Conversion of glutathione to glutathione disulfide, a catalytic function of gamma-glutamyl transpeptidase.

A purification procedure, based on that previously used for rat kidney gamma-glutamyl transpeptidase, was used for the purification of glutathione oxidase (which converts glutathione to gluthathione disulfide). The two activities co-purified, the ratio of the activities remaining constant through all steps of the isolation procedure. The purified enzyme was separable into 12 isozymic species by isoelectric focusing. All 12 isozymes exhibited a constant ratio of transpeptidase to glutathione oxidase activities, strongly supporting the conclusion that conversion of glutathione to glutathione disulfide is a catalytic function of gamma-glutamyl transpeptidase. Modulation of oxidase activity by inhibitors and acceptor substrates of transpeptidase is discussed in relation to the possible glutathione binding sites involved in gamma-glutamyl transfer and oxidase activities of the enzyme.     

Stimulation of intralysosomal proteolysis by cysteinyl-glycine, a product of the action of gamma-glutamyl transpeptidase on glutathione

The mechanism of the stimulatory effect of glutathione on proteolysis in mouse kidney lysosomes and a lack of an effect in lysosomes from the liver was investigated. The stimulation in kidney lysosomes was inhibited by serine plus borate, a reversibly inhibitor of gamma-glutamyl transpeptidase. Treatment of mouse kidney lysosome suspensions with L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin), an irreversibly inhibitor of the transpeptidase, also inhibited the effect of glutathione, but this inhibition was completely relieved by washing and addition of freshly prepared kidney membranes or purified gamma-glutamyl transpeptidase to the incubation mixtures. Cysteinyl-glycine, a product of the action of gamma-glutamyl transpeptidase, stimulated proteolysis in acivicin-inhibited kidney lysosome preparations similarly to glutathione, and cysteine had no effect at equivalent concentrations. Glutathione also stimulated proteolysis in liver lysosomes in the presence of washed kidney membranes or gamma-glutamyl transpeptidase, but the effect was similar to that produced by equivalent concentrations of cysteine. These results suggest that the stimulatory effect of glutathione was mediated by the action of gamma-glutamyl transpeptidase present in contaminating cell membrane fragments in the lysosome preparations, and that glutathione does not take part in intralysosomal proteolysis. However, the possibility that cysteinyl-glycine is a physiological intralysosomal disulfide reductant in kidney lysosomes has not been excluded.     

The apparent glutathione oxidase activity of gamma-glutamyl transpeptidase. Chemical mechanism

The apparent glutathione oxidase activity of gamma-glutamyl transpeptidase is due to nonenzymatic oxidation and transhydrogenation reactions of cysteinylglycine, an enzymatic product formed from glutathione by hydrolysis or autotranspeptidation. Since cysteinylglycine reacts with oxygen more rapidly than does glutathione, the rate of disulfide formation is increased and either cystinyl-bis-glycine or the mixed disulfide of cysteinylglycine and glutathione forms as an intermediate product. Nonenzymatic transhydrogenation reactions of these disulfides with glutathione yield glutathione disulfide and thus account for the apparent glutathione oxidase activity of gamma-glutamyl transpeptidase. A sensitive assay for glutathione oxidation is described, and it is shown that covalent inhibitors of gamma-glutamyl transpeptidase abolish the oxidase activity of the purified enzyme and of crude homogenates of mouse and rat kidney.     

Ammonia production by IMR-90 fibroblast cultures: effects of ammonia on glutathione, gamma-glutamyl transpeptidase, lysosomal enzymes, and cell division

gamma-Glutamyl transpeptidase has multi-catalytic activities. It degrades glutathione and can produce ammonia from glutamine. The present study was designed to examine whether the decreased cell proliferation, cellular glutathione content and concurrent increase in ammonia production in senescent cells in culture are the result of increased gamma-glutamyl transpeptidase activity. We used IMR-90 fibroblast and 3T3 LI preadipocyte cultures. The cellular glutathione content depended upon cell proliferation and cell density. The glutathione content was higher in cells at logarithmic growth, and lower at stationary growth or post confluency; dead cells had no detectable glutathione by the method currently used. The glutathione content was minimal in "old" IMR-90 cells, regardless of cell density. On the other hand, an increase occurred in the unit number of molecules of bound 5-iodoacetoamidofluorescein, an active-site directed stoichiometric inhibitor of transpeptidase. That result corresponded favorably with the increased enzyme activity, suggesting that the number of enzyme molecules per cell was increased. The inhibition of ammonia production of the cultures by inhibition of gamma-glutamyl transpeptidase by 5-iodoacetoamidofluorescein and reversible inhibition of ammonia production by a serine-borate mixture were consistent with our postulate. Addition of NH4Cl (0.1 mM) to IMR-90 cultures caused increased activities of transpeptidase and some of the lysosomal enzymes; concurrently, the amount of cellular glutathione and the number of cell divisions decreased. This suggests that the increased ammonia production presumably resulting from glutaminase activity of the observed increase of transpeptidase may profoundly affect certain cellular functions.     

Inevitable glutathione,then and now

Glutathione a predominant tripeptide thiol compound of many prokaryotes and eukaryotes, is synthesized from its precursor amino acids eg. gamma-glutamate, cysteine and glycine. It is mainly involved in detoxication mechanisms through conjugation reactions. Other functions include thiol transfer, destruction of free radicals and metabolism of various exogenous and endogenous compounds. It becomes mandatory for a cell to manage high concentration of intracellular GSH to protect itself from chemical/dug abuse. Glutathione dependent enzymes viz: glutathione-S-transferases, glutathione peroxidase, glutathione reductase and gamma-glutamate transpeptidase facilitate protective manifestations. Liver serves as a glutathione-generating factor which supplies the kidney and intestine with other constituents of glutathione resynthesis. The principal mechanism of hepatocyte glutathione turnover appears to be cellular efflux. Kidney too plays an important role in organismic GSH homeostasis. Role of GSH in organs like lung, intestine and brain has recently been described. GSH involvement in programmed cell death has also been indicated. Immense interest makes the then "thee glutathione" as "inevitable glutathione". This article describes the role of this vital molecule in cell physiology and detoxication mechanisms in particular.     

Perturbation of hepatic glutathione level and glutathione-related enzyme activities by repeated administration of aminopyrine in rats

The influence of repeated administration of aminopyrine on the tissue glutathione level and related enzyme activities was investigated in rats. Reduced glutathione level in the liver was not changed after 5 days of treatment but a significant increase was seen after 15 days of aminopyrine treatment. Oxidized glutathione level was unaltered throughout the experiment. Repeated administration of aminopyrine for 5 days caused a marked increase in gamma-glutamyl transpeptidase activities in liver whole homogenates as well as in the nuclear fraction, but not in liver microsomes. These results suggest that gamma-glutamyl transpeptidase located in plasma membrane may be induced by repeated administration of aminopyrine for 5 days. The activities of cytosolic glutathione peroxidase, which modulates glutathione level, were also significantly increased by aminopyrine treatment. Under the same conditions, glutathione peroxidase activity with H2O2 as a substrate was unaltered, while a time-dependent increase in the activity was found when cumene hydroperoxide was used as a substrate, even after a single administration of aminopyrine. The intracellular cysteine level was increased accompanying the increased gamma-glutamyl transpeptidase activities. Therefore, induced gamma-glutamyl transpeptidase may play a role in the reclamation of extracellular oxidized glutathione.     

The alternative pathway of glutathione degradation is mediated by a novel protein complex involving three new genes in Saccharomyces cerevisiae

Glutathione (GSH), L-gamma-glutamyl-L-cysteinyl-glycine, is the major low-molecular-weight thiol compound present in almost all eukaryotic cells. GSH degradation proceeds through the gamma-glutamyl cycle that is initiated, in all organisms, by the action of gamma-glutamyl transpeptidase. A novel pathway for the degradation of GSH that requires the participation of three previously uncharacterized genes is described in the yeast Saccharomyces cerevisiae. These genes have been named DUG1 (YFR044c), DUG2 (YBR281c), and DUG3 (YNL191w) (defective in utilization of glutathione). Although dipeptides and tripeptides with a normal peptide bond such as cys-gly or glu-cys-gly required the presence of only a functional DUG1 gene that encoded a protein belonging to the M20A metallohydrolase family, the presence of an unusual peptide bond such as in the dipeptide, gamma-glu-cys, or in GSH, required the participation of the DUG2 and DUG3 gene products as well. The DUG2 gene encodes a protein with a peptidase domain and a large WD40 repeat region, while the DUG3 gene encoded a protein with a glutamine amidotransferase domain. The Dug1p, Dug2p, and Dug3p proteins were found to form a degradosomal complex through Dug1p-Dug2p and Dug2p-Dug3p interactions. A model is proposed for the functioning of the Dug1p/Dug2p/Dug3p proteins as a specific GSH degradosomal complex.     

Enhanced gamma-glutamyl transpeptidase expression and superoxide production in Mpv17-/- glomerulosclerosis mice.

Recently, gamma-glutamyl transpeptidase, which initiates cleavage of extracellular glutathione, has been shown to promote oxidative damage to cells. Here we examined a murine disease model of glomerulosclerosis, involving loss of the Mpv17 gene coding for a peroxisomal protein. In Mpv17-/- cells, enzyme activity and mRNA expression (examined by quantitative RT-PCR) of membrane-bound gamma-glutamyl transpeptidase were increased, while plasma glutathione peroxidase and superoxide dismutase levels were lowered. Superoxide anion production in these cells was increased as documented by electron spin resonance spectroscopy. In the presence of Mn(III)tetrakis(4-benzoic acid)porphyrin, the activities of gamma-glutamyl transpeptidase and plasma glutathione peroxidase were unchanged, suggesting a relationship between enzyme expression and the amount of reactive oxygen species. Inhibition of gamma-glutamyl transpeptidase by acivicin reverted the lowered plasma glutathione peroxidase and superoxide dismutase activities, indicating reciprocal control of gene expression for these enzymes.     

Metabolism of glutamine and glutathione via gamma-glutamyltranspeptidase and glutamate transport in Helicobacter pylori: possible significance in the pathophysiology of the organism

gamma-Glutamyltranspeptidase (GGT) is a periplasmic enzyme of Helicobacter pylori implicated in its pathogenesis towards mammalian cells. We have cloned and expressed the H. pylori strain 26695 recombinant GGT protein in Escherichia coli and purified it to homogeneity. The purified protein exhibited hydrolysis activity with very high affinities for glutamine and glutathione shown by apparent K(m) values lower than 1 muM. H. pylori cells were unable to take up extracellular glutamine and glutathione directly. Instead, these substances were hydrolysed to glutamate by the action of GGT outside the cells. The glutamate produced was then transported by a Na(+)-dependent reaction into H. pylori cells, where it was mainly incorporated into the TCA cycle and partially utilized as a substrate for glutamine synthesis. These observations show that one of the principle physiological functions of H. pylori GGT is to enable H. pylori cells to utilize extracellular glutamine and glutathione as a source of glutamate. As glutamine and glutathione are important nutrients for maintenance of healthy gastrointestinal tissue, their depletion by the GGT enzyme is hypothesized to account for the damaging of mammalian cells and the pathophysiology of H. pylori.     

Transport of amino acids in gamma-glutamyl transpeptidase-implanted human erythrocytes

gamma-Glutamyl transpeptidase purified from hog kidney cortex was implanted in the human erythrocyte membrane by incubation of erythrocytes at 37 degrees c with gamma-glutamyl transpeptidase-incorporated dipalmitoyl phosphatidylcholine vesicles. Membranes prepared from these implanted cells exhibited 4- to 5-fold increase in gamma-glutamyl transpeptidase activity. The association/insertion of gamma-glutamyl transpeptidase into erythrocyte membrane was further demonstrated by antibody to gamma-glutamyl transpeptidase. Implantation of gamma-glutamyl transpeptidase into erythrocyte membrane led to stimulation of uptake of glutamate and alanine, which are normally transported at a slow rate in human erythrocytes. The uptake of these amino acids in the implanted system was inhibited by inhibitors (serine-borate and azaserine) of transpeptidase activity as well as by antibody to gamma-glutamyl transpeptidase. These results in the implanted human erythrocytes demonstrate that gamma-glutamyl transpeptidase enzyme can mediate the translocation of amino acids and provide further evidence in support of its postulated role in the transport of amino acids in natural membranes.     

Accumulation of cystine from glutathione-cysteine mixed disulfide in cystinotic fibroblasts; blockade by an inhibitor of gamma-glutamyl transpeptidase

Cystinotic and normal skin fibroblasts in tissue culture were treated with varying concentrations of reduced glutathione, oxidized glutathione and glutathione-cysteine mixed disulfide, substrates of gamma-glutamyl transpeptidase, the catabolic enzyme of the gamma-glutamyl cycle. Cystine accumulated more rapidly and to a greater extent from the glutathione-cysteine mixed disulfide in cystinotic than in normal cells. Inhibition of gamma-glutamyl transpeptidase activity by serine in a borate buffer partially blocked this accumulation of cystine. Reduced glutathione and oxidized glutathione have lesser effects on cystine accumulation. Stored cystine in cystinotic tissues may derive in part from glutathione-cysteine mixed disulfide via transpeptidation.     

gamma-Glutamyl transpeptidase in rat ascites tumor cell LY-5. Lack of functional correlation of its catalytic activity with the amino acid transport.

gamma-Glutamyl transpeptidase activity was detected in rat ascites tumor cells (LY-5) suspended in Hanks' balanced saline solution using L-gamma-glutamyl-p-nitroanilide as a substrate. Whole-cell suspension of the tumor cells exhibited full activity of the enzyme without detectable cell disruption under the conditions examined. Various amino acids, transported through specific membrane carriers, did not affect the accessibility of substrate for the enzyme. An inhibitor of sodium-dependent transport systems of amino acids caused no significant change in the rate of enzyme catalysis. Like glutathione or S-methylglutathione, S-acetyldextran (mol. wt 215000) derivative of glutathione, which is believed to be unable to penetrate into intact cells, caused marked inhibition of the rate of p-nitroaniline release from the synthetic substrate by the tumor cells. These data indicated that the active site of the enzyme faced to the outer surface of the cells. gamma-Glutamyl transpeptidase of the tumor cells was successfully affinity-labeled by 6-diazo-5-oxo-L-norleucine, a glutamine analog, without causing detectable change in the viability of the cells under the conditions examined. The rate of transport of alanine, leucine, glycine and glutamine into cells was not affected by the inactivation of this enzyme with the affinity label. Thus, the activity of gamma-glutamyl transpeptidase located on the outer surface of tumor cell membrane does not seem to be requisite for the transport process of amino acids.     

Glutathione and gamma-glutamyl transpeptidase in the adult female rat brain after intraventricular injection of LHRH and somatostatin

Glutathione content and glutamyl transpeptidase activity in different regions of adult female rat brain were determined at 10 and 30 min following intraventricular injection of LHRH and somatostatin. Hypothalamic glutathione levels were significantly elevated at 10 and 30 min after a single injection of a 0.1 micrograms dose of LHRH. On the contrary, glutathione levels significantly decreased in the hypothalamus, cerebral cortex and cerebellum at 10 and 30 min after 0.5 or 1 microgram dose. However, significant decrease in brain stem glutathione was evident at 30 min after 0.5 microgram and 10 min after the 1 microgram dose. Somatostatin at doses of 0.5 microgram and 1 microgram significantly decreased glutathione levels in all four brain regions both at 10 and 30 min following injection into the 3rd ventricle. Gamma-glutamyl transpeptidase activity in the hypothalamus and cerebral cortex was significantly elevated after intraventricular injection of LHRH. However, a significant increase in gamma-glutamyl transpeptidase activity in cerebellum and brain stem was seen only with 0.5 and 1 micrograms doses of LHRH. Somatostatin also significantly increased gamma-glutamyl transpeptidase activity in hypothalamus, cerebral cortex, brain stem and cerebellum. The decrease in glutathione levels with corresponding increase in gamma-glutamyl transpeptidase activity after intraventricular administration of LHRH and somatostatin suggests a possible interaction between glutathione and hypothalamic peptides.     

Distribution and some properties of the glutathione S-transferase and gamma-glutamyl transpeptidase activities of rainbow trout

1. Gills, kidney, intestinal caeca and liver of trout have glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene (200 500 nmol/min/mg protein), and reduced glutathione (0.5 2.0 mmol/kg tissue). 2. Only kidney and intestinal caeca have substantial gamma-glutamyl transpeptidase activity with gamma-glutamyl-rho-nitroanilide (2-9 nmol/min/mg protein). 3. Renal gamma-glutamyl transpeptidase is membrane-bound and has similar kinetic properties to its mammalian counterparts. 4. The data are consistent with the presence of a mercapturic acid pathway in trout.     

Dietary influence of selenium on the incidence of N-nitrosodiethylamine-induced hepatoma with reference to drug and glutathione metabolizing enzymes

The dietary administration of selenium (sodium selenite; 4 p.p.m.) daily has been found to be highly effective in reducing the incidence of cancer induced by N-nitrosodiethylamine (DEN) in Wistar strain rats. Selenium treatment either before initiation, during initiation and selection/phenobarbital promotion phases of hepatocarcinogenesis has been found to be effective in elevating hepatic microsomal cytochrome b(5), NADPH-cytochrome C reductase and cytosolic aryl hydrocarbon hydroxylase activities to a statistically significant level measured either in the hyperplastic nodule or in the surrounding liver tissues compared to control animals. Moreover, selenium treatment throughout the study, decreases the cytosolic glutathione S-transferase and microsomal UDP-glucuronyl transferase activities by a significant degree when compared to control rats. Alterations in glutathione metabolizing enzyme activities (glutathione reductase, gamma-glutamyl transpeptidase, gamma-glutamylcysteine synthetase and glucose-6-phosphate dehydrogenase) were also observed in selenium-treated groups. Our results confirm the fact that selenium is particularly protective in limiting the action of DEN during the initiation phase of hepatocarcinogenesis.     

Novel kinetics of mammalian glutathione synthetase: characterization of gamma-glutamyl substrate cooperative binding

Glutathione (GSH) synthetase [L-gamma-glutamyl-L-cysteinyl:glycine ligase (ADP-forming), EC 6.3.2.3] catalyzes the final step in GSH biosynthesis. Mammalian glutathione synthetase is a homodimer with each subunit containing an active site. We report the detailed kinetic data for purified recombinant rat glutathione synthetase. It has the highest specific activity (11 micromol/min/mg) reported for any mammalian glutathione synthetase. The apparent K(m) values for ATP and glycine are 37 and 913 microM, respectively. The Lineweaver-Burk double reciprocal plot for gamma-glutamyl substrate binding revealed a departure from linearity indicating cooperative binding. Quantitative analysis of the kinetic results for gamma-glutamyl substrate binding gives a Hill coefficient (h) of 0. 576, which shows the negative cooperativity. Neither ATP, the other substrate involved in forming the enzyme-bound gamma-glutamyl phosphate intermediate, nor glycine, which attacks this intermediate to form GSH, exhibit any cooperativity. The cooperative binding of gamma-glutamyl substrate is not affected by ATP concentration. Thus, mammalian glutathione synthetase is an allosteric enzyme.     

Studies in the enzymology of glutathione metabolism in human erythrocytes

Spectrophotometric assay methods are described for glutathione synthetase, gamma-glutamylcysteine synthetase and gamma-glutamyl transpeptidase of erythrocytes. The contents of these enzymes in normal human erythrocytes are reported. Erythrocyte glutathione synthetase is inhibited by ADP; this inhibition is competitive with respect to ATP. gamma-Glutamylcysteine synthetase is subject to feedback inhibition by GSH, and is also inhibited by NADH, and to a lesser extent by NAD(+) and NADPH. This enzyme is irreversibly inactivated by cysteamine.     

The role of glutathione in cancer

Glutathione is an abundant natural tripeptide found within almost all cells. Glutathione is highly reactive and is often found conjugated to other molecules via its sulfhydryl moiety. It instils several vital roles within a cell including antioxidation, maintenance of the redox state, modulation of the immune response and detoxification of xenobiotics. With respect to cancer, glutathione metabolism is able to play both protective and pathogenic roles. It is crucial in the removal and detoxification of carcinogens, and alterations in this pathway, can have a profound effect on cell survival. However, by conferring resistance to a number of chemotherapeutic drugs, elevated levels of glutathione in tumour cells are able to protect such cells in bone marrow, breast, colon, larynx and lung cancers. Here we present a number of studies investigating the role of glutathione in promoting cancer, impeding chemotherapy, and the use of glutathione modulation to enhance anti-neoplastic therapy.     

Effects of inhibition of cystathionase activity on glutathione and metallothionein levels in the adult rat

The effects of alterations in sulfur metabolism on hepatic and renal metallothionein and glutathione metabolism were studied in the adult rat using inhibition of two enzymes of these pathways, hepatic cystathionase and renal gamma-glutamyl transpeptidase. Rats were fed a diet containing both methionine (0.66%) and cystine (0.20%) for 1 week before receiving three consecutive daily intraperitoneal injections of propargylglycine, a selective cystathionase inhibitor, at various doses (2.5–375 μmol/kg). When hepatic cystathionase was inhibited greater than 90% (≥50 μmol propargylglycine/kg), renal and hepatic metallothionein and hepatic glutathione were unaltered except at the highest dose. On the other hand, renal glutathione was increased twofold with a concomitant decrease in renal gamma-glutamyl transpeptidase activity (50% of control). In another experiment, when renal gamma-glutamyl transpeptidase was inhibited greater than 90% with three consecutive daily injections of acivicin, a selective gamma-glutamyl transpeptidase inhibitor (10 mg/kg IP), renal glutathione content was unaltered while hepatic glutathione was decreased. Renal and hepatic metallothionein were not changed. Thus, the cysteine pools for metallothionein and glutathione appear unrelated under the present experimental conditions. In addition, following either propargylglycine or acivicin injections, renal and hepatic glutathione pools appear to be altered differently. These results suggest that renal glutathione may be preferentially maintained even when hepatic glutathione is decreased.