Kinetic measurement by LC/MS of gamma-glutamylcysteine ligase activity.
gamma-Glutamylcysteine ligase (GCL) combines cysteine and glutamate through its gamma carboxyl moiety as the first step for glutathione (GSH) synthesis and is considered to be the rate-limiting enzyme in this pathway. The enzyme is a heterodimer, with a heavy catalytic and a light regulatory subunit, which plays a critical role in the anti-oxidant response. Besides the original method of Seelig designed for the measurement of a purified enzyme, few endpoint methods, often unrefined, are available for measuring it in complex biological samples. We describe a new, fast and reliable kinetic LC/MS method which enabled us to optimize its detection. l-2-Aminobutyrate is used instead of cysteine (to avoid glutathione synthetase interference) as triggering substrate with saturating concentrations of glutamate and ATP; the gamma glutamylaminobutyrate formed is measured at m/z=233 at regular time intervals. Reaction rate is maximum because ATP is held constant by enzymatic recycling of ADP by pyruvate kinase and phosphoenolpyruvate. The repeatability of the method is good, with CV% of 6.5 and 4% for catalytic activities at, respectively 0.9 and 34 U/l. The affinities of rat and human enzymes for glutamate and aminobutyrate are in good agreement with previous published data. However, unlike the rat enzyme, human GCL is not sensitive to reduced glutathione and displays a more basic optimum pH.
Chikh
Karim
K
UF 21455 Stress Oxydant et Vitamines, Fédération de Biochimie, Biochemistry Federation, Hôpital E. Herriot, 5 Place d'Arsonval, F-69437 Lyon, France.
Chik
Karim
K
Flourie
Françoise
F
Arab
Khelifa
K
Steghens
Jean Paul
JP
D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods; D004151:Dipeptides / Q000032:analysis; D005347:Fibroblasts / Q000378:metabolism; D005721:Glutamate-Cysteine Ligase / Q000378:metabolism*; D005978:Glutathione / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods; D008862:Microsomes, Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D051381:Rats; D015203:Reproducibility of Results; D016415:Sequence Alignment
D004151:Dipeptides; C042262:gamma-glutamyl-alpha-aminobutyrate; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione