Protein-glutathione Mixed Disulfides Research Articles

Evolution based on domain combinations: the case of glutaredoxins

BackgroundProtein domains represent the basic units in the evolution of proteins. Domain duplication and shuffling by recombination and fusion, followed by divergence are the most common mechanisms in this process. Such domain fusion and recombination events are predicted to occur only once for a given multidomain architecture. However, other scenarios may be relevant in the evolution of specific proteins, such as convergent evolution of multidomain architectures. With this in mind, we study glutaredoxin (GRX) domains, because these domains of approximately one hundred amino acids are widespread in archaea, bacteria and eukaryotes and participate in fusion proteins. GRXs are responsible for the reduction of protein disulfides or glutathione-protein mixed disulfides and are involved in cellular redox regulation, although their specific roles and targets are often unclear.ResultsIn this work we analyze the distribution and evolution of GRX proteins in archaea, bacteria and eukaryotes. We study over one thousand GRX proteins, each containing at least one GRX domain, from hundreds of different organisms and trace the origin and evolution of the GRX domain within the tree of life.ConclusionOur results suggest that single domain GRX proteins of the CGFS and CPYC classes have, each, evolved through duplication and divergence from one initial gene that was present in the last common ancestor of all organisms. Remarkably, we identify a case of convergent evolution in domain architecture that involves the GRX domain. Two independent recombination events of a TRX domain to a GRX domain are likely to have occurred, which is an exception to the dominant mechanism of domain architecture evolution.

The thioredoxin reductase‐glutaredoxins‐ferredoxin crossroad pathway for selenate tolerance in Synechocystis PCC6803

Most organisms use two systems to maintain the redox homeostasis of cellular thiols. In the thioredoxin (Trx) system, NADPH sequentially reduces thioredoxin reductases (NTR), Trxs and protein disulfides. In the glutaredoxin (Grx) system, NADPH reduces the glutathione reductase enzyme occurring in most organisms, glutathione, Grxs, and protein disulfides or glutathione-protein mixed disulfides. As little is known concerning these enzymes in cyanobacteria, we have undertaken their analysis in the model strain Synechocystis PCC6803. We found that Grx1 and Grx2 are active, and that Grx2 but not Grx1 is crucial to tolerance to hydrogen peroxide and selenate. We also found that Synechocystis has no genuine glutathione reductase and uses NTR as a Grx electron donor, in a novel integrative pathway NADPH-NTR-Grx1-Grx2-Fed7 (ferredoxin 7), which operates in protection against selenate, the predominant form of selenium in the environment. This is the first report on the occurrence of a physical interaction between a Grx and a Fed, and of an electron transfer between two Grxs. These findings are discussed in terms of the (i) selectivity of Grxs and Feds (Synechocystis possesses nine Feds), (ii) crucial importance of NTR for cell fitness and (iii) resistance to selenate, in absence of a Thauera selenatis-like selenate reductase.

Attenuation of doxorubicin-induced cardiac injury by mitochondrial glutaredoxin 2

While the cardiotoxicity of doxorubicin (DOX) is known to be partly mediated through the generation of reactive oxygen species (ROS), the biochemical mechanisms by which ROS damage cardiomyocytes remain to be determined. This study investigates whether S-glutathionylation of mitochondrial proteins plays a role in DOX-induced myocardial injury using a line of transgenic mice expressing the human mitochondrial glutaredoxin 2 (Glrx2), a thiotransferase catalyzing the reduction as well as formation of protein–glutathione mixed disulfides, in cardiomyocytes. The total glutaredoxin (Glrx) activity was increased by 76% and 53 fold in homogenates of whole heart and isolated heart mitochondria of Glrx2 transgenic mice, respectively, compared to those of nontransgenic mice. The expression of other antioxidant enzymes, with the exception of glutaredoxin 1, was unaltered. Overexpression of Glrx2 completely prevents DOX-induced decreases in NAD- and FAD-linked state 3 respiration and respiratory control ratio (RCR) in heart mitochondria at days 1 and 5 of treatment. The extent of DOX-induced decline in left ventricular function and release of creatine kinase into circulation at day 5 of treatment was also greatly attenuated in Glrx2 transgenic mice. Further studies revealed that heart mitochondria overexpressing Glrx2 released less cytochrome c than did controls in response to treatment with tBid or a peptide encompassing the BH3 domain of Bid. Development of tolerance to DOX toxicity in transgenic mice is also associated with an increase in protein S-glutathionylation in heart mitochondria. Taken together, these results imply that S-glutathionylation of heart mitochondrial proteins plays a role in preventing DOX-induced cardiac injury.

Kinetic and Mechanistic Characterization and Versatile Catalytic Properties of Mammalian Glutaredoxin 2: Implications for Intracellular Roles

Glutaredoxin (Grx)-catalyzed deglutathionylation of protein-glutathione mixed disulfides (protein-SSG) serves important roles in redox homeostasis and signal transduction, regulating diverse physiological and pathophysiological events. Mammalian cells have two Grx isoforms: Grx1, localized to the cytosol and mitochondrial intermembrane space, and Grx2, localized primarily to the mitochondrial matrix [Pai, H. V., et al. (2007) Antioxid. Redox Signaling 9, 2027-2033]. The catalytic behavior of Grx1 has been characterized extensively, whereas Grx2 catalysis is less well understood. We observed that human Grx1 and Grx2 exhibit key catalytic similarities, including selectivity for protein-SSG substrates and a nucleophilic, double-displacement, monothiol mechanism exhibiting a strong commitment to catalysis. A key distinction between Grx1- and Grx2-mediated deglutathionylation is decreased catalytic efficiency ( k cat/ K M) of Grx2 for protein deglutathionylation (due primarily to a decreased k cat), reflecting a higher p K a of its catalytic cysteine, as well as a decreased enhancement of nucleophilicity of the second substrate, GSH. As documented previously for hGrx1 [Starke, D. W., et al. (2003) J. Biol. Chem. 278, 14607-14613], hGrx2 catalyzes glutathione-thiyl radical (GS (*)) scavenging, and it also mediates GS transfer (protein S-glutathionylation) reactions, where GS (*) serves as a superior glutathionyl donor substrate for formation of GAPDH-SSG, compared to GSNO and GSSG. In contrast to its lower k cat for deglutathionylation reactions, Grx2 promotes GS-transfer to the model protein substrate GAPDH at rates equivalent to those of Grx1. Estimation of Grx1 and Grx2 concentrations within mitochondria predicts comparable deglutathionylation activities within the mitochondrial subcompartments, suggesting localized regulatory functions for both isozymes.

Parallel intestinal and liver injury during early cholestasis in the rat: Modulation by bile salts and antioxidants

Whereas long-term cholestasis results in intestinal alterations and increased permeability to hepatotoxins, the effect of short-term cholestasis is less known and was investigated in bile duct ligated (BDL) rats. In the intestinal mucosa, at Day 7 BDL, total glutathione and protein sulfhydryl contents had decreased, oxidized glutathione levels increased ( P < 0.05 vs baseline), and a reduced epithelium thickness with dissolving crypt phenomena was observed in 40% of rats. At Day 10, total protein content, glutathione-related enzyme activities, and the transmural electrophysiological activity had decreased (−50%); by contrast, oxidized proteins doubled ( P < 0.05), and histological changes were extended to 70% of rats. In vitro exposure to taurodeoxycholate at micellar concentrations determined dysepithelization in normal gut but dissolving crypt phenomena and necrosis in cholestatic bowels. In the liver, ongoing cholestasis was associated with early oxidative changes especially in mitochondria, where protein sulfhydryls were decreased and negatively correlated with glutathione-protein mixed disulfides ( r = −0.807, P < 0.001). Daily oral administration of tauroursodeoxycholate, a hydrophilic bile salt, and glutathione to BDL rats improved intestinal histology, function, and redox state. In conclusion, short-term cholestasis results in distinctive functional, oxidative, and morphological changes of intestinal mucosa, determined increased vulnerability to toxic injury, and parallel hepatic oxidative damage.

Protein and non-protein sulfhydryls and disulfides in gastric mucosa and liver after gastrotoxic chemicals and sucralfate: Possible new targets of pharmacologic agents

To investigate the role of major non-protein and protein sulfhydryls and disulfides in chemically induced gastric hemorrhagic mucosal lesions (HML) and the mechanism of gastroprotective effect of sucralfate. Rats were given 1 mL of 75% ethanol, 25% NaCl, 0.6 mol/L HCl, 0.2 mol/L NaOH or 1% ammonia solutions intragastrically (i.g.) and sacrificed 1, 3, 6 or 12 min later. Total (reduced and oxidized) glutathione (GSH + GSSG), glutathione disulfide (GSSG), protein free sulfhydryls (PSH), protein-glutathione mixed disulfides (PSSG) and protein cystine disulfides (PSSP) were measured in gastric mucosa and liver. Reduced glutathione (GSH) was depleted in the gastric mucosa after ethanol, HCl or NaCl exposure, while oxidized glutathione (GSSG) concentrations increased, except by HCl and NaOH exposure. Decreased levels of PSH after exposure to ethanol were observed, NaCl or NaOH while the total protein disulfides were increased. Ratios of reduced to oxidized glutathione or sulfhydrils to disulfides were decreased by all chemicals. No changes in thiol homeostasis were detected in the liver after i.g. abbreviation should be spelled out the first time here administration of ethanol. Sucralfate increased the concentrations of GSH and PSH and prevented the ethanol-induced changes in gastric mucosal thiol concentrations. Our modified methods are now suitable for direct measurements of major protein and non-protein thiols/disulfides in the gastric mucosa or liver. A common element in the pathogenesis of chemically induced HML and in the mechanism of gastroprotective drugs seems to be the decreased ratios of reduced and oxidized glutathione as well as protein sulfhydryls and disulfides.

Down-regulation of glutaredoxin by estrogen receptor antagonist renders female mice susceptible to excitatory amino acid mediated complex I inhibition in CNS

β- N-oxalyl-amino- l-alanine, ( l-BOAA), an excitatory amino acid, acts as an agonist of the AMPA subtype of glutamate receptors. It inhibits mitochondrial complex I in motor cortex and lumbosacral cord of male mice through oxidation of critical thiol groups, and glutaredoxin, a thiol disulfide oxido-reductase, helps maintain integrity of complex I. Since incidence of neurolathyrism is less common in women, we examined the mechanisms underlying the gender-related effects. Inhibition of complex I activity by l-BOAA was seen in male but not female mice. Pretreatment of female mice with estrogen receptor antagonist ICI 182,780 or tamoxifen sensitizes them to l-BOAA toxicity, indicating that the neuroprotection is mediated by estrogen receptors. l-BOAA triggers glutathione (GSH) loss in male mice but not in female mice, and only a small but significant increase in oxidized glutathione (GSSG) was seen in females. As a consequence, up-regulation of γ-glutamyl cysteinyl synthase (the rate-limiting enzyme in glutathione synthesis) was seen only in male mouse CNS but not in females. Both glutathione reductase and glutaredoxin that reduce oxidized glutathione and protein glutathione mixed disulfides, respectively, were constitutively expressed at higher levels in females. Furthermore, glutaredoxin activity in female mice was down-regulated by estrogen antagonist indicating its regulation by estrogen receptor. The higher constitutive expression of glutathione reductase and glutaredoxin could potentially confer neuroprotection to female mice.

Membrane skeletal protein S-glutathionylation and hemolysis in human red blood cells

In this work, protein–glutathione mixed disulfide formation in human red blood cells (RBCs) was evaluated in vitro by using the thiol-specific reagent diamide. We investigated what mechanism could lead to S-glutathionylation of membrane skeletal proteins, what are the main target proteins, and the correlation between protein S-glutathionylation and RBC hemolysis. Diamide caused a decrease in the reduced form of glutathione (GSH), which was accompanied by an increase in the basal level of glutathione disulfide (GSSG) and in S-glutathionylation of protein 4.2 and spectrin. The increase in membrane skeletal protein S-glutathionylation was correlated with a lower susceptibility of RBCs to osmotic hemolysis, suggesting that S-glutathionylation of protein 4.2 and spectrin could contribute to regulate RBC membrane stability.

Modulation of Glutaredoxin-1 Expression in a Mouse Model of Allergic Airway Disease

Glutaredoxins (GRX) are antioxidant enzymes that preferentially catalyze the reduction of protein-glutathione mixed disulfides. The formation of mixed disulfides with GSH is known as S-glutathionylation, a post-translational modification that is emerging as an important mode of redox signaling. Since asthma is a disease that is associated with increased oxidative stress and altered antioxidant defenses, we investigated the expression of GRX in a murine model of allergic airway disease. Sensitization and challenge of C57BL/6 mice with ovalbumin resulted in increased expression of GRX1 mRNA, as well as increased amounts of GRX1 protein and total GRX activity in the lung. Because GRX1 expression is prominent in bronchial epithelium, we isolated primary epithelial cells from mouse trachea to investigate the presence of GRX. Primary tracheal epithelial cells were found to express both GRX1 and 2 mRNA and detectable GRX activity. Treatment with IFN-gamma increased the expression of GRX1 and overall GRX activity, resulting in attenuation of protein S-glutathionylation. In contrast, TGF-beta1 caused decreased GRX1 expression and overall GRX activity, leading to markedly enhanced protein S-glutathionylation. GRX1 joins the cadre of antioxidant defenses known to be modulated during allergic airway inflammation.

Protein S-glutathionylation and platelet anti-aggregating activity of disulfiram

Blood platelets are central to haemostasis, and reactions in platelets involving sulfhydryl groups play important roles in platelet function. Reduced glutathione (GSH) plays an important role in platelet aggregation and glutathione-depleting chemicals inhibit platelet aggregation. The lipophilic drug disulfiram, because of its affinity for sulfhydryl groups, is a highly thiol-reacting agent. As a consequence, GSH and sulfhydryl groups of protein cysteines in human platelets, in analogy to other components of human blood, are a potential target of disulfiram. In the present study, we have shown that exposure of human platelets to disulfiram causes the depletion of platelet GSH and augmentation of mixed disulfides between GSH and protein sulfhydryl groups to form protein-glutathione mixed disulfides ( S-glutathionylated proteins). The depletion of platelet GSH and the increase in S-glutathionylated proteins occurred at concentrations of disulfiram that inhibited platelet aggregation, suggesting that protein S-glutathionylation is involved in the inhibition of platelet aggregation caused by disulfiram.

Molecular mechanism of glutathione-mediated protection from oxidized low-density lipoprotein-induced cell injury in human macrophages: Role of glutathione reductase and glutaredoxin

Macrophage death is a hallmark of advanced atherosclerotic plaque, and oxidized low-density lipoprotein (OxLDL) found in these lesions is believed to contribute to macrophage injury. However, the underlying mechanisms of this phenomenon are only poorly understood. Here we show that in human monocyte-derived macrophages, OxLDL depleted intracellular glutathione (GSH) and inhibited glutathione reductase, resulting in a marked diminution of the glutathione/glutathione disulfide ratio. In the absence of OxLDL, an 80% depletion of intracellular GSH levels did not affect cell viability, but glutathione depletion dramatically increased OxLDL-induced cell death. Conversely, supplementation of intracellular GSH stores with glutathione diethyl ester substantially diminished OxLDL toxicity. OxLDL also promoted protein- S-glutathionylation, which was increased in macrophages pretreated with the glutathione reductase inhibitor BCNU. Knockdown experiments with siRNA directed against glutathione reductase and glutaredoxin showed that both enzymes are essential for the protection of macrophages against OxLDL. Finally, the peroxyl-radical scavenger Trolox did not prevent GSH depletion but completely blocked OxLDL-induced protein- S-glutathionylation and cell death. These data suggest that OxLDL promotes ROS formation and protein- S-glutathionylation by a mechanism independent from its effect on GSH depletion. Neither mechanism was sufficient to induce macrophage injury, but when stimulated concurrently, these pathways promoted the accumulation of protein-glutathione mixed disulfides and cell death.

Hemoglobin S-thiolation during peroxide-induced oxidative stress in chicken blood

Protein S-thiolation or protein–glutathione mixed disulfide (PSSG) occurs when cells are exposed to oxidative stress, and has been implicated in several cellular functions. The S-thiolation of hemoglobin as well as other abundant proteins is proposed to participate as a redox buffer, being part of the antioxidant protection system of the cell during the oxidative challenge. We studied the oxidative stress caused by peroxides (H2O2, cumene and tert-butyl hydroperoxide) on chicken blood by measuring the thiol/disulfide status. Chicken blood under peroxide treatment showed a time- and concentration-dependent increase in glutathione disulfide (GSSG) and PSSG. GSSG peaked immediately after treatment (1 min), while PSSG increased progressively over time, showing a maximum after about 30 min. The system recovered after 140 min of incubation, with GSSG and PSSG then barely reaching control values. The S-thiolation of hemoglobin was monitored under nondenaturing PAGE, and the fraction of S-thiolated hemoglobin, or Hb A1, rose in a dose-dependent fashion and was proportional to total S-thiolation, measured as PSSG. This significant correlation indicates that hemoglobin is the major S-thiolated protein in chicken erythrocytes treated with peroxides. The present work shows the behavior of chicken blood under peroxide treatment; it anticipated that chicken hemoglobin thiol groups can actively participate in the redox processes of erythrocytes exposed to oxidative stress, and that hemoglobin is the major S-thiolated protein. This further corroborates the hypothesis that abundant proteins, such as hemoglobin, may take part in the cellular antioxidant defense system.

Thiol–Disulfide Balance: From the Concept of Oxidative Stress to that of Redox Regulation

Originally, small thiols, including glutathione, were viewed as protective antioxidants, acting as free radical scavengers in the context of oxidative damage. Recently, there is a growing literature showing that protein glutathionylation (formation of protein-glutathione mixed disulfides) and other forms of cysteine oxidation may be a means of redox regulation under physiological conditions. This review discusses the importance of protein oxidation in redox regulation in view of the recent data originating from the application of redox proteomics to identify redox-sensitive targets.

Catalysis of Thiol/Disulfide Exchange: GLUTAREDOXIN 1 AND PROTEIN-DISULFIDE ISOMERASE USE DIFFERENT MECHANISMS TO ENHANCE OXIDASE AND REDUCTASE ACTIVITIES

Glutaredoxin (Grx) and protein-disulfide isomerase (PDI) are members of the thioredoxin superfamily of thiol/disulfide exchange catalysts. Thermodynamically, rat PDI is a 600-fold better oxidizing agent than Grx1 from Escherichia coli. Despite that, Grx1 is a surprisingly good protein oxidase. It catalyzes protein disulfide formation in a redox buffer with an initial velocity that is 30-fold faster than PDI. Catalysis of protein and peptide oxidation by the individual catalytic domains of PDI and by a Grx1-PDI chimera show that differences in active site chemistry are fundamental to their oxidase activity. Mutations in the active site cysteines reveal that Grx1 needs only one cysteine to catalyze rapid substrate oxidation, whereas PDI requires both cysteines. Grx1 is a good oxidase because of the high reactivity of a Grx1-glutathione mixed disulfide, and PDI is a good oxidase because of the high reactivity of the disulfide between the two active site cysteines. As a protein disulfide reductase, Grx1 is also superior to PDI. It catalyzes the reduction of nonnative disulfides in scrambled ribonuclease and protein-glutathione mixed disulfides 30-180 times faster than PDI. A multidomain structure is necessary for PDI to catalyze effective protein reduction; however, placing Grx1 into the PDI multidomain structure does not enhance its already high reductase activity. Grx1 and PDI have both found mechanisms to enhance active site reactivity toward proteins, particularly in the kinetically difficult direction: Grx1 by providing a reactive glutathione mixed disulfide to supplement its oxidase activity and PDI by utilizing its multidomain structure to supplement its reductase activity.

S-glutathionylation in human platelets by a thiol–disulfide exchange-independent mechanism

Protein–glutathione mixed disulfide formation was investigated in vitro by exposure of human platelets to the thiol-specific oxidant azodicarboxylic acid-bis-dimethylamide (diamide). We found that diamide causes a decrease in the reduced form of glutathione (GSH), paralleled by an increase in protein–GSH mixed disulfides (S-glutathionylated proteins), which was not accompanied by any significant increase in the basal level of glutathione disulfide (GSSG). The increase in the appearance of S-glutathionylated proteins was inversely correlated with ADP-induced platelet aggregation. Platelet cytoskeleton was analyzed by SDS–PAGE followed by Western immunoblotting with anti-GSH antibody. The main S-glutathionylated cytoskeletal protein proved to be actin, which accounts for 35% of the platelet total protein content. Our results suggest that neither GSSG formation nor a consequent thiol–disulfide exchange mechanism is involved in actin S-glutathionylation of human platelets exposed to diamide. Instead, a mechanism involving the initial oxidative activation of actin thiol groups, which then react with GSH to the protein–GSH mixed disulfides, makes it likely that platelet actin is S-glutathionylated without any significant increase in the GSSG content.

Enhanced Dephosphorylation of cAMP-dependent Protein Kinase by Oxidation and Thiol Modification

The catalytic subunit of cAMP-dependent protein kinase (PKA) is phosphorylated at threonine 197 and serine 338. Phosphorylation of threonine 197, located in the activation loop, is required for coordinating the active site conformation and optimal enzymatic activity. However, this phosphorylation has not been widely appreciated as a regulatory site because of the apparent constitutive nature of the phosphorylation and the general resistance of the kinase to phosphatase treatment. We demonstrate here that the observed resistance of the catalytic subunit to dephosphorylation is due, in part, to the presence of the highly nucleophilic cysteine 199 located proximal to the phosphate on threonine 197. Experiments performed in vitro demonstrated that mutation (cysteine 199 to alanine), oxidation, such as by glutathionylation or internal disulfide bond formation, or alkylation of the C-subunit enhanced its ability to be dephosphorylated. Furthermore, rephosphorylation of reduced C-subunit by PDK1 created a cycle whereby the inactive kinase could be reactivated. To demonstrate that thiol modification of PKA can lead to enhanced dephosphorylation in vivo, PC12 cells were treated with N-ethylmaleimide (NEM). Such treatment resulted in complete PKA inactivation and dephosphorylation of threonine 197. This effect of NEM was contingent upon prior treatment of the cells with PKA activators, demonstrating the resistance of the holoenzyme to thiol alkylation-mediated dephosphorylation. Our results also demonstrated that NEM treatment of PC12 cells enhanced the dephosphorylation of the protein kinase Calpha activation loop, suggesting a common mechanism of regulation among members of the AGC family of kinases.

Changes in the glutathione thiol-disulfide status in wheat grain by foliar sulphur fertilization: consequences for the rheological properties of dough

Storage proteins and glutathione in wheat play an important role in gluten network formation and can be modified by supplementation of nitrogen (N) and sulphur (S) in wheat plants. The glutathione thiol-disulfide status and its relationship to the molecular weight distribution wheat polymeric protein and dough rheological properties have been examined after different foliar S fertilizations (S derived from micronized elemental S and NS, a mixture of N urea and elemental S) applied at the post-anthesis stage. Changes in levels of reduced glutathione (GSH), glutathione disulfide (GSSG), polymeric protein-glutathione mixed disulfide (PPSSG) were analysed by reversed phase high performance liquid chromatography, during grain development using the wheat cultivars, Soissons and Trémie. During the grain desiccation phase, S supplementation (i) increased the GSSG/GSH ratio by 23–25% (ii) induced PPSSG accumulation, and (iii) decreased the formation of SDS-unextractable polymeric protein (UPP) and its molecular mass distribution. However, simultaneous N and S supplementation results in: (i) a decrease in PPSSG formation by 20–30% and (ii) an increase of UPP by 7–18% by enhancing both the branching of the aggregated proteins and their molecular weight. The mixograph parameters show that all forms of endogenous glutathione are linked to dough weakening and are negatively correlated with dough mixing tolerance, dough strength and consistency, while UPP is positively correlated with dough strength and consistency. These findings indicate that S nutrition influences dynamics of the glutathione forms in the grain and results in modification the degree of polymerization of storage protein. Thus both the changes in the form of glutathione and protein polymerization influence the rheological properties of dough.

The effects of age and hyperhomocysteinemia on the redox forms of plasma thiols

We assayed the redox forms of cysteine (reduced [CSH], oxidized [CSSC], and bound to protein [CS-SP]), cysteinylglycine (CGSH; cysteinylgycine disulfide [CGSSGC] and cysteinylglycine-protein mixed disulfide [CGS-SP]), glutathione (GSH; glutathione disulfide [GSSG] and glutathione-protein mixed disulfide [GS-SP]), homocysteine (Hcy; homocystine [HcyS] and homocystine-protein mixed disulfides [bHcy]), and protein sulfhydryls in the plasma of healthy subjects (divided into 8 groups ranging in age from birth to 70 years) and patients with mild hyperhomocysteinemia associated with cardiovascular disease (heart-transplant patients) or vascular atherosclerosis, with or without renal failure. In healthy individuals, levels of disulfides and protein-mixed disulfides were more abundant than those of thiols, and those of protein-thiol mixed disulfides were higher than disulfides. Concentrations of CSH, GSH, and CGSH in the various groups had profiles characterized by a maximum over time. The concentration of Hcy was unchanged up to the age of 30 years, after which it increased. CSSC concentration increased gradually with age, whereas concentrations of the other disulfides were essentially unchanged. By contrast, the concentrations of all protein-thiol mixed disulfides, especially those with CSH, increased gradually with age. Ranks of distribution of the reduced forms changed with age (at birth, CSH > CGSH > GSH > Hcy; in 1- to 2-year-olds, CSH > GSH > CGSH > Hcy; and in 51- to 70-year-olds, CSH > CGSH = GSH > Hcy), whereas those of disulfides and protein-thiol mixed disulfides were substantially unchanged (in all age groups, CSSC > CGSSGC > GSSG = HcyS and CS-SP > CGS-SP > bHcy > GS-SP). In patients with pathologic conditions, plasma levels of disulfide forms CSSC, HcyS, CS-SP, and bHcy were significantly increased, whereas other redox forms of thiols were unchanged or showed variations opposite (increasing or decreasing) to control values. Maximal increases in disulfides and protein-thiol mixed disulfides were associated with renal failure. Our data suggest that increases in plasma bHcy concentrations in subjects with pathologic conditions were more likely the result of activation of thiol-disulfide exchange reactions between free reduced Hcy and CS-SP than of a direct action of reactive oxygen species.

Effect of tea catechins on the change of glutathione levels caused by Pb(++) in PC12 cells.

Our previous research showed that tea catechins could significantly increase the viability of lead-exposed PC12 cells. Whereas the cellular thiol status is known to be responsible for protecting against lead-induced toxicity, whether the role of tea catechins on lead-induced PC12 cell toxicity is related to the metabolism of intracellular thiol compounds remained vague. In the present study, it was found that Pb(2+) significantly decreased reduced glutathione (GSH)/oxidative glutathione (GSSG) and protein sulfhydryl groups (PSH)/glutathione-protein mixed disulfide (GSSP) ratios as well as glutathione reductase activities in a concentration-dependent manner. Both (-)-epicatechin and (-)-epicatechin gallate (ECG) supplementation resulted in an increased GSH/GSSG ratio and glutathione reductase activities. The galloylated catechins (ECG or (-)-epigallocatechin gallate) treatment significantly decreased the GSSP levels and increased the intracellular PSH/GSSP ratio in lead-exposed PC12 cells. To our surprise, as compared with the group treated by lead acetate, 100 microM EGC treatment following lead exposure significantly decreased GSH/GSSG and PSH/GSSP ratios, as well as glutathione reductase activities. The results suggested that the effect of tea catechins on the intracellular thiols status in PC12 cells was different, which may be related to their chemical structures and/or regulation of special gene expression properties.

Changes in the glutathione thiol–disulfide status during wheat grain development

Glutathione status and its relationship to molecular weight distribution (MWD) of wheat polymeric protein (PP) during the grain development have been examined. Changes in reduced glutathione (GSH), glutathione disulfide (GSSG), and protein-glutathione mixed disulfide (PSSG) levels were followed through reversed phase high performance liquid chromatography (RP-HPLC) analysis. By applying this procedure to two varieties of wheat, differing in high molecular weight glutenin subunit (HMW-GS) composition (2*, 7 + 8, 5 + 10 and 0, 6 + 8, 2 + 12, respectively, for Soissons and Thésée), we showed that during the grain desiccation (after 33 d after anthesis) which coincide with the formation of SDS-unextractable polymeric protein (UPP), glutathione reductase activity declines, the ratio of GSSG to GSH increases and PSSG occurs. Moreover, approximately 85% of PSSG in mature wheat grains are represented by polymeric protein conjugated to glutathione (PP-S-S-G). As asymmetrical flow field flow fractionation–multi-angle laser light scattering (AFFFF–MALLS) analysis showed, the PP-S-S-G formation during the grain desiccation can induce an increase in the SDS solubility of the polymeric protein by reducing both their molecular weight distribution and the branching of these aggregates. We speculate that glutathione may play a crucial role in controlling the degree of polymerisation of the polymeric protein, which has been shown to be extremely important in determining baking performance.