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

Authors
Journal
Analytical Biochemistry
0003-2697
Publisher
Elsevier
Publication Date
Volume
318
Issue
2
Identifiers
DOI: 10.1016/s0003-2697(03)00143-x
Keywords
  • Glutamate–Cysteine Ligase
  • Glutathione
  • Microtiter Plate
  • Fluorescence
Disciplines
  • Biology
  • Design

Abstract

Abstract Glutamate–cysteine ligase (GCL; also known as γ-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 γ-glutamylcysteine (γ-GC) product formation. We took advantage of the reaction of naphthalene dicarboxaldehyde (NDA) with GSH or γ-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.

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