Fluorescence-based microtiter plate assay for glutamate-cysteine ligase activity

被引：243

作者：

White, CC ^{[1
]}

Viernes, H ^{[1
]}

Krejsa, CM ^{[1
]}

Botta, D ^{[1
]}

Kavanagh, TJ ^{[1
]}

机构：

[1] Univ Washington, NIEHS, Ctr Ecogenet & Environm Hlth, Dept Environm Hlth, Seattle, WA 98195 USA

来源：

ANALYTICAL BIOCHEMISTRY | 2003年 / 318卷 / 02期

关键词：

glutamate-cysteine ligase; glutathione; microtiter plate; fluorescence;

D O I：

10.1016/S0003-2697(03)00143-X

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

Glutamate-cysteine ligase (GCL; also known as gamma-glutamyleysteine synthetase) is the rate-limiting enzyme in glutathione (GSH) synthesis. Traditional assays for the activity of this enzyme are based either on coupled reactions with other enzymes or on high-performance liquid chromatography (HPLC) assessment of gamma-glutamylcysteine (gamma-GC) product formation. We took advantage of the reaction of naphthalene dicarboxaldehyde (NDA) with GSH or gamma-GC to form cyclized products that are highly fluorescent. Hepa-1 cells which were designed to overexpress mouse GCL and mouse liver homogenates were used to evaluate and compare the utility of the NDA method with an assay based on monobromobimane derivatization and HPLC analysis with fluorescence detection. Excellent agreement was found between GCL activities measured by HPLC and NDA-microtiter plate analyses. This assay should be useful for high-throughput GCL activity analyses. (C) 2003 Elsevier Science (USA). All rights reserved.

引用

页码：175 / 180

页数：6

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