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

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.

Phorbol ester regulation of the human gamma-glutamyltransferase gene promoter.

In the present study the molecular mechanisms underlying tetradecanoylphorbol-13-acetate (TPA) mediated regulation of the human gamma-glutamyltransferase (GGT) gene were examined. TPA challenge of HeLa cells resulted in an increase of GGT mRNA and enzyme activity. Deletion analysis of the promoter revealed that the -348 to +60 fragment was able to mediate TPA induced expression. Gel shift and supershift analyses showed that TPA treatment increased nuclear protein binding to a putative AP-1 site (-225 to -214) and that c-Jun was part of the complex. This AP-1 element, when cloned either in its native arrangement or as tandem repeat 5' of the minimal thymidine kinase promoter, mediated a significant increase of luciferase activity after TPA treatment of transfected HeLa cells, while its mutated counterpart abolished the induction. The same AP-1 element was able to mediate TPA induced expression in HepG2 cells. Collectively these results indicate that like other GSH metabolising enzymes, GGT too is a target for AP-1 mediated regulation.