Determination of glutamate-cysteine ligase (gamma-glutamylcysteine synthetase) activity by high-performance liquid chromatography and electrochemical detection.
The tripeptide glutathione (gamma-glutamylcysteinylglycine; GSH) is the predominant low molecular mass thiol in cells. The function of GSH is of considerable interest, with the molecule being implicated in numerous cellular processes in addition to being a major cellular antioxidant. The enzyme glutamate-cysteine ligase (GCL) is the rate-limiting step in GSH synthesis. The GCL assay described here is based on high-performance liquid chromatography and exploits the electrochemically active nature of gamma-glutamylcysteine (gamma-GC), the product of GCL activity. This method allows for the direct detection of gamma-GC rather than relying on derivatization of the molecule or linked assays. The sensitivity of the assay is sufficient to allow for the measurement of GCL activity in cultured cells. The specific activity of GCL in rat primary culture astrocytes was 9.7 +/- 1.7 nmol gamma-GC synthesized/min/mg protein.
Gegg
Matthew E
ME
Department of Molecular Pathogenesis, Division of Neurochemistry, Institute of Neurology, UCL, Queen Square, London, WC1N 3BG, United Kingdom.
Clark
John B
JB
Heales
Simon J R
SJ
D000818:Animals; D001253:Astrocytes / Q000201:enzymology; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000592:standards; D004151:Dipeptides / Q000032:analysis; D004563:Electrochemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005721:Glutamate-Cysteine Ligase / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D005978:Glutathione / Q000032:analysis; D007700:Kinetics; D051381:Rats; D012015:Reference Standards
D004151:Dipeptides; D004791:Enzyme Inhibitors; D019328:Buthionine Sulfoximine; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione; C017341:gamma-glutamylcysteine