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

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.