Intermediates of the gamma-Glutamyl Cycle in Mouse Tissues. Influence of Administration of Amino Acids on Pyrrolidone Carboxylate and gamma-Glutamyl Amino Acids

Abstract

y-Glutamyl transpeptidase, y-glutamyl cyclotransferase, L-pyrrolidone carboxylate hydrolase, y-glutamylcysteine synthetase and glutathione synthetase, the enzymes of the y-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 y-glutamyl cyclotransferase from mouse tissues was similar to that from rat tissues. The concentration of pyrrolidone carboxylate and y-glutamyl amino acids, intermediates of the y-glutamyl cycle, was determined by a gas chromatographic procedure coupled with electron capture detection. Administration of ~-2-aminobutyrate, an amino acid that is utilized as substrate in the reaction catalyzed by y-glutamylcysteine synthetase, led to a large accumulation of y-glutamyl-2-aminobutyrate and pyrrolidone carboxylate in mouse tissues. L-Methionine-RS-sulfoximine, an inhibitor of y-glutamylcysteine 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 y-glutamyl derivatives (e.g. y-glutamylmethionine and y-glutamylphenylalanine) was detected. These intermediates are probably formed by transpeptidation between glutathione and the corresponding amino acid, catalyzed by y-glutamyl transpeptidase. 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 y-glutamyl amino acids and pyrrolidone carboxylate exist in vivo. One of these results from the function of y-glutamylcysteine synthetase in glutathione synthesis. The other pathway involves the amino-acid-dependent degradation of glutathione, mediated by y-glutamyl transpeptidase. Only very small amounts of free intermediates are apparently derived from the latter pathway, suggesting that the y-glutamyl amino acids formed in this pathway are either enzyme-bound or are directly hydrolyzed to glutamate and free amino acid. y-glutamyl amino acids with the concomitant release of free amino acid. The enzyme is widely distributed in animal tissues [4] and is part of the y-glutamyl cycle which was proposed as a transport system for amino acids [5]. Further reactions of the cycle include: (a) conversion of L-pyrrolidone carboxylate to L-glutamate in an ATP-requiring reaction [6,7]; (b) the ATPdependent synthesis of glutathione catalyzed in sequence by y-glutamylcysteine synthetase and glutathione synthetase [8]; and (c) transfer of the y-glutamyl moiety of GSH to an amino acid to form the correEur. J. Biochem. 53 (1975)

Metabolism of γ‐Glutamyl Amino Acids and Peptides in Mouse Liver and Kidney in vivo

“…There was also a moderate, but significant, increase in the concentration of pyrrolidone carboxylate. Such an increase has been previously observed after administration of L-2-aminobutyrate alone [9]. It is notable that this increase was much lower than that found in the kidney.…”

“…The radioactive peaks were isolated using a fraction collector and counted in a Nuclear Chicago isocap-300 scintillation counter. Pyrrolidone carboxylate was determined by gas-liquid chromatography [9,23] or by conversion to glutamate followed by amino acid analysis, as described previously [24]. and aspartate coupled with a concomitant large accumulation of free L-2-aminobutyrate indicates that uptake of the y-glutamyl derivative is accompanied to a significant extent by hydrolysis of the peptide to its component amino acids.…”

“…y-Glutamyl transpeptidase or (y-g1utamyl)-peptide: amino acid y-glutamyltransferase (EC 2.3.2.2); y-Glutamylcyclotransferase (EC 2.3.2.4); y-Glutamylcysteine synthetase (EC 6.3.2.2); Glutathione synthetase (EC 6.3.2.3); Glutamine synthetase (EC 6.3.1.2). y-Glutamyl amino acids are formed in cico by two separate pathways [9]. In the first glutamate reacts with cysteine or several related amino acids to give the corresponding 7)-glutamyl amino acid.…”

“…y-Glutamyl derivatives of a number of amino acids have been also found in brain [6] and human urine [7,8]. y-Glutamyl amino acids are formed in cico by two separate pathways [9]. In the first glutamate reacts with cysteine or several related amino acids to give the corresponding 7)-glutamyl amino acid.…”

Molecular Mechanism of Decreased Glutathione Content in Human Immunodeficiency Virus Type 1 Tat-transgenic Mice

“…Obviously, if GS activity falls significantly, it could become the rate-limiting step. ␥-Glutamylcysteine, produced by GCS, has been suggested to be channeled efficiently to GS (63). It is known that physical interaction or close spatial arrangement of proteins that participate in a given signal transduction or biochemical pathway enables an efficient relay of signals to effector molecules as well as the channeling of intermediates between interacting metabolic enzymes (64).…”