Fluorescence-based microtiter plate assay for glutamate-cysteine ligase activity.
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 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.
White
Collin C
CC
NIEHS Center Ecogenetics and Environmental Health, Department of Environmental Health, University of Washington, Seattle, WA 98195, USA.
Viernes
Hannah
H
Krejsa
Cecile M
CM
Botta
Dianne
D
Kavanagh
Terrance J
TJ
D000818:Animals; D002478:Cells, Cultured; D005453:Fluorescence; D005721:Glutamate-Cysteine Ligase / Q000032:analysis* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000166:cytology / Q000201:enzymology; D008110:Liver Extracts; D051379:Mice; D008832:Microchemistry / Q000379:methods; D013997:Time Factors
D008110:Liver Extracts; D005721:Glutamate-Cysteine Ligase