The presence of glutathione and glutathione reductase in chloroplasts: A proposed role in ascorbic acid metabolism

Both glutathione and an NADPH-dependent glutathione reductase are present in spinach (Spinacia oleracea L.) chloroplasts. It is proposed that glutathione functions to stabilise enzymes of the Calvin cycle, and it may also act to keep ascorbic acid in chloroplasts in the reduced form.Abbreviations GSH tripeptide glutathione - GSH reduced form of glutathione - GSSG oxidised form of glutathione     

Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple

Redox state is a term used widely in the research field of free radicals and oxidative stress. Unfortunately, it is used as a general term referring to relative changes that are not well defined or quantitated. In this review we provide a definition for the redox environment of biological fluids, cell organelles, cells, or tissue. We illustrate how the reduction potential of various redox couples can be estimated with the Nernst equation and show how pH and the concentrations of the species comprising different redox couples influence the reduction potential. We discuss how the redox state of the glutathione disulfide-glutathione couple (GSSG/2GSH) can serve as an important indicator of redox environment. There are many redox couples in a cell that work together to maintain the redox environment; the GSSG/2GSH couple is the most abundant redox couple in a cell. Changes of the half-cell reduction potential (E(hc)) of the GSSG/2GSH couple appear to correlate with the biological status of the cell: proliferation E(hc) approximately -240 mV; differentiation E(hc) approximately -200 mV; or apoptosis E(hc) approximately -170 mV. These estimates can be used to more fully understand the redox biochemistry that results from oxidative stress. These are the first steps toward a new quantitative biology, which hopefully will provide a rationale and understanding of the cellular mechanisms associated with cell growth and development, signaling, and reductive or oxidative stress.     

In vitro oxidation of ascorbic acid and its prevention by GSH

The interaction of glutathione (GSH) with ascorbic acid and dehydroascorbic acid was examined in in-vitro experiments in order to examine the role of GSH in protecting against the autoxidation of ascorbic acid and in regenerating ascorbic acid by reaction with dehydroascorbic acid. If a buffered solution (pH 7.4) containing 1.0 mM ascorbic acid was incubated at 37 degrees C, there was a rapid loss of ascorbic acid in the presence of oxygen. When GSH was added to this solution, ascorbic acid did not disappear. Maximum protection against ascorbic acid autoxidation was achieved with as little as 0.1 mM GSH. Cupric ions (0.01 mM) greatly accelerated the rate of autoxidation of ascorbic acid, an effect that was inhibited by 0.1 mM GSH. Other experiments showed that GSH complexes with cupric ions, resulting in in a lowering of the amount of GSH in solution as measured in GSH standard curves. These results suggest that the inhibition of ascorbic acid autoxidation by GSH involves complexation with cupric ions that catalyze the reaction. When ascorbic acid was allowed to autoxidize at 37 degrees C the subsequent addition of GSH (up to 10 mM) did not lead to the regeneration of ascorbic acid. This failure to detect a direct reaction between GSH and the dehydroascorbic acid formed by oxidation of ascorbic acid under this condition was presumably due to the rapid hydrolysis of dehydroascorbic acid. When conditions were chosen, i.e., low temperature, that promote stability of dehydroascorbic acid, the direct reaction between GSH and dehydroascorbic acid to form ascorbic acid was readily detected. The marked instability of dehydroascorbic acid at 37 degrees C raises questions regarding the efficiency of the redox couple between GSH and dehydroascorbic acid in maintaining the concentration of ascorbic acid in mammalian cells exposed to an oxidative challenge.     

Impact of sex,age and diet on the cysteine/cystine and glutathione/glutathione disulfide plasma redox couples in mice

Age, sex and diet are well-established risk factors for several diseases. In humans, each of these variables has been linked to differences in plasma redox potentials (E_h) of the glutathione/glutathione disulfide (GSH/GSSG) and cysteine/cystine (Cys/CySS) redox couples. Mice have been very useful for modeling human disease processes, but it is unknown if age, sex and diet affect redox couples in mice as they do in humans. The purpose of the present study was to examine the effects of these factors on plasma redox potentials in C57BL/6J mice. We found that age had no effect on either redox couple in either sex. Plasma E_h Cys/CySS and E_h GSH/GSSG were both more oxidized (more positive) in females than in males. A 24-hour fast negated the sex differences in both redox potentials by oxidizing both redox couples in male mice, while having no effect on E_h Cys/CySS and a smaller effect on E_h GSH/GSSG in female mice. A diet with excess sulfur amino acids reduced the plasma E_h Cys/CySS in females to a level comparable to that seen in male mice. Thus, sex-specific differences in plasma E_h Cys/CySS could be normalized by two different dietary interventions. Some of these findings are consistent with reported human studies, while others are not. Most strikingly, mice do not exhibit age-dependent oxidation of plasma redox potentials. Care must be taken when designing and interpreting mouse studies to investigate redox regulation in humans.     

A comparative study of glutathione and ascorbate metabolism during germination of Pinus pinea L. seeds

The ascorbate and glutathione systems have been studied during the first stages of germination in orthodox seeds of the gymnosperm Pinus pinea L. (pine). The results indicate that remarkable changes in the content and redox balance of these metabolites occur in both the embryo and endosperm; even if with different patterns for the two redox pairs. Dry seeds are devoid of the ascorbate reduced form (ASC) and contain only dehydroascorbic acid (DHA). By contrast, glutathione is present both in the reduced (GSH) and in the oxidized (GSSG) forms. During imbibition the increase in ASC seems to be mainly caused by the reactivation of its biosynthesis. On the other hand, the GSH rise occurring during the first 24 h seems to be largely due to GSSG reduction, even if GSH biosynthesis is still active in the seeds. The enzymes of the ascorbate--glutathione cycle also change during germination, but in different ways. ASC peroxidase (EC 1.11.1.11) and glutathione reductase (EC 1.6.4.2) activities progressively rise both in the embryo and in endosperm. These changes are probably required for counteracting production of reactive oxygen species caused by recovery of oxidative metabolism. The two enzymes involved in the ascorbate recycling, ascorbate free radical (AFR) reductase (EC 1.6.5.4) and DHA reductase (EC 1.8.5.1), show different behaviour: the DHA reductase activity decreases, while that of AFR reductase remains unchanged. The relationship between ascorbate and glutathione metabolism and their relevance in the germination of orthodox seeds are also discussed.     

Thiram and dimethyldithiocarbamic acid interconversion in Saccharomyces cerevisiae: a possible metabolic pathway under the control of the glutathione redox cycle.

A rapid decrease of intracellular glutathione (GSH) was observed when exponentially growing cells of Saccharomyces cerevisiae were treated with sublethal concentrations of either dimethyldithiocarbamic acid or thiram [bis(dimethylthiocarbamoyl) disulfide]. The underlying mechanism of this effect possibly involves the intracellular oxidation of dimethyldithiocarbamate anions to thiram, which in turn oxidizes GSH. Overall, a linear relationship was found between thiram concentrations up to 21 microM and production of oxidized GSH (GSSG). Cytochrome c can serve as the final electron acceptor for dimethyldithiocarbamate reoxidation, and it was demonstrated in vitro that NADPH handles the final electron transfer from GSSG to the fungicide by glutathione reductase. These cycling reactions induce transient alterations in the intracellular redox state of several electron carriers and interfere with the respiration of the yeast. Thiram and dimethyldithiocarbamic acid also inactivate yeast glutathione reductase when the fungicide is present within the cells as the disulfide. Hence, whenever the GSH regeneration rate falls below its oxidation rate, the GSH:GSSG molar ratio drops from 45 to 1. Inhibition of glutathione reductase may be responsible for the saturation kinetics observed in rates of thiram elimination and uptake by the yeast. The data suggest also a leading role for the GSH redox cycle in the control of thiram and dimethyldithiocarbamic acid fungitoxicity. Possible pathways for the handling of thiram and dimethyldithiocarbamic acid by yeast are considered with respect to the physiological status, the GSH content, and the activity of glutathione reductase of the cells.     

Catalytic selenols couple the redox cycles of metallothionein and glutathione.

Co-ordination of zinc to the thiol group of cysteine allows mobilization of zinc through oxidation of its ligand. This molecular property links the binding and release of zinc in metallothionein (MT) to the cellular redox state [Maret W. & Vallee B.L. (1998) Proc. Natl Acad. Sci. USA 95, 3483-3488]. Biological disulfides such as glutathione disulfide (GSSG) oxidize MT with concomitant release of zinc, while glutathione (GSH) reduces the oxidized protein to thionein, which then binds to available zinc. Neither of these two redox processes is very efficient, even at high concentrations of GSSG or GSH. However, the GSH/GSSG redox pair can efficiently couple with the MT/thionein system in the presence of a selenium compound that has the capacity to form a catalytic selenol(ate). This coupling provides a very effective means of modulating oxidation and reduction. Remarkably, selenium compounds catalyze the oxidation of MT even under overall reducing conditions such as those prevailing in the cytosol. In this manner, the binding and release of zinc from zinc-thiolate co-ordination sites is linked to redox catalysis by selenium compounds, changes in the glutathione redox state, and the availability of either a zinc donor or a zinc acceptor. The results also suggest that the pharmacological actions of selenium compounds in cancer prevention and other antiviral and anti-inflammatory therapeutic applications, as well as unknown functions of selenium-containing proteins, may relate to coupling between the thiol redox state and the zinc state.     

Regulation of apoptosis by glutathione redox state in PC12 cells exposed simultaneously to iron and ascorbic acid

We previously reported that the levels of non-protein-bound iron (NPBI) and ascorbic acid (AA) are markedly increased in the cerebrospinal fluid of infants with perinatal asphyxia. The present study showed that FeSO4 and AA synergistically induced apoptosis of PC12 cells, which was prevented by alpha-tocopherol and glutathione (GSH) ethyl ester. Markers of free radical damage, such as ortho-tyrosine, meta-tyrosine, and F(2alpha)-isoprostane, showed a gradual increase. AA and ferrous NPBI disappeared rapidly from the culture medium, but exposure for only a few hours was sufficient to trigger apoptosis. Intracellular GSH decreased progressively along with a concomitant increase of glutathione disulfide (GSSG). The baseline half-cell reduction potential (Ehc) for GSSG, 2H+/2GSH couple was -246 mV and an Ehc of -200 mV was the critical level to switch on apoptosis, although some cells escaped this fate by transient increase of intracellular GSH. Once Ehc reached around -165 mV (81 mV oxidation from the baseline), all cells lost the ability to maintain an adequate intracellular GSH level and subsequently underwent apoptosis. These findings at least partly explain the mechanism of Fe-AA cytotoxicity, in that ferrous iron catalyzes hydroxyl radical generation and induces lipid peroxidation, after which subsequent depletion of GSH raises Ehc to the critical level for triggering or potentiating the apoptotic cascade.     

Changes in ascorbate, glutathione, and related enzyme activities during thidiazuron-induced bud break of apple

The changes of ascorbic acid, dehydroascorbic acid, and glutathione content and related enzyme activities were studied in apple buds during dormancy and thidiazuron-induced bud break. An increase in ascorbic acid, reduced form of glutathione (GSH), total glutathione, total non-protein thiol (NPSH) and non-glutathione thiol (RSH) occurred as a result of induction by thidiazuron during bud break, whereas dehydroascorbic acid and oxidized glutathione (GSSG) decreased during the same period. Thidiazuron also enhanced the ratio of GSH/GSSG, and activities of ascorbate free radical reductase (AFR; EC 1.6.5.4), ascorbate peroxidase (EC 1.11.1.11). dehydroascorbate reductase (DHAR; EC 1.8.5.1) and glutathione reductase (GR; EC 1.6.4.2). The ascorbic acid content and the activities of AFR, ascorbate peroxidase, and DHAR peaked when buds were in the side green or green tip stage just prior to the start of rapid expansion, and declined thereafter. The GSH, NPSH, RSH, ratio of GSH/GSSG, and activities of GR increased steadily during bud development.     

Preferential transport of glutathione versus glutathione disulfide in rat liver microsomal vesicles

A bi-directional, saturable transport of glutathione (GSH) was found in rat liver microsomal vesicles. GSH transport could be inhibited by the anion transport blockers flufenamic acid and 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid. A part of GSH taken up by the vesicles was metabolized to glutathione disulfide (GSSG) in the lumen. Microsomal membrane was virtually nonpermeable toward GSSG; accordingly, GSSG generated in the microsomal lumen could hardly exit. Therefore, GSH transport, contrary to previous assumptions, is preferred in the endoplasmic reticulum, and GSSG entrapped and accumulated in the lumen creates the oxidized state of its redox buffer.     

Determination of glutathione in biological material by flow-injection analysis using an enzymatic recycling reaction

A sensitive and specific assay for glutathione using a recycling reaction followed by spectrophotometric detection in a flow-injection analysis system is presented. The proposed method provides specific amplification of the response to glutathione by combined use of the enzyme GSSG reductase and the chromogenic reagent 5,5'-dithiobis(2-nitrobenzoic acid). Both oxidized (GSSG) and reduced (GSH) glutathione are detected, so that GSSG must be determined separately after alkylation of the GSH with N-ethylmaleimide. The sensitivity is controlled by the number of times the cycle occurs and therefore by the residence time of the sample in the reactor. This time depends on the reactor length and the flow rate. The influence of residence time, temperature, and enzyme concentration on the response has been studied and the optimum reaction conditions have been selected. The sample throughput is as high as 30 h(-1) and the detection limit is 1 pmol GSH at a signal-to-noise ratio of 3. The method has been evaluated by the quantification of GSH and GSSG in isolated hepatocytes. A high correlation between the new flow-injection analysis method and the original spectrophotometric batch assay has been found (slope = 1.039, intercept = 0.6, n = 216, r = 0.977). The main advantages of the proposed method are high sample throughout, high sensitivity, and good reproducibility.     

Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method

The spectrophotometric/microplate reader assay method for glutathione (GSH) involves oxidation of GSH by the sulfhydryl reagent 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) to form the yellow derivative 5'-thio-2-nitrobenzoic acid (TNB), measurable at 412 nm. The glutathione disulfide (GSSG) formed can be recycled to GSH by glutathione reductase in the presence of NADPH. The assay is composed of two parts: the preparation of cell cytosolic/tissue extracts and the detection of total glutathione (GSH and GSSG). The method is simple, convenient, sensitive and accurate. The lowest detection for GSH and GSSG is 0.103 nM in a 96-well plate. This method is rapid and the whole procedure takes no longer than 15 min including reagent preparation. The method can assay GSH in whole blood, plasma, serum, lung lavage fluid, cerebrospinal fluid, urine, tissues and cell extracts and can be extended for drug discovery/pharmacology and toxicology protocols to study the effects of drugs and toxic compounds on glutathione metabolism.     

Genetic analysis of tissue glutathione concentrations and redox balance

Glutathione redox balance—defined as the ratio GSH/GSSG—is a critical regulator of cellular redox state, and declines in this ratio are closely associated with oxidative stress and disease. However, little is known about the impact of genetic variation on this trait. Previous mouse studies suggest that tissue GSH/GSSG is regulated by genetic background and is therefore heritable. In this study, we measured glutathione concentrations and GSH/GSSG in liver and kidney of 30 genetically diverse inbred mouse strains. Genetic background caused an approximately threefold difference in hepatic and renal GSH/GSSG between the most disparate strains. Haplotype association mapping determined the loci associated with hepatic and renal glutathione phenotypes. We narrowed the number of significant loci by focusing on those located within protein-coding genes, which we now consider to be candidate genes for glutathione homeostasis. No candidate genes were associated with both hepatic and renal GSH/GSSG, suggesting that genetic regulation of GSH/GSSG occurs predominantly in a tissue-specific manner. This is the first quantitative trait locus study to examine the genetic regulation of glutathione concentrations and redox balance in mammals. We identified novel candidate genes that have the potential to redefine our knowledge of redox biochemistry and its regulation and inform future therapeutic applications.     

Sequential opening of mitochondrial ion channels as a function of glutathione redox thiol status

Mitochondrial membrane potential (DeltaPsi(m)) depolarization contributes to cell death and electrical and contractile dysfunction in the post-ischemic heart. An imbalance between mitochondrial reactive oxygen species production and scavenging was previously implicated in the activation of an inner membrane anion channel (IMAC), distinct from the permeability transition pore (PTP), as the first response to metabolic stress in cardiomyocytes. The glutathione redox couple, GSH/GSSG, oscillated in parallel with DeltaPsi(m) and the NADH/NAD(+) redox state. Here we show that depletion of reduced glutathione is an alternative trigger of synchronized mitochondrial oscillation in cardiomyocytes and that intermediate GSH/GSSG ratios cause reversible DeltaPsi(m) depolarization, although irreversible PTP activation is induced by extensive thiol oxidation. Mitochondrial dysfunction in response to diamide occurred in stages, progressing from oscillations in DeltaPsi(m) to sustained depolarization, in association with depletion of GSH. Mitochondrial oscillations were abrogated by 4'-chlorodiazepam, an IMAC inhibitor, whereas cyclosporin A was ineffective. In saponin-permeabilized cardiomyocytes, the thiol redox status was systematically clamped at GSH/GSSG ratios ranging from 300:1 to 20:1. At ratios of 150:1-100:1, DeltaPsi(m) depolarized reversibly, and a matrix-localized fluorescent marker was retained; however, decreasing the GSH/GSSG to 50:1 irreversibly depolarized DeltaPsi(m) and induced maximal rates of reactive oxygen species production, NAD(P)H oxidation, and loss of matrix constituents. Mitochondrial GSH sensitivity was altered by inhibiting either GSH uptake, the NADPH-dependent glutathione reductase, or the NADH/NADPH transhydrogenase, indicating that matrix GSH regeneration or replenishment was crucial. The results indicate that GSH/GSSG redox status governs the sequential opening of mitochondrial ion channels (IMAC before PTP) triggered by thiol oxidation in cardiomyocytes.     

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.     

Thiol/disulfide redox equilibrium and kinetic behavior of chicken liver fatty acid synthase

Chicken liver fatty acid synthase is rapidly inactivated and cross-linked at pH 7.2 and 8.0 by incubation with low concentrations of common biological disulfides including glutathione disulfide, coenzyme A disulfide, and glutathione-coenzyme A-mixed disulfide. Glutathione disulfide inactivation of the enzyme is accompanied by the oxidation of a total of 4-5 enzyme thiols per monomer. Only one glutathione equivalent is incorporated per monomer as a protein-mixed disulfide, and its rate of incorporation is significantly slower than the rate of inactivation. The formation of protein-SS-protein disulfides results in significant cross-linking of enzyme subunits. The inactive enzyme is rapidly and completely reactivated, and the cross-linking is completely reversed by incubation of the enzyme with thiols (10-20 mM) including dithiothreitol, mercaptoethanol, and glutathione. In a glutathione redox buffer (GSH + GSSG), disulfide bond formation comes to equilibrium. The enzyme activity at equilibrium is dependent both on the ratio of glutathione to glutathione disulfide and on the total glutathione concentration. The equilibrium constant for the redox equilibration of fatty acid synthase in a glutathione redox buffer is 15 mM (Ered + GSSG in equilibrium Eox + 2GSH). The formation of at least one protein-protein disulfide per monomer dominates the redox properties of the enzyme while the formation of one protein-mixed disulfide with glutathione (Kmixed = 0.45) has little effect on activity. The oxidation equilibrium constant suggests that there would be no significant cycling between the reduced and the oxidized enzyme in response to likely physiological variations in the hepatic glutathione status. The possibility that changes in the concentration of cellular glutathione may act as a mechanism for metabolic control of other enzymes is discussed.     

Direct and simultaneous determination of reduced and oxidized glutathione and homoglutathione by liquid chromatography-electrospray/mass spectrometry in plant tissue extracts

A simple, highly selective, sensitive, and reproducible liquid chromatography-electrospray ionization/mass spectrometry (time of flight) method has been developed for the direct and simultaneous determination of glutathione and related compounds such as homoglutathione in different plant tissues. These compounds are low-molecular mass antioxidants involved in cellular redox homeostasis in plants, and efforts are being made to develop methods to determine the concentrations of oxidized and reduced forms of these compounds and their ratio. Many of the methodologies developed so far, however, are time-consuming and complex; therefore, analytes can decompose and their redox status can change during the analysis process. The method we have developed allows the simultaneous determination of reduced forms (glutathione [GSH] and homoglutathione [hGSH]) and oxidized forms (glutathione disulfide [GSSG]) of these compounds and is also suitable for the determination of ascorbic acid (ASA) and S-nitrosoglutathione (GSNO). Quantification was done using isotopically labeled GSH and ASA as internal standards. All compounds were base peak resolved in less than 6 min, and limits of detection were 60 pmol for GSH, 30 pmol for hGSH, 20 pmol for GSSG, 100 pmol for ASA, and 30 pmol for GSNO. The intraday repeatability values were approximately 0.4 and 7% for retention time and peak area, respectively, whereas the interday repeatability values were approximately 0.6 and 9% for retention time and peak area, respectively. Analyte recoveries found were between 92 and 105%. The method was used to determine the concentrations of GSH, GSSG, hGSH, and ASA in extracts from several plant tissues.     

Ageing of tomato seeds involves glutathione oxidation

The effect of seed ageing on the oxidation of reduced glutathione (GSH) and the role of GSH oxidation in ageing-induced deterioration were studied in seeds of tomato ( Lycopersicon esculentum Mill. cv. Lerica, Moneymaker and Cromco). Both long-term storage at 15°C/30% relative humidity (RH) and artificial ageing at 20°C/75% RH, 30°C/45% RH and 60°C/45% RH resulted in a marked loss of GSH and a simultaneous, though not proportional, increase in its oxidized form GSSG. The glutathione thiol-disulfide status shifted towards a highly oxidized form, while the total glutathione pool decreased. The extent of GSH oxidation differed between ageing conditions and was not directly related to the extent of seed deterioration. Thiobarbituric acid-reactive substances did not increase in ageing tomato seeds, suggesting that lipid peroxidation did not take place. Hydration of seeds, either upon imbibition in water or by priming in an osmotic solution, resulted in a rapid decrease in GSSG, a shift of the glutathione redox couple to a mainly reduced status and an increase in the glutathione pool, in both control and aged seeds. The results indicate that, in tomato seeds, (1) seed ageing involves GSH oxidation into GSSG, which is indicative of oxidative stress, (2) ageing does not affect the GSSG reduction capacity upon subsequent imbibition, and (3) the lowered viability of aged seeds cannot directly be ascribed to the decreased GSH pool or To the highly oxidized glutathione redox status.