A spectrophotometric assay of gamma-glutamylcysteine synthetase and glutathione synthetase in crude extracts from tissues and cultured mammalian cells.

Tipo de material: Texto

TextoSeries ; Chemico-Biological Interactions, 140(1), p.49-65, 2002Trabajos contenidos:

Volohonsky, G
Tuby, C.N
Porat, N
Wellman-Rousseau, M
Visvikis, A
Leroy, P
Rashi, S
Rashi, S
Stark, A.A

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Resumen: An assay of gamma-glutamylcysteine synthetase (gamma-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 gamma-GCS, or from gamma-glutamylcysteine and glycine for the assay of GS. GSH is then quantified by the Tietze recycling method. Assay mixtures contain the gamma-glutamyl transpeptidase (GGT)inhibitor acivicin in order to prevent the degradation of gamma-glutamylcysteine and of the accumulating GSH, and dithiothreitol in order to prevent the oxidation of cysteine and gamma-glutamylcysteine. gamma-GCS and GS levels determined by this method are comparable to those determined by others. The method is suitable for the rapid determination of gamma-GCS GS in GGT-containing tissues and for the studies of induction of gamma-GCS and GS in tissue cultures.

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An assay of gamma-glutamylcysteine synthetase (gamma-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 gamma-GCS, or from gamma-glutamylcysteine and glycine for the assay of GS. GSH is then quantified by the Tietze recycling method. Assay mixtures contain the gamma-glutamyl transpeptidase (GGT)inhibitor acivicin in order to prevent the degradation of gamma-glutamylcysteine and of the accumulating GSH, and dithiothreitol in order to prevent the oxidation of cysteine and gamma-glutamylcysteine. gamma-GCS and GS levels determined by this method are comparable to those determined by others. The method is suitable for the rapid determination of gamma-GCS GS in GGT-containing tissues and for the studies of induction of gamma-GCS and GS in tissue cultures.

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