Glutathione metabolism in primate lenses: A phylogenetic study of glutathione synthesis and glutathione redox cycle enzyme activities

Lens wet weights, soluble protein, and activities of γ-glutiamylcysteine synthetase, glutathione synthetase, glutathione peroxidase, and glutathione reductase were determined in primate lenses. The primary sources of lenses were middle-aged adult animals. The Primates, from 23 genera, were categorized into six superfamilies: hominoids (five species), Old World monkeys (seven species), New World monkeys (five species), tarsiers (two species), lemurs (six species), and lorisids (three species). Significant differences between various groups or combinations of groups were noted for γ-glutamylcysteine synthetase, glutathione peroxidase, and glutathione reductase activities. Lenticular γ-glutamylcysteine synthetase activity was very low in the Old World simian lenses and highest in the prosimians. Glutathione peroxidase activity was extraordinarily high in lenses of Old World monkeys. Glutathione reductase activity was low in all the prosimians but tenfold higher in hominoid lenses with intermediate values in monkeys of both the Old World and New World. Glutathione synthetase activity was variable, and no clear pattern which might be useful for primate classification was noted. Lenticular activity ratios of glutathione synthetase:γ-glutamylcysteine synthetase were highest in the Old World simians and lowest in the prosimians. These data with emphasis upon Aotus and the tarsiers were examined with regard to phylogenetic relationships. © 1994 Wiley-Liss, Inc.     

Treatment of glutathione synthetase deficient fibroblasts by inhibiting gamma-glutamyl transpeptidase activity with serine and borate.

Glutathione synthetase deficiency results in decreased cellular glutathione content and consequent overproduction of 5-oxoproline. L-serine in borate buffer inhibits γ-glutamyl transpeptidase, the major catabolic enzyme for glutathione. Treatment of glutathione synthetase deficient fibroblasts with 40mM serine and borate for 24 hours produced more than a 2-fold increase in cellular glutathione content. L-serine alone led to a smaller increase in glutathione level, and borate alone was without effect. On exposure to serine and borate, 5-oxoproline formation from L-glutamate was decreased to normal levels in glutathione synthetase deficient fibroblasts, presumably secondary to feedback inhibition of γ-glutamylcysteine synthetase by the increased intracellular glutathione concentration. Cellular free amino acid content was generally unaffected by such exposure although increases were observed in serine and phosphoserine. This model system suggests that γ-glutamyl transpeptidase inhibition may be a rational approach to alleviating the effects of glutathione synthetase deficiency.     

The Effect of Phosphinothricin (Glufosinate) on Glutathione Synthesis in Plants

The inhibitory effect of DL-phosphinothricin (glufosinate) on glutathione synthesis was studied in vivo and in vitro. The influence of phosphinothricin on γ-glutamylcysteine synthetase was compared with the already known effects of l -buthionine sulfoximine and l -methionine sulfoximine. The results showed that phosphinothricin and buthionine sulfoximine are inhibitors of γ-glutamylcysteine synthetase of plants. With both substances the enzyme was inhibited by 50 % at a concentration of 7 . 10^?4M (pI₅₀ = 3.15). Methionine sulfoximine reduced the enzyme activity by 50% at 5 . 10^?2 M (pI₅₀ = 1.30). It is discussed that the target enzyme of phosphinothricin is the glutamine synthetase whereas the γ-glutamylcysteine synthetase is only an accessory target.     

Subcellular distribution of glutathione precursors in Arabidopsis thaliana

Glutathione is an important antioxidant and has many important functions in plant development, growth and defense. Glutathione synthesis and degradation is highly compartment-specific and relies on the subcellular availability of its precursors, cysteine, glutamate, glycine and γ-glutamylcysteine especially in plastids and the cytosol which are considered as the main centers for glutathione synthesis. The availability of glutathione precursors within these cell compartments is therefore of great importance for successful plant development and defense. The aim of this study was to investigate the compartment-specific importance of glutathione precursors in Arabidopsis thaliana. The subcellular distribution was compared between wild type plants (Col-0), plants with impaired glutathione synthesis (glutathione deficient pad2-1 mutant, wild type plants treated with buthionine sulfoximine), and one complemented line (OE3) with restored glutathione synthesis. Immunocytohistochemistry revealed that the inhibition of glutathione synthesis induced the accumulation of the glutathione precursors cysteine, glutamate and glycine in most cell compartments including plastids and the cytosol. A strong decrease could be observed in γ-glutamylcysteine (γ-EC) contents in these cell compartments. These experiments demonstrated that the inhibition of γ-glutamylcysteine synthetase (GSH1) - the first enzyme of glutathione synthesis - causes a reduction of γ-EC levels and an accumulation of all other glutathione precursors within the cells.     

A fluorescence-based microtiter plate assay for γ-glutamylcyclotransferase

γ-Glutamylcyclotransferase (GGCT) is a component of the γ-glutamyl cycle and specifically cleaves γ-glutamyl-amino acid dipeptides to release the free amino acid and 5-oxoproline. The action of GGCT in glutathione synthetase-deficient patients results in the accumulation of high levels of 5-oxoproline. In addition, GGCT may be a biomarker in some cancers. GGCT inhibitors, therefore, may be of value in the treatment of glutathione synthetase deficiency or in the elucidation of the role of GGCT in cancer cells. Published GGCT assays are not suitable for high-throughput screening for inhibitory molecules. We have developed a fluorescence-based 96-well plate assay for the determination of the rate of γ-glutamylcysteine cleavage by GGCT. After the reaction, the residual γ-glutamylcysteine is determined fluorometrically after derivatization with 2,3-naphthalenedicarboxaldehyde. The method has sufficient sensitivity to detect low-affinity competitive inhibitors and, as a result of its simplicity and microtiter plate format, can be readily used in high-throughput inhibitor screens.     

Purification and evaluation of the glutathione-synthesizing enzymes from Candida boidinii for cell-free synthesis of glutathione

To examine the application of the glutathione-synthesizing enzymes for cell-free synthesis of this tripeptide we carried out a limited screening of yeast strains to find organisms with high glutathione-synthesizing activity. We used an improved, rapid and sensitive HPLC method for the determination of nmol levels of γ-glutamylcysteine (the first product in synthesis) and glutathione (the endproduct of the enzymatic reaction). High enzyme activities were found in Candida boidinii grown in mineral salt-medium supplemented with trace element and vitamin solutions and methanol as carbon source and Hansenula polymorpha grown under the same conditions except that glucose was used as sole carbon source. Candida boidinii was chosen for further investigations. Determination of enzyme formation during growth showed that the specific activities of the two glutathione-synthesizing enzymes remained almost constant during the whole growth phase while the intracellular glutathione content increased markedly. The enzymes were purified by DEAE-cellulose column chromatography, ultrafiltration and gel chromatography to apparent homogeneity. Properties of the enzymes including stability, molecular weight and subunit composition, substrate and inhibitor kinetics and the ability of ATP, bound to polyethylene glycol, to serve as coenzyme in the two enzymatic reactions were investigated in detail. Due to the limited stability of the purified γ-glutamylcysteine synthetase and the inability of the glutathione synthetase to utilize ATP derivatives as coenzyme, presently, immobilized cells appear to be more favourable for glutathione synthesis.     

IN VIVO INHIBITION OF γ-GLUTAMYLCYSTEINE SYNTHETASE BY l-METHIONINE-RS-SULFOXIMINE; INFLUENCE ON INTERMEDIATES OF THE γ-GLUTAMYL CYCLE

—The inhibition of γ-glutamylcysteine synthetase and its influence on the concentration of intermediates associated with the metabolism of glutathione was studied in mice receiving methionine sulfoximine, a convulsant agent. The activity of the enzyme decreased significantly in the liver and kidney 1-4 h after administration of methionine sulfoximine; the activity of the enzyme in the brain was unchanged after 1 and 2 h but decreased significantly after 4 h. There was a rapid and sharp decrease in the concentration of glutathione in the kidney and a slower decrease in the liver. Brain glutathione concentrations were unaffected. Methionine sulfoximine in vivo, inhibited the synthesis of l -γ-glutamyl-l -α-aminobutyrate after administration of l -α-aminobutyrate, a reaction catalyzed by γ-glutamylcysteine synthetase. The inhibitor also lowered the concentration of pyrrolidone carboxylate in mouse tissues and prevented the accumulation of this intermediate after administration of l -α-aminobutyrate. The results show that methionine sulfoximine in vivo affects the metabolism of glutathione and that this action may contribute to its convulsive properties.     

Comparative effects of antioxidants on enzymes involved in glutathione metabolism

The present study was designed to examine changes in glutathione metabolism in the liver of mice as influenced by supplementation of their diet with 1 of 4 antioxidants: butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), vitamin E and selenium. In addition to determination of the acid-soluble thiol levels, 5 different enzymes involved with glutathione utilization and synthesis were measured: glutathione transferase, γ-glutamyl transpeptidase, selenium-dependent glutathione peroxidase, γ-glutamylcysteine synthetase and glutathione reductase. All 4 antioxidants produced significant increases in glutathione transferase activity, with BHA and BHT being much more effective than the other two. With the exception of vitamin E, BHA, BHT and selenium all resulted in a slight enhancement in the activity of glutathione reductase as well as in the acid-soluble thiol level. On the other hand, the induction of γ-glutamyl transpeptidase and γ-glutamylcysteine synthetase was responsive to only vitamin E and selenium supplementation, respectively. Although the influence of each of these antioxidants in glutathione metabolism appears to be specific and somewhat compartmentalized, the overall impression is that of an increased capacity for glutathione-conjugate formation and recovery of reduced glutathione. These biochemical changes in glutathione metabolism may be relevant to the anticarcinogenic effects observed with BHA, BHT and selenium.     

Regulation of glutathione synthesis in leaves of transgenic poplar (Populus tremula X P. alba) overexpressing glutathione synthetase

The poplar hybrid Populus tremula X P. alba was transformed with the Escherichia coli gene for glutathione synthetase ( gsh II ) targetted to the cytosol. Leaves of five lines of transgenic plants exhibited glutathione synthetase activities 15- to 60-fold higher than those of wild-type plants. Total glutathione levels and GSH/GSSG ratios were similar in transgenic and wild-type plants. Precursor feeding experiments with cysteine and γ-glutamylcysteine suggest that glutathione synthesis in the cytoplasm is controlled by a multistep procedure that includes (i) the availability of cysteine, (ii) the availability of γ-glutamylcysteine, and (iii) regulation of the activities of both γ-glutamylcysteine synthetase and glutathione synthetase. However step (ii) may set an upper limit for the cellular glutathione content.     

γ-Glutamylcysteinylserine — A New Homologue of Glutathione in Plants of the Family Poaceae*

In addition to glutathione (γ-GluCysGly), many species of the family Poaceae have another tripeptide which has the amino acid sequence γ-GluCysSer. This thiol was isolated from etiolated leaves of wheat (Triticum aestivum L. cv. Star). Its structure was elucidated by quantitative amino acid analysis after total hydrolysis and by partial hydrolysis with carboxypeptidase A and γ-glutamyltranspeptidase. The content of γ-GluCysSer in the leaves of T. aestivum is increased by incubation with sulfate and is severely diminished by incubation with buthionine sulfoximine, a specific inhibitor of γ-glutamylcysteine synthetase. Oxidized γ-GluCysSer is reduced by yeast glutathione reductase with a rate somewhat lower than for glutathione, but the new tripeptide is not a substrate of glutathione-S-transferase from equine liver. Besides homoglutathione (γ-GluCysßAla), a tripeptide found in plants of the order Fabales, the tripeptide γ-GluCysSer is the second homologue of glutathione detected in plants.     

Heavy Metal Tolerance and Accumulation in Indian Mustard (Brassica Juncea L.) Expressing Bacterial γ-Glutamylcysteine Synthetase or Glutathione Synthetase

The overexpression of either γ-glutamylcysteine synthetase (γ-ECS) or glutathione synthetase (GS) in Brassica juncea transgenics was shown previously to result in higher accumulation of glutathione (GSH) and phytochelatins (PCs), as well as enhanced Cd tolerance and accumulation. The present study was aimed at analyzing the effects of γ-ECS or GS overexpression on tolerance to and accumulation of other metal/loids supplied individually in agar medium (seedlings) or in hydroponics (mature plants). Also, as pollution in nature generally consists of mixtures of metals, glutamylcysteine synthetase (ECS) and GS seedlings were tested on combinations of metals. Compared to wild-type plants, ECS and GS transgenics exhibited a significantly higher capacity to tolerate and accumulate a variety of metal/loids (particularly As, Cd, and Cr) as well as mixed-metal combinations (As, Cd, Zn/As, Pb, and Zn). This enhanced metal tolerance and accumulation of the ECS and GS transgenics may be attributable to enhanced production of PCs, sustained by a greater availability of GSH as substrate, as suggested by their higher concentrations of GSH, PC2, PC3, and PC4 as compared to wild-type plants. Overexpression of GS and γ-ECS may represent a promising strategy for the development of plants with an enhanced phytoremediation capacity for mixtures of metals.     

Construction of self-cloning, indigenous wine strains of Saccharomyces cerevisiae with enhanced glycerol and glutathione production

To improve wine taste and flavor stability, a novel indigenous strain of Saccharomyces cerevisiae with enhanced glycerol and glutathione (GSH) production for winemaking was constructed. ALD6 encoding an aldehyde dehydrogenases of the indigenous yeast was replaced by a GPD1 and CUP1 gene cassette, which are responsible for NAD-dependent glycerol-3-phosphatase dehydrogenase and copper resistance, respectively. Furthermore, the α-acetohydroxyacid synthase gene ILV2 of the indigenous yeast was disrupted by integration of the GSH1 gene which encodes γ-glutamylcysteine synthetase and the CUP1 gene cassette. The fermentation capacity of the recombinant was similar to that of the wild-type strain, with an increase of 21 and 19?% in glycerol and GSH production. No heterologous DNA was harbored in the recombinant in this study.     

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.     

Isolation and initial characterization of glutathione-deficient mutants of Escherichia coli K 12

The thiol-oxidizing agent “diamide” (CH₃)₂NCON=NCON(CH₃)₂ was used to isolate mutants of Escherichia coli K 12 deficient in the biosynthesis of glutathione. A colony-colour technique has been developed for identification of colonies of these mutants. Four glutathione-deficient mutants were isolated. They show normal growth rates in minimal medium without GSH supplementation, indicating that glutathione is not involved in essential metabolic processes. In one mutant, glutathione synthetase was entirely inactive. Three mutants were deficient in γ-glutamylcysteine synthetase; in two of them, this resulted in a complete lack of GSH. These mutants were found to be more susceptible than their parent strains to a wide range of chemical agents, but did not show a greater sensitivity to X-rays. It must be concluded that the protective role of glutathione is only significant when a chemical challenge is present.     

Localization of [gamma]-Glutamylcysteine Synthetase and Glutathione Synthetase Activity in Maize Seedlings

Fresh weight, protein, cysteine, [gamma]-glutamylcysteine, glutathione, and the extractable activity of the enzymes of glutathione biosynthesis, [gamma]-glutamylcysteine synthetase (EC 6.3.2.2) and glutathione synthetase (EC 6.3.2.3), were measured in roots, scutella, endosperms, and shoots of 3-, 7-, and 11-d-old maize (Zea mays L. cv LG 9) seedlings. In 3-d-old seedlings, the scutella represented 14% of the seedling fresh weight, containing 43% of total protein and 63 and 55% of the activity of [gamma]-glutamylcysteine synthetase and glutathione synthetase, respectively; in 11-d-old seedlings, the corresponding values were 4.5% for fresh weight, 8.0% for protein content, and 14 and 20% for the enzyme activities. The highest concentrations of thiols were found for cysteine (0.27 mM) in the roots, for glutathione (4.4 mM) in the shoots, and for [gamma]-glutamylcysteine (13 [mu]M) in the scutella of 3-d-old seedlings. The enzyme activities of roots were localized in subcellular fractions after sucrose density gradient centrifugation. Nearly half of the [gamma]-glutamylcysteine synthetase activity was detected in the root proplastids of 4-d-old seedlings, whereas <10% of the glutathione synthetase activity was localized in this organelle. Our results demonstrate the importance of scutella in glutathione synthesis in the early stage of seedling development. Unlike chloroplasts, root plastids show only a small proportion of glutathione synthetase activity.     

Using Lactococcus lactis for glutathione overproduction

Glutathione and -glutamylcysteine were produced in Lactococcus lactis using a controlled expression system and the genes gshA and gshB from Escherichia coli encoding the enzymes -glutamylcysteine synthetase and glutathione synthetase. High levels of -glutamylcysteine were found in strains growing on chemically defined medium and expressing either gshA alone or both gshA and gshB. As anticipated, glutathione was found in a strain expressing gshA and gshB. The level of glutathione production could be increased by addition of the precursor amino acid cysteine to the medium. The addition of cysteine led to an increased activity of glutathione synthetase, which is remarkable because the amino acid is not a substrate of this enzyme. The final intracellular glutathione concentration attained was 358 nmol mg^–1 protein, which is the highest concentration reported for a bacterium, demonstrating the suitability of engineered L. lactis for fine-chemical production and as a model for studies of the impact of glutathione on flavour formation and other properties of food.     

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

After rats were injected with the convulsant methionine sulfoximine, there was a rapid decrease in the glutathione concentrations of the kidney and liver, but there was no measurable effect (within 5 hours) on brain glutathione. The maximum decreases in the glutathione concentrations of kidney and liver were observed 1 hr after injection and were about 60 and 40%, respectively, of the control levels. The findings suggest that there may be at least two pools of tissue glutathione. Studies in which other amino acids were injected, and earlier $\text{in vitro}$ studies, are consistent with the conclusion that methionine sulfoximine affects glutathione synthesis $\text{in vivo}$ by inhibiting γ-glutamylcysteine synthetase. Injection of glycylglycine also decreased glutathione levels, an effect probably mediated by γ-glutamyltranspeptidase.     

Beta-aminoisobutyrate in the coupled enzymic assay of bovine lens gamma-glutamylcysteine synthetase

The formation of ADP, a product of bovine lens γ-glutamylcysteine synthetase activity, was determined by measurement of NADH oxidation in the pyruvate kinase-lactate dehydrogenase coupled assay system. Using α-aminobutyrate in place of cysteine, the time course of the spectrophotometric procedure was shown to be identical with the formation of [¹⁴C]-labeled dipeptide from [U¹⁴C]-l-glutamate. The assay for γ-glutamylcysteine synthetase was used to demonstrate that β-(+)-aminoisobutyrate was utilized at a rate two to three times that of the (?) isomer. The ability of the enzyme to distinguish between isomers suggests the binding site for the α-methyl group is a relatively broad area within the enzyme's active site.     

Relationship between cell age,glutathione and cation concentrations in sheep erythrocytes with a normal and defective transport system for amino acids

Percoll density gradients were used to separate sheep erythrocytes according to cell age. Erythrocytes with low intracellular levels of glutathione (GSH) caused by an inherited deficiency of the System C amino acid transporter exhibited large age-realted decreases in GSH and K⁺ content. In contrast, there was no age-related loss of intracellular GSH in normal sheep erythrocytes or in sheep erythrocytes with low GSH resulting from a diminished activity of γ-glutamylcysteine synthetase. Loss of GSH from amino acid transport-deficient erythrocytes was parallel by the progressive appearance of Heinz bodies in the cells, indicating an increased susceptibility to oxidative damage.