A spectrophotometric assay of gamma-glutamylcysteine synthetase and glutathione synthetase in crude extracts from tissues and cultured mammalian cells

An assay of gamma-glutamylcysteine synthetase (gamma-GCS) and glutathione synthetase (GS) in crude extracts of cultured cells and tissues is described. It represents a novel combination of known methods, and is based on the formation of glutathione (GSH) from cysteine, glutamate and glycine in the presence of rat kidney GS for the assay of gamma-GCS, or from gamma-glutamylcysteine and glycine for the assay of GS. GSH is then quantified by the Tietze recycling method. Assay mixtures contain the gamma-glutamyl transpeptidase (GGT) inhibitor acivicin in order to prevent the degradation of gamma-glutamylcysteine and of the accumulating GSH, and dithiothreitol in order to prevent the oxidation of cysteine and gamma-glutamylcysteine. gamma-GCS and GS levels determined by this method are comparable to those determined by others. The method is suitable for the rapid determination of gamma-GCS GS in GGT-containing tissues and for the studies of induction of gamma-GCS and GS in tissue cultures.     

Dissecting the role of glutathione biosynthesis in Plasmodium falciparum

Glutathione (γ-glutamylcysteinyl-glycine, GSH) has vital functions as thiol redox buffer and cofactor of antioxidant and detoxification enzymes. Plasmodium falciparum possesses a functional GSH biosynthesis pathway and contains mM concentrations of the tripeptide. It was impossible to delete in P. falciparum the genes encoding γ-glutamylcysteine synthetase (γGCS) or glutathione synthetase (GS), the two enzymes synthesizing GSH, although both gene loci were not refractory to recombination. Our data show that the parasites cannot compensate for the loss of GSH biosynthesis via GSH uptake. This suggests an important if not essential function of GSH biosynthesis pathway for the parasites. Treatment with the irreversible inhibitor of γGCS L-buthionine sulfoximine (BSO) reduced intracellular GSH levels in P. falciparum and was lethal for their intra-erythrocytic development, corroborating the suggestion that GSH biosynthesis is important for parasite survival. Episomal expression of γgcs in P. falciparum increased tolerance to BSO attributable to increased levels of γGCS. Concomitantly expression of glutathione reductase was reduced leading to an increased GSH efflux. Together these data indicate that GSH levels are tightly regulated by a functional GSH biosynthesis and the reduction of GSSG.     

Restoration of glutathione levels in vascular smooth muscle cells exposed to high glucose conditions.

Hyperglycemia-induced oxidative stress may play a key role in the pathogenesis of diabetic vascular disease. The purpose of this study was to determine the effects of glucose on levels of glutathione (a major intracellular antioxidant), the expression of gamma-glutamylcysteine synthetase (the rate-limiting enzyme in glutathione de novo synthesis), and DNA damage in human vascular smooth muscle cells in vitro. High glucose conditions and buthionine sulphoximine, an inhibitor of gamma-glutamylcysteine synthetase, reduced intracellular glutathione levels in vascular smooth muscle cells. This reduction was accompanied by a decrease in the mRNA expression of both subunits of gamma-glutamylcysteine synthetase as well as an increase in DNA damage. In high glucose conditions, incubation of the vascular smooth muscle cells with alpha-lipoic acid and L-cystine restored glutathione levels. We suggest that the decrease in GSH levels seen in high glucose conditions is mediated by the availability of cysteine (rate-limiting substrate in de novo glutathione synthesis) and the gene expression of the gamma-glutamylcysteine synthetase enzyme. Glutathione depletion is associated with an increase in DNA damage, which can be reduced when glutathione levels are restored.     

Metal(loid)s and radionuclides cytotoxicity in Saccharomyces cerevisiae. Role of YCF1, glutathione and effect of buthionine sulfoximine

The presence of heavy metal(loid)s in soils and waters is an important issue with regards to human health. Taking into account speciation problems, in the first part of this report, we investigated under identical growth conditions, yeast tolerance to a set of 15 cytotoxic metal(loid)s and radionuclides. The yeast cadmium factor 1 (YCF1) is an ATP-Binding Cassette transporter mediating the glutathione detoxification of heavy metals. In the second part, metal(loid)s that could be handled by YCF1 and a possible re-localisation of the transporter after heavy metal exposure were evaluated. YCF1 and a C-terminal GFP fusion, YCF1-GFP, were overexpressed in wild-type and Deltaycf1 strains. Both forms were functional, conferring a tolerance to Cd, Sb, As, Pb, Hg but not to Ni, Zn, Cu, Ag, Se, Te, Cr, Sr, Tc, U. Confocal experiments demonstrated that during exposure to cytotoxic metals, the localisation of YCF1-GFP was restricted to the yeast vacuolar membrane. In the last part, the role of glutathione in this resistance mechanism to metal(loid)s was studied. In the presence of heavy metals, application of buthionine sulfoximine (BSO), a well-known inhibitor of gamma-glutamylcysteine synthetase, led to a decrease in the cytosolic pool of GSH and to a limitation of yeast growth. Surprisingly, BSO was able to phenocopy the deletion of gamma-glutamylcysteine synthetase after exposure to Cd but not to Sb or As. In the genetic context of gsh1 and gsh2 yeast mutants, the critical role of GSH for Cd, As, Sb and Hg tolerance was compared to that of wild-type and Deltaycf1.     

Glutathione is required for growth and prespore cell differentiation in Dictyostelium

Glutathione (GSH) is the most abundant non-protein thiol in eukaryotic cells and acts as reducing equivalent in many cellular processes. We investigated the role of glutathione in Dictyostelium development by disruption of gamma-glutamylcysteine synthetase (GCS), an essential enzyme in glutathione biosynthesis. GCS-null strain showed glutathione auxotrophy and could not grow in medium containing other thiol compounds. The developmental progress of GCS-null strain was determined by GSH concentration contained in preincubated media before development. GCS-null strain preincubated with 0.2 mM GSH was arrested at mound stage or formed bent stalk-like structure during development. GCS-null strain preincubated with more than 0.5 mM GSH formed fruiting body with spores, but spore viability was significantly reduced. In GCS-null strain precultured with 0.2 mM GSH, prestalk-specific gene expression was delayed, while prespore-specific gene and spore-specific gene expressions were not detected. In addition, GCS-null strain precultured with 0.2 mM GSH showed prestalk tendency and extended G1 phase of cell cycle. Since G1 phase cells at starvation differentiate into prestalk cells, developmental defect of GCS-null strain precultured with 0.2 mM GSH may result from altered cell cycle. These results suggest that glutathione itself is essential for growth and differentiation to prespore in Dictyostelium.     

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.     

Rapid alteration of cellular redox homeostasis upon exposure to cadmium and mercury in alfalfa seedlings

Here, the kinetics of oxidative stress responses of alfalfa (Medicago sativa) seedlings to cadmium (Cd) and mercury (Hg) (0, 3, 10 and 30 microm) exposure, expanding from a few minutes to 24 h, were studied. Intracellular oxidative stress was analysed using 2',7'-dichlorofluorescin diacetate and extracellular hydrogen peroxide (H(2)O(2)) production was studied with Amplex Red. Growth inhibition, concentrations of ascorbate, glutathione (GSH), homoglutathione (hGSH), Cd and Hg, ascorbate peroxidase (APX) activity, and expression of genes related to GSH metabolism were also determined. Both Cd and Hg increased cellular reactive oxygen species (ROS) production and extracellular H(2)O(2) formation, but in different ways. The increase was mild and slow with Cd, but more rapid and transient with Hg. Hg treatments also caused a higher cell death rate, significant oxidation of hGSH, as well as increased APX activity and transient overexpression of glutathione reductase 2, glutamylcysteinyl synthetase, and homoglutathione synthetase genes. However, Cd caused minor alterations. Hg accumulation was one order of magnitude higher than Cd accumulation. The different kinetics of early physiological responses in vivo to Cd and Hg might be relevant to the characterization of their mechanisms of toxicity. Thus, high accumulation of Hg might explain the metabolism poisoning observed in Hg-treated seedlings.     

Peroxisome proliferator perfluorodecanoic acid alters glutathione and related enzymes

Previously we have shown that treatment with the peroxisome proliferator perfluorodecanoic acid (PFDA) significantly increased hepatic reduced glutathione (GSH) content without altering the activity of selenium-glutathione peroxidase. In this study we examined some potential mechanisms by which PFDA treatment increases GSH levels. Male Sprague-Dawley rats were given a single injection of 0, 8.8, 17.5, and 35 mg PFDA in corn oil per kg body weight. Twelve days later the effects of PFDA on the activities of enzymes associated with GSH synthesis, utilization, and regeneration were assessed. The results showed that in a dose-dependent manner, PFDA treatment significantly decreased the activity of gamma-glutamylcysteine synthetase, while the activities of NADPH-generating enzymes, malic enzyme, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase were increased. PFDA treatment also dose dependently decreased cytosolic, but not microsomal, glutathione S-transferase activity, and the activity of glutathione reductase was decreased by the highest dose of PFDA. The data obtained suggest that increased hepatic GSH levels following PFDA treatment may result from increased regeneration and/or decreased utilization.     

Coordinate induction of glutathione biosynthesis and glutathione-metabolizing enzymes is correlated with salt tolerance in tomato

The acclimation of reduced glutathione (GSH) biosynthesis and GSH-utilizing enzymes to salt stress was studied in two tomato species that differ in stress tolerance. Salt increased GSH content and GSH:GSSG (oxidized glutathione) ratio in oxidative stress-tolerant Lycopersicon pennellii (Lpa) but not in Lycopersicon esculentum (Lem). These changes were associated with salt-induced upregulation of gamma-glutamylcysteine synthetase protein, an effect which was prevented by preincubation with buthionine sulfoximine. Salt treatment induced glutathione peroxidase and glutathione-S-transferase but not glutathione reductase activities in Lpa. These results suggest a mechanism of coordinate upregulation of synthesis and metabolism of GSH in Lpa, that is absent from Lem.     

Chromatographic and mass spectrometric analysis of glutathione in biological samples

Biological thiol compounds are classified into high-molecular-mass protein thiols and low-molecular-mass free thiols. Endogenous low-molecular-mass thiol compounds, namely, reduced glutathione (GSH) and its corresponding disulfide, glutathione disulfide (GSSG), are very important molecules that participate in a variety of physiological and pathological processes. GSH plays an essential role in protecting cells from oxidative and nitrosative stress and GSSG can be converted into the reduced form by action of glutathione reductase. Measurement of GSH and GSSG is a useful indicator of oxidative stress and disease risk. Many publications have reported successful determination of GSH and GSSG in biological samples. In this article, we review newly developed techniques, such as liquid chromatography coupled with mass spectrometry and tandem mass spectrometry, for identifying GSH bound to proteins, or for localizing GSH in bound or free forms at specific sites in organs and in cellular locations.     

Effects of lanthanum on the ascorbate and glutathione metabolism of Vigna radiata seedlings under salt stress

In order to elucidate the role of lanthanum (La) in response of Vigna radiata to a salt stress, we investigated the effects of La on the ascorbate and glutathione metabolism. The results show that in comparison with a control, the salt stress increased the activities of ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), γ-glutamylcysteine synthetase (γ-ECS), and L-galactono-1,4-lactone dehydrogenase (GalLDH), and the content of ascorbic acid (AsA) and glutathione (GSH). It also increased the malondialdehyde content (MDA) and electrolyte leakage. The salt stress significantly decreased the ratios of AsA/dehydroascorbate (DHA) and GSH/glutathione disulphide (GSSG) compared with the control. The pretreatment with La not only significantly increased the activities of the above enzymes, the content of AsA, GSH, and the ratios of AsA/DHA and GSH/GSSG, but also significantly reduced the MDA content and electrolyte leakage compared with the salt stress alone. Our results suggest that La could up-regulate the ascorbate and glutathione metabolisms and could have an important role for acquisition of salt stress tolerance in Vigna radiata.     

Effect of osmotic stress on glutathione and hydroxymethylglutathione accumulation in wheat

The effect of osmotic stress on glutathione and hydroxymethylglutathione levels was compared in three wheat genotypes and two 5A chromosome substitution lines. Freezing-tolerant genotypes seemed also to be tolerant to osmotic stress induced by polyethylene glycol (PEG), since their fresh weight was not affected by the treatment. However, the growth of freezing-sensitive genotypes was reduced by 7-day PEG treatment and they had greater injuries after osmotic stress. The reduced forms of the two glutathione precursors, cysteine and gamma-glutamylcysteine, and of hydroxymethylglutathione (hmGSH) and glutathione (GSH) were present in greater quantities after PEG treatment in the two tolerant genotypes than in the sensitive ones. Similarly, osmotic stress resulted in a higher ratio of the reduced to the oxidised form of these thiols and in greater activity of gamma-glutamylcysteine synthetase and glutathione reductase in the tolerant genotypes compared to the sensitive ones. Following in vivo glutathione synthesis, a greater incorporation of radioactivity from [35S]sulphate into the four thiols was found in the tolerant genotypes than in the sensitive ones during osmotic stress. The present results indicate that hmGSH and GSH may contribute to the improvement of tolerance against osmotic stress in wheat and that the 5A chromosome influences the stress-induced changes in GSH and hmGSH levels.     

谷胱甘肽生物合成及代谢相关酶的研究进展

谷胱甘肽是广泛存在于生物体内的一个含有γ-肽键的生物活性三肽,其中游离的巯基是其活性中心。在生物体内谷胱甘肽主要是由GSH I和GSH II两个酶依次催化合成,而GSH I和GSH II的进化过程复杂,由此衍生出多条生物合成途径,其代谢过程在不同生物体内也复杂多样。本文主要综述了谷胱甘肽生物合成及代谢相关酶的研究进展和利用基因工程手段提高胞内谷胱甘肽含量的策略。     

Supplementation with N-acetylcysteine and taurine failed to restore glutathione content in liver of streptozotocin-induced diabetics rats but protected from oxidative stress

Rats were rendered diabetic with streptozotocin and supplemented or not with N-acetylcysteine (NAC) and taurine (TAU). The liver was examined for the quantity of glutathione (GSH), both total and oxidised (GSSG), by HPLC assay. Moreover, the liver expression of gamma-glutamyl-cysteine synthetase, cysteine dioxygenase and heme oxygenase 1 was evaluated. Streptozotocin-diabetic rats showed decreased levels of liver glutathione (GSH); dietary supplementation with the antioxidants NAC and TAU failed to restore liver GSH to the level of control rats. Gamma-glutamyl-cysteine synthetase expression was not reduced in the diabetic rats, so the low hepatic GSH level in the supplemented diabetic rats cannot be ascribed to decreased expression of the biosynthetic key enzyme. Moreover, the diabetic rats showed no evidence of increased expression of cysteine dioxygenase, which could have indicated that NAC-derived cysteine was consumed in metabolic pathways different from GSH synthesis. However, NAC+TAU treatment provided partial protection from glutathione oxidation in the liver of diabetic rats; moreover, the antioxidant treatment reduced the hepatic overexpression of heme oxygenase 1 (HO-1) mRNA which was detected in the diabetic rats. In conclusion, although NAC was not able to restore liver GSH levels, the antioxidant treatment restrained GSH oxidation and HO-1 overexpression, which are markers of cellular oxidative stress: diabetic rats probably exploit NAC as an antioxidant itself rather than as a GSH precursor.     

Protective role of glutathione synthesis in response to oxidized low density lipoprotein in human vascular endothelial cells

Impairment of endothelial cells by oxidized low density lipoprotein (OxLDL) is believed to be the first step in atherogenesis. It is also believed that oxidative stress/antioxidant imbalance is involved in the cell damage by OxLDL. However, little is known about the interaction between OxLDL and antioxidants. In this study, we show that treatment of human vascular endothelial cells with OxLDL caused a gradual increase of glutathione (gamma-glutamylcysteinyl glycine, GSH) levels in 24 h. OxLDL increased the intracellular levels of reactive oxygen species (ROS) and stimulated the expression of gamma-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme for the GSH synthesis, the mitogen-activated protein kinase (MAPK) activity, and the AP-1-DNA binding activity. The luciferase activity of gamma-GCS promoter containing AP-1 site was activated by OxLDL. Collectively, OxLDL induces gamma-GCS expression mediated by AP-1 resulting in an increase of GSH levels. The MAPK activity stimulated by ROS may be involved in the activation of AP-1. The increase in GSH by OxLDL may afford cellular protection against OxLDL-induced oxidative stress.     

Glutathiolation of the proteasome is enhanced by proteolytic inhibitors

The proteasome inhibitors lactacystin, clastro lactacystin beta-lactone, or tri-leucine vinyl sulfone (NLVS), in the presence of [(35)S]cysteine/methionine, caused increased incorporation of (35)S into cellular proteins, even when protein synthesis was inhibited by cycloheximide. This effect was blocked by incubation with the glutathione synthesis inhibitor buthionine sulfoximine. Proteasome inhibitors also enhanced total glutathione levels, increased reduced/oxidized glutathione ratio (GSH/GSSG) and upregulated gamma-glutamylcysteine synthetase (rate-limiting in glutathione synthesis). Micromolar concentrations of GSH, GSSG, or cysteine stimulated the chymotrypsin-like activity of purified 20S proteasome, but millimolar GSH or GSSG was inhibitory. Interestingly, GSH did not affect 20S proteasome's trypsin-like activity. Enhanced proteasome glutathiolation was verified when purified preparations of the 20S core enzyme complex were incubated with [(35)S]GSH after pre-incubation with any of the inhibitors. NLVS, lactacystin or clastro lactacystin beta-lactone may promote structural modification of the 20S core proteasome, with increased exposure of cysteine residues, which are prone to S-thiolation. Three main conclusions can be drawn from the present work. First, proteasome inhibitors alter cellular glutathione metabolism. Second, proteasome glutathiolation is enhanced by inhibitors but still occurs in their absence, at physiological GSH and GSSG levels. Third, proteasome glutathiolation seems to be a previously unknown mechanism of proteasome regulation in vivo.     

Inhibition of acrylamide genotoxicity in human liver-derived HepG2 cells by the antioxidant hydroxytyrosol

The chemoprotective effect of hydroxytyrosol (HT), a strong antioxidant compound from extra virgin olive oil, against acrylamide (AA)-induced genotoxicity was investigated in a human hepatoma cell line, HepG2. The micronucleus test (MNT) assay was used to monitor genotoxicity. In MNT, we found that HT at all tested concentrations (12.5-50 microM) significantly reduced the micronuclei frequencies in a concentration-dependent manner caused by AA. In order to clarify the underlying mechanisms we measured the intracellular reactive oxygen species (ROS) formation using 2,7-dichlorofluorescein diacetate (DCFH-DA) as a fluorescent probe. Intracellular glutathione (GSH) level was estimated by fluorometric methods. The rate-limiting enzyme in GSH synthesis is gamma-glutamylcysteine synthetase (gamma-GCS) and gamma-GCS was measured using Western blotting. The results showed that HT significantly concentration-dependent reduced the genotoxicity caused by AA. Furthermore, HT was able to reduce intracellular ROS formation and attenuate GSH depletion caused by AA in a concentration-dependent manner. It was also found that HT enhanced the expression of gamma-GCS in HepG2 cells treated with 10 mM AA using immunoblotting in a concentration-dependent manner. The results showed that HT reduced the AA-induced genotoxicity by decreasing the ROS level and increasing the GSH level. The data strongly suggest that HT have significant protective ability against AA-induced genotoxicity in vitro.