Intermediates of the γ‐Glutamyl Cycle in Mouse Tissues: Influence of Administration of Amino Acids on Pyrrolidone Carboxylate and γ‐Glutamyl Amino Acids

γ‐Glutamyl transpeptidase, γ‐glutamyl cyclotransferase, l‐pyrrolidone carboxylate hydrolase, γ‐glutamyleysteine synthetase and glutathione synthetase, the enzymes of the γ‐glutamyl cycle, were found in mouse brain, liver and kidney. The activity of l‐pyrrolidone carboxylate hydrolase was many times lower than the activities of the other enzymes, and thus the conversion of l‐pyrrolidone carboxylate to l‐glutamate is likely to be the rate‐limiting step of the cycle. The specificity of γ‐glutamyl cyclotransferase from mouse tissues was similar to that from rat tissues. The concentration of pyrrolidone carboxylate and γ‐glutamyl amino acids, intermediates of the γ‐glutamyl cycle, was determined by a gas chromatographic procedure coupled with electron capture detection. Administration of l‐2‐aminobutyrate, an amino acid that is utilized as substrate in the reaction catalyzed by γ‐glutamyleysteine synthetase, led to a large accumulation of γ‐glutamyl‐2‐aminobutyrate and pyrrolidone carboxylate in mouse tissues. l‐Methionine‐RS‐sulfoximine, an inhibitor of γ‐glutamyl‐cysteine synthetase, abolished the increase in concentration of pyrrolidone carboxylate. No accumulation of pyrrolidone carboxylate was observed after l‐cysteine. The separate administration of several protein amino acids had little effect on the concentration of pyrrolidone carboxylate; however formation of small amounts of the corresponding γ‐glutamyl derivatives (e.g., γ‐glutamylmethionine and γ‐glutamylphenylalanine) was detected. These intermediates are probably formed by trans‐peptidation between glutathione and the corresponding amino acid, catalyzed by γ‐glutamyl trans‐peptidase. The concentration of pyrrolidone carboxylate increased significantly after administration of a mixture containing all protein amino acids, the highest increase occurring in the kidney. The results suggest that two separate pathways for the formation of γ‐glutamyl amino acids and pyrrolidone carboxylate exist in vivo. One of these results from the function of γ‐glutamylcysteine synthetase in glutathione synthesis. The other pathway involves the amino‐acid‐dependent degradation of glutathione, mediated by γ‐glutamyl transpeptidase. Only very small amounts of free intermediates are apparently derived from the latter pathway, suggesting that the γ‐glutamyl amino acids formed in this pathway are either enzyme‐bound or are directly hydrolyzed to glutamate and free amino acid.