Decrease in glutathione levels of kidney and liver after injection of methionine sulfoximine into rats

105

y-Glutamyl transpeptidase, which consists of two nonidentical subunits, is rapidly inactivated with respect to its transpeptidase and hydrolase activities by the y-Yglutamyl analogs 6-diazo-5-oxo-L-norleucine and L-azaserine. Inactivation, which is prevented by oy-glutamyl substrates (but not by acceptor substrates), is accelerated by maleate, which was previously shown to enhance utilization of glutamine by transpeptidase. 6-Diazo-5-oxo-norleucine reacts specifically, covalently, and stoichiometrically at the 'y-glutamyl site of the enzyme, which was localized through studies with 6-diazo-5-oxo-[4C]norleucine to the light subunits of both the transpeptidase of rat kidney (which has subunits of molecular weights 22,000 and 46,000) and the transpeptidase of human kidney (which has subunits of molecular weights 22,000 and 62,000). The findings, which indicate that these enzymes have similar 7-glutamyl binding subunits, are relevant to the structurefunction relationships of this membrane-bound enzyme and its physiological role.'y-Glutamyl transpeptidase, an enzyme that interacts with glutathione and other y-glutamyl compounds, is found in the plasma membranes of cells that are intimately involved in rapid absorptive and secretory processes (1). Its proposed role in transmembrane transport is consistent with its potential for transferring the y-glutamyl moiety of intracellular glutathione to a wide range of extracellular amino acids and peptides (2-4).Highly purified rat kidney y-glutamyl transpeptidase is a glycoprotein (molecular weight, 68,000) composed of two subunits having molecular weights of 46,000 and 22,000, respectively (5). The enzyme also catalyzes the transfer of the y-glutamyl moiety to water as well as to hydroxylamine (6, 7). Maleate enhances hydrolysis and y-glutamylhydroxamate formation, simultaneously inhibiting transpeptidation (6, 7). Thus, the utilization of L-glutamine by the transpeptidase (which occurs at a very low rate compared to that of glutathione), especially its hydrolysis, is increased about 10-fold by maleate. These findings and kinetic studies support the hypothesis that the reaction mechanism involves a y-glutamylenzyme intermediate (6,8). Consideration of the differences between the subunit compositions and acceptor specificities of transpeptidases obtained from various mammalian kidneys led to the speculation that the y-glutamyl binding site is located on the light subunit (5). It seems evident that data on the locations of the -y-glutamyl donor and acceptor sites on the enzyme subunits and on the orientation of the enzyme in the plasma membrane are crucial to an understanding of the enzyme's physiological function.The studies reported here, in which covalent binding of certain y-glutamyl analogs to the enzyme has been demonstrated, support the hypothesis that the reaction mechanism involves intermediate y-glutamyl enzyme formation. TheAbbreviations: DON, 6-diazo-5-oxo-L-norleucine; DONV, 5-diazo-4-oxo-L-norvaline; CONV, 5-chloro-4-oxo-L-norvaline (L-2-amino-4-oxo-5-c...

Section: Resultsmentioning

confidence: 99%

Affinity labeling of gamma-glutamyl transpeptidase and location of the gamma-glutamyl binding site on the light subunit.

Tate¹,

Meister²

1977

105

“…Glutathione levels also decrease when there is an increase in transpeptidation. Thus, administration to rats of glycylglycine, a good transpeptidase acceptor substrate, leads to marked decreases 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.…”

mentioning

confidence: 98%

“…Thus, administration to rats of glycylglycine, a good transpeptidase acceptor substrate, leads to marked decreases 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.…”

mentioning

confidence: 98%

“…The steady-state level of glutathione in a tissue such as kidney is a function of the -yglutamyl cycle and thus reflects a balance between the synthesis of glutathione (catalyzed by y-glutamylcysteine and glutathione 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. Glutathione levels also decrease when there is an increase in transpeptidation.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Evidence that the gamma-glutamyl cycle functions in vivo using intracellular glutathione: effects of amino acids and selective inhibition of enzymes.

Griffith¹,

Bridges²,

Meister³

1978

122

The function of the y-glutamyl cycle was explored in in vivo studies in which amino acids and specific inhibitors of cycle enzymes ('y-glutamyl transpeptidase, y-glutamyl cyclotransferase, y-glutamylcysteine synthetase, and 5-oxoprolinase) 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 intracellular 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 -yglutamyl cycle and thus reflects a balance between the synthesis of glutathione (catalyzed by y-glutamylcysteine and glutathione 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. Glutathione levels also decrease when there is an increase in transpeptidation. Thus, administration to rats of glycylglycine, a good transpeptidase acceptor substrate, leads to marked decreases 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 transpeptidase in vivo also (i) decreases the rate of glutathione disappearance 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 accumulation 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-oxoprolinase 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 utiliz...

“…Administration of methionine sulfoximine to animals leads to markedly decreased levels of glutathione in the kidney and liver (31). The convulsant activity of methionine sulfoximine is probably due to inhibition of glutamine synthetase or ,y-glutamyl-cysteine synthetase or both (32,33).…”

Section: -Oxoprolinasementioning

confidence: 99%

Selective inhibition of gamma-glutamyl-cycle enzymes by substrate analogs.

Griffith¹,

Meister²

1977

Substrate analogs have been obtained that selectively inhibit the reactions of the -glutamyl cycle or that are susceptible to only limited metabolism by the cycle. Thus, glutathione synthesis may be inhibited and analogs of glutathione may be synthesized that do not participate in transpeptidation. Specific inhibitors of "y-glutamylcyclotransferase and 5-oxoprolinase have been obtained. The findings offer new approaches to the in vivo study of the cycle and also to the design of more specifically directed analogs of inhibitors such as methionine sulfoximine and 6-diazo-5-oxonorleucine.The enzymic reactions of the y-glutamyl cycle [which accounts for the synthesis and degradation of glutathione ( Fig. 1) (1)] include synthesis of the "y-glutamyl peptide bond, transfer of the y-glutamyl group to an acceptor, cyclization of the -y-glutamyl group to form 5-oxoproline, and energy-dependent decyclization of 5-oxoproline to glutamate. In an approach to further understanding of the function of the cycle, we have sought specific inhibitors of its individual reactions and also substrate analogs that would function in some but not all of the reactions. Such goals might be difficult to achieve because all of these reactions involve the y-carboxyl group of glutamate, and a given substrate analog might therefore be expected to interact with more than one enzyme. However, the several enzymic reaction pathways are clearly different and are presumably associated with differences in the enzyme-bound conformations of the glutamate carbon chain at the active sites of the several enzymes.In the present work, we have attempted to exploit such expected differences among the enzymes by manipulation of substrate structure.* We have found that suitable modification of the glutamyl moiety of the substrates yields the desired results: effective and specific inhibitors or nonmetabolizable analogs at each of the steps of the 'y-glutamyl cycle. The present findings thus offer several potentially new approaches to the study of the cycle in vvo and also provide information relevant to further investigations on the mechanisms of action of these metabolically related enzymes. The data reported here may be applied to the modification of potent (but relatively nonspecific) inhibitors such as methionine sulfoximine, azaserine, and 6-diazo-5-oxonorleucine so as to achieve one-enzyme specificity. The findings also make possible improved assay procedures for y-glutamyl-cysteine synthetase, glutathione synthetase, and y-glutamylcyclotransferase in which the substrates and products are not degraded by other enzymes present in unpurified tissue extracts. D-y-Glutamyl-L-a-aminobutyric acid, 'y-(y-methyl)glutamyl-L-a-aminobutyric acid, and fl-aminoglutaryl-L-a-aminobutyric acid were prepared via, the corresponding Nphthaloyl glutamic anhydride analogs as described (7). L-'y-glutamyl-La-aminobutyric acid, and f3-aminoglutaryl-L-a-aminobutyric acid were prepared enzymatically with -y-glutamyl-cysteine synthetase by using phosphoenolpyruvate and pyruv...