analysis of the changes of kinetic parameters to characterize an inhibitor of glutathione-S-transferase(glutathione-S-transferases

GST).Methods:The acidic GST isozyme was purified from the porcine liver via anion-exchange chromatography and affinity chromatography.The reaction of glutathione(glutathione

GSH) and 1-chloro-2

4-dinitrobenzene(1

-chloro-2

4-dinitrobenzene

CDNB) gave S-(2

4-dinitrobenzyl)-glutathione(GS-DNB) as a candidate inhibitor.Michaelis-Menten constant(Km) and maximal reaction rate(Vm) were estimated to determine the inhibition constant(Ki) of GS-DNB.Results:Specific activity of GST was increased by more than 146 times with overall activity yield of about 30%.GST followed random bi-substrate kinetics and had Km of 42 μmol/L for GSH

and Km of 0.86 mmol/L for CDNB.The competitive Ki of GS-DNB was(21±1) μmol/L(n=2) against CDNB

and(17±1) μmol/L(n=2) against GSH.Conclusion:GS-DNB is an effective competitive inhibitor of GST;the estimation of Ki from responses of Km and Vm to inhibitor concentrations can be a conventional method to screen GST inhibitors.

关键词

谷胱甘肽-S-转移酶抑制剂还原型谷胱甘肽

Keywords

glutathione-S-transferasesinhibitorglutathione

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