publication . Article . 2003

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

Dianne Botta; Collin C. White; Terrance J. Kavanagh; Hannah Malia A Viernes; Cecile M. Krejsa;

Open Access

Published: 01 Jul 2003 Journal: Analytical Biochemistry, volume 318, pages 175-180 (issn: 0003-2697,

Publisher: Elsevier BV

Summary

Abstract

Glutamate-cysteine ligase (GCL; also known as gamma-glutamylcysteine 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...

doi: 10.1016/s0003-2697(03)00143-x

Subjects

free text keywords: DNA ligase, chemistry.chemical_classification, chemistry, Chromatography, Enzyme, Microtiter plate, Glutathione, chemistry.chemical_compound, Biochemistry, Fluorescence, High-performance liquid chromatography, Glutamate-cysteine ligase activity, Derivatization

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