US EPA

1490066 

Journal Article 

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

White, CC; Viernes, H; Krejsa, CM; Botta, D; Kavanagh, TJ 

2003 

Yes 

Analytical Biochemistry
ISSN: 0003-2697
EISSN: 1096-0309 

318 

2 

175-180 

English 

12814619 

10.1016/S0003-2697(03)00143-X 

WOS:000183785000001 

http://://WOS:000183785000001
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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. 

glutamate-cysteine ligase; glutathione; microtiter plate; fluorescence