Peptide transport in bacteria: methods, mutants and energy coupling.

yglutamyl amino acids and for glutathione. Transport of y-glutamylmethionine is associated with markedly increased renal 5-oxoproline formation, indicating that transported dipeptide serves as a substrate of intracellular yglutamylcyclotransferase (R. J. Bridges & A. Meister, unpublished work). Previous investigations showed that renal 5-oxoproline levels are significantly elevated after administration of a competitive inhibitor of 5-oxoprolinase: when amino acids were administered together with the inhibitor, the formation of 5-oxoproline was substantially increased (Van Der Werf et al., 1974). These findings were interpreted to indicate a pathway involving transpeptidation between glutathione and the administered amino acid, transport of y-glutamyl amino acids and conversion of the latter into 5-oxoproline. In other studies, administration of moderate amounts of amino acids were found to decrease intracellular glutathione levels substantially and this effect was prevented by administration of an inhibitor of transferase. The transport of y-glutamyl amino acids into the kidney in intact mice is inhibited by administration of inhibitors of glutathione biosynthesis, inhibitors of y-glutamyltransferase, and by high levels of extracellular glutathione. Inhibition by inhibitors of transferase may be ascribed, at least in part, to the associated increase in extracellular glutathione levels. Inhibition by inhibitors of glutathione synthesis may reflect a requirement for glutathione in the transport process, and also a direct effect of such inhibitors (e.g., buthionine sulphoximine) on the transport of y-glutamyl amino acids. Administration to mice of y-glutamylcystine or of yglutamylcysteine disulphide leads to higher renal levels of glutathione than found in control animals or in animals given a mixture of the constituent amino acids (Anderson & Meister, 1983). This result seems to reflect a by-pass of the feedback-regulated step of glutathione synthesis. Studies with yglutamylcystine labelled selectively with 35S in either the internal or external sulphur atom indicate preferential utilization of the yglutamylcysteine moiety of this compound for glutathione synthesis. When mice were depleted of glutathione by treatment with buthionine sulphoximine, the disulphides y-glutamylcystine and yglutamylcysteine disulphide did not increase glutathione levels. However, under these conditions, y-glutamylcysteine was effectively utilized for glutathione synthesis. These observations suggest a pathway in which y-glutamylcystine, formed by transpeptidation between glutathione and cystine, is transported and reduced by transhydrogenation with glutathione to form cysteine and y-glutamylcysteine, and in which yglutamylcysteine is used directly by glutathione synthetase for glutathione synthesis. These results provide additional strong evidence for transport of y-glutamyl amino acids and indicate an alternative pathway of glutathione synthesis. Administration of yglutamylcysteine (or similar compounds) may provide a means for increasing kidney glutathione levels. The hypothesis that glutathione, via the reactions of the y-glutamyl cycle, may participate in one of the pathways that mediates transport of amino acids is thus supported by a number of observations and experimental approaches. These include the findings that y-glutamyl amino acids are formed by transpeptidase under physiological conditions and that y-glutamyl amino acids are effectively transported by a system not shared by free amino acids. Furthermore, a number of studies in viuo indicate that there is a significant relationship between glutathione metabolism and the transport of amino acids. The studies reported above reflect current progress in the elucidation of the enzymic and transport phenomena associated with glutathione metabolism. which form the basis for detailed understanding of the physiological functions of this tripeptide. Recent research indicates that selective modification of glutathione metabolism may be of considerable value in chemotherapy, radiation therapy, inhibition of thermotolerance and in the selective destruction of certain parasites, Thus, buthionine sulphoximine-induced depletion of glutathione may be usefully harnessed for various therapeutic purposes (Meister, 1983). In addition. treatment with L-2-oxothiazolidine 4carboxylate, a substrate of 5-oxoprolinase (Williamson & Meister. 1982), appears to be useful as an efficient intracellular cysteine delivery system (which stimulates glutathione formation) for the treatment and prevention of drug toxicity and for protection against radiation and oxygen toxicity.