The ectoenzyme, ␥-glutamyl transpeptidase (GGT, EC 2.3.2.2) cleaves glutathione (GSH) to facilitate the recapture of cysteine for synthesis of intracellular GSH. The impact of GGT expression on cell survival during oxidative stress was investigated using the human B cell lymphoblastoid cell line, Ramos. Ramos cells did not express surface GGT and exhibited no GGT enzyme activity. In contrast, Ramos cells stably transfected with the human GGT cDNA expressed high levels of surface GGT and enzymatic activity. GGT-transfected Ramos cells were protected from apoptosis when cultured in cyst(e)ine-deficient medium. The GGT-expressing cells also had lower levels of intracellular reactive oxygen species (ROS). Homocysteic acid and alanine, inhibitors of cystine and cysteine uptake, respectively, caused increased ROS content and diminished viability of GGT expressing cells. Exogenous GSH increased the viability of the GGT-transfected cells more effectively than that of control cells, whereas the products of GSH metabolism prevented death of both the control and GGT-transfected cells comparably. These data indicate that GGT cleavage of GSH and the subsequent recapture of cysteine and cystine allow cells to maintain low levels of cellular ROS and thereby avoid apoptosis induced by oxidative stress. Glutathione (GSH)1 is the major intracellular nonprotein thiol defense against free radicals (1). It is a tripeptide consisting of glutamic acid, cysteine, and glycine. The antioxidant effect of GSH is dependent on the reduced sulfhydryl moiety of cysteine. Levels of intracellular GSH are maintained by a series of synthetic enzymes that regulate production of this tripeptide. Because of its relatively high intracellular concentration, GSH is transported out of cells into the extracellular environment. Once in the extracellular environment, there is little or no cellular uptake of intact glutathione. Rather, GSH is metabolized by ␥-glutamyl transpeptidase (GGT, EC 2.3.2.2), a plasma membrane enzyme whose catalytic site faces the extracellular environment (reviewed in Ref. 2). GGT removes the ␥-glutamyl group from GSH, after which cysteinylglycine can be cleaved by a membrane dipeptidase. The released cysteine can then be transported into the cell and used as a substrate for de novo synthesis of GSH. This ␥-glutamyl cycle is thought to maintain cellular GSH levels. Despite reports of GGT-related enzymes (3) or direct uptake of intact GSH by cells (4), the ␥-glutamyl cycle involving GGT is generally thought to be the major pathway by which cells utilize extracellular GSH for the de novo synthesis of intracellular GSH (5).We have recently shown that primary human memory T cells express higher level of GGT than naive T cells (6). Because these cells are specialized to access extravascular inflammatory sites (7), this observation suggests that increased GGT expression may provide an adaptive advantage in permitting these cells to resist oxidative stress and/or grow more effectively. This possibility is supported by the finding that T c...

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