Proc. Natl. Acad. Sci. USA Vol. 75, No. 11, pp. 5405-5408, November 1978 Biochemistry Evidence that the y-glutamyl cycle functions in vivo using intracellular glutathione: Effects of amino acids and selective inhibition of enzymes ('y-glutamyl transpeptidase/5-oxoproline/prothionine sulfoximine/amino acid transport/jB-aminoglutaryl-a-aminobutyrate) OWEN W. GRIFFITH, RICHARD J. BRIDGES, AND ALTON MEISTER Department of Biochemistry, Cornell University Medical College, New York, New York 10021 Contributed by Alton Meister, August 28, 1978 ABSTRACT The function of the y-glutamyl cycle was ex- plored in in vivo studies in which amino acids and specific in- hibitors of cycle enzymes ('y-glutamyl transpeptidase, y-glutamyl cyclotransferase, y-glutamylcysteine synthetase, and 5-ox- oprolinase) were administered to mice. The findings, which show that the y-glutamyl cycle functions in vivo, support the conclusion that 'y-glutamyl amino acids formed by yglutamyl transpeptidase from externally supplied amino acids and in- tracellular glutathione are translocated into the cell and thus indicate that there is a significant physiological connection between the metabolism of glutathione and the transport of amino acids. Glutathione occurs intracellularly in millimolar concentrations whereas the level of extracellular glutathione (e.g., blood plasma) is in the micromolar range. The steady-state level of glutathione in a tissue such as kidney is a function of the -y- glutamyl cycle and thus reflects a balance between the synthesis of glutathione (catalyzed by y-glutamylcysteine and gluta- thione synthetases) and the utilization of glutathione (catalyzed by y-glutamyl transpeptidase) (1). When glutathione synthesis is inhibited, the glutathione level falls rapidly (2, 3), reflecting the substantial normal rate of glutathione utilization. Gluta- thione levels also decrease when there is an increase in trans- peptidation. Thus, administration to rats of glycylglycine, a good transpeptidase acceptor substrate, leads to marked de- creases in the levels of glutathione in the kidney and liver (2). Such a decrease in glutathione did not occur after equivalent doses of glycine or other amino acids (2), presumably because after small doses of amino acids the amount of glutathione used for transpeptidation can be rapidly regenerated by synthesis. However, the very rapid transpeptidation reaction that occurs after glycylglycine administration uses glutathione at a much faster rate than that of its resynthesis, and the glutathione level therefore decreases. We have now found that administration of a large amount of amino acid to mice also decreases renal glutathione and that this effect (which is accompanied by increased 5-oxoproline formation) is markedly diminished by administration of an inhibitor of y-glutamyl transpeptidase. Inhibition of trans- peptidase in vivo also (i) decreases the rate of glutathione dis- appearance that occurs after inhibition of glutathione synthesis, and (ii) leads to a moderate increase in kidney glutathione levels in otherwise untreated animals. Previous studies showed that in vivo inhibition of 5-oxoprolinase led to 5-oxoproline accu- mulation and that such accumulation was greater after amino acid administration (4). In the present work we found that in vivo inhibition of y-glutamylcyclotransferase decreased the accumulation of 5-oxoproline found after inhibition of 5-ox- oprolinase and also decreased the normal steady-state levels of 5-oxoproline. This indicates that y-glutamyl cyclotransferase and 5-oxoprolinase are major in vivo catalysts for the formation and utilization, respectively, of 5-oxoproline. EXPERIMENTAL Materials. Amino acids were obtained from Sigma. L-2- Imidazolidone-4-carboxylate was obtained from Bachem (Torrance, CA). f3-Aminoglutaryl-L-a-amino[14C]butyrate was prepared enzymatically (5). NCS strain male mice, 6-7 weeks old (25-30 g), were obtained from The Rockefeller Universi- ty. Methods. Mice, fasted 24 hr with free access to water, were injected intraperitoneally (two-thirds of dose) and subcuta- neously on the back (one-third of dose). The doses, 32 mmol/kg, were given as 0.4 M solutions; lower doses were proportionately diluted. Multicompound injections were made with mixed so- lutions. Animals were killed by decapitation and the kidneys were removed and homogenized in 5 vol of 1% picric acid. After centrifugation, an aliquot was treated with 2-vinylpyri- dine and analyzed for amino acids and glutathione (6). Tissue 5-oxoproline was isolated (7) and then hydrolyzed and quan- titated by amino acid analysis (4, 6). Data given are average values based on 4-20 separate determinations. RESULTS When methionine or certain amino acids were given to mice, there was a substantial decrease in the level of glutathione in the kidney (Table 1). At 30 min after L-methionine was given at a dose of 32 mmol/kg, the glutathione level decreased to about half of the control (Table 1; Fig. 1), and this effect was accompanied by a marked increase in the level of 5-oxoproline. Increases in 5-oxoproline levels were found after administration of serine or alanine but not glycylglycine, the y-glutamyl de- rivative of which is not a substrate of 'y-glutamylcyclotrans- ferase. Because administration of methionine was accompanied by decreases in the levels of glutamate, glycine, and cysteine (Table 2), a study was done in which glutamate, glycine, and cysteine were also given. Administration of the amino acid constituents of glutathione increased the glutathione level somewhat (Exp. 3) but, when these amino acids and methionine were given together, the net decrease of the glutathione levels [3.7 (Exp. 3) minus 2.1 (Exp. 4) = 1.6] was about the same as found when methionine was given alone [3.0 (Exp. 1) minus 1.24 (Exp. 2) = 1.76]. The effects of giving a transpeptidase inhibitor [serine plus borate (8, 9)] on the phenomena described above are given in Table 3. Injection of serine and borate led to a moderate in- crease in the glutathione level, whereas borate had no effect. O-Methylserine [which does not serve in place of serine in in- hibiting transpeptidase (unpublished data)] did not replace 5405 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Downloaded by guest on June 16, 2020