TY - JOUR
T1 - A spectrophotometric assay of γ-glutamylcysteine synthetase and glutathione synthetase in crude extracts from tissues and cultured mammalian cells
AU - Volohonsky, Gloria
AU - Tuby, Chen N.Y.H.
AU - Porat, Noga
AU - Wellman-Rousseau, Maria
AU - Visvikis, Athanase
AU - Leroy, Pierre
AU - Rashi, Sharon
AU - Steinberg, Pablo
AU - Stark, Avishay Abraham
N1 - Funding Information:
We wish to thank Professor H.C. Pitot, McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, for the cDNA of human GGT. This research was supported by the Eizen foundation, the Tel-Aviv University Cancer Research Center; the Rekanati Foundation for Medical Research; the Ella Kodesz Institute for Research on Cancer Development and Prevention, the France-Israel Arc-en-Ciel program (project No. 58), and by a generous donation by the late Mr Abe Feinberg, New York, and his family. P.S. is a recipient of the Heisenberg Scholarship from the Deutsche Forschungsgemeinschaft.
PY - 2002/4/20
Y1 - 2002/4/20
N2 - An assay of γ-glutamylcysteine synthetase (γ-GCS) and glutathione synthetase (GS) in crude extracts of cultured cells and tissues is described. It represents a novel combination of known methods, and is based on the formation of glutathione (GSH) from cysteine, glutamate and glycine in the presence of rat kidney GS for the assay of γ-GCS, or from γ-glutamylcysteine and glycine for the assay of GS. GSH is then quantified by the Tietze recycling method. Assay mixtures contain the γ-glutamyl transpeptidase (GGT) inhibitor acivicin in order to prevent the degradation of γ-glutamylcysteine and of the accumulating GSH, and dithiothreitol in order to prevent the oxidation of cysteine and γ-glutamylcysteine. γ-GCS and GS levels determined by this method are comparable to those determined by others. The method is suitable for the rapid determination of γ-GCS GS in GGT-containing tissues and for the studies of induction of γ-GCS and GS in tissue cultures.
AB - An assay of γ-glutamylcysteine synthetase (γ-GCS) and glutathione synthetase (GS) in crude extracts of cultured cells and tissues is described. It represents a novel combination of known methods, and is based on the formation of glutathione (GSH) from cysteine, glutamate and glycine in the presence of rat kidney GS for the assay of γ-GCS, or from γ-glutamylcysteine and glycine for the assay of GS. GSH is then quantified by the Tietze recycling method. Assay mixtures contain the γ-glutamyl transpeptidase (GGT) inhibitor acivicin in order to prevent the degradation of γ-glutamylcysteine and of the accumulating GSH, and dithiothreitol in order to prevent the oxidation of cysteine and γ-glutamylcysteine. γ-GCS and GS levels determined by this method are comparable to those determined by others. The method is suitable for the rapid determination of γ-GCS GS in GGT-containing tissues and for the studies of induction of γ-GCS and GS in tissue cultures.
KW - Enzymatic assays
KW - GSH synthetase
KW - γ-Glutamylcysteine synthetase
UR - http://www.scopus.com/inward/record.url?scp=0037140394&partnerID=8YFLogxK
U2 - 10.1016/S0009-2797(02)00017-0
DO - 10.1016/S0009-2797(02)00017-0
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AN - SCOPUS:0037140394
SN - 0009-2797
VL - 140
SP - 49
EP - 65
JO - Chemico-Biological Interactions
JF - Chemico-Biological Interactions
IS - 1
ER -