IDENTIFICATION, ISOLATION AND CHARACTERIZATION OF gamma-Glutamyl transpeptidase related enzyme

γ-Glutamyltranferase (γGT) is a key enzyme of the γ-glutamyl cycle, able to transfer the γ glutamyl moiety of glutathione (GSH) to acceptor molecules such as amino acids, peptides or simply to water. On the basis of molecular biology data only, the existence of another enzyme, distinct from γGT, capable of splitting the isopeptide bond of GSH, has been reported; this enzyme has been defined γGT-related (γGT-rel). γGT-rel has never been purified nor characterized, so that its existence as a functional enzyme, as well as its possible specific role in GSH homeostasis and hence in the mechanism responsible for the maintenance of cell redox status, are still to be assessed. A peculiar feature associated to γGT-rel is its strict specificity for GSH as substrate that appears to discriminate this enzyme from γGT. The latter is in fact able to act on a series of chromogenic synthetic substrates which are normally used to measure γGT activity in biological samples. Thus, taking advantage of an original specific γGT-rel assay combined with the well known chromogenic assay for γGT with γ-gluthamyl-p-nitroanilide as substrate, we were able to monitor changes in the relative specific activity (γ-glutamyl-p-nitroanilide/GSH activity) following some protein purification steps from bovine spleen. Results obtained are indicative of the actual presence of a GSH specific enzyme distinct from γGT, likely γGT-rel, whose activity is apparently elicited by incubation with oxidized glutathione.