Glutaredoxin 2 catalyzes the reversible oxidation and glutathionylation of mitochondrial membrane thiol proteins: implications for mitochondrial redox regulation and antioxidant DEFENSE

The redox poise of the mitochondrial glutathione pool is central in the response of mitochondria to oxidative damage and redox signaling, but the mechanisms are uncertain. One possibility is that the oxidation of glutathione (GSH) to glutathione disulfide (GSSG) and the consequent change in the GSH/GSSG ratio causes protein thiols to change their redox state, enabling protein function to respond reversibly to redox signals and oxidative damage. However, little is known about the interplay between the mitochondrial glutathione pool and protein thiols. Therefore we investigated how physiological GSH/GSSG ratios affected the redox state of mitochondrial membrane protein thiols. Exposure to oxidized GSH/GSSG ratios led to the reversible oxidation of reactive protein thiols by thiol-disulfide exchange, the extent of which was dependent on the GSH/GSSG ratio. There was an initial rapid phase of protein thiol oxidation, followed by gradual oxidation over 30 min. A large number of mitochondrial proteins contain reactive thiols and most of these formed intraprotein disulfides upon oxidation by GSSG; however, a small number formed persistent mixed disulfides with glutathione. Both protein disulfide formation and glutathionylation were catalyzed by the mitochondrial thiol transferase glutaredoxin 2 (Grx2), as were protein deglutathionylation and the reduction of protein disulfides by GSH. Complex I was the most prominent protein that was persistently glutathionylated by GSSG in the presence of Grx2. Maintenance of complex I with an oxidized GSH/GSSG ratio led to a dramatic loss of activity, suggesting that oxidation of the mitochondrial glutathione pool may contribute to the selective complex I inactivation seen in Parkinson's disease. Most significantly, Grx2 catalyzed reversible protein glutathionylation/deglutathionylation over a wide range of GSH/GSSG ratios, from the reduced levels accessible under redox signaling to oxidized ratios only found under severe oxidative stress. Our findings indicate that Grx2 plays a central role in the response of mitochondria to both redox signals and oxidative stress by facilitating the interplay between the mitochondrial glutathione pool and protein thiols.     

Biological disulfides: the third messenger? Modulation of phosphofructokinase activity by thiol/disulfide exchange

Rabbit muscle phosphofructokinase is rapidly inactivated at pH 8.0 by incubation with low concentrations of oxidized glutathione, Coenzyme A glutathione mixed disulfide, and oxidized Coenzyme A. The inactivation is first order in disulfide concentration over the concentration ranges examined (50-200 microM), and is approximately 8-fold slower at pH 7.0 than at pH 8.0. The substrates ATP and fructose 6-phosphate protect against inactivation while effector molecules such as AMP, cAMP, and citrate do not. The oxidation of the enzyme by disulfides is fully reversible. The equilibrium constant for the reaction Ered + GSSG in equilibrium Eox + GSH at pH 8.0 is 7.1 in the absence of substrates and 2.5 in the presence of 0.1 mM ATP. For comparison, the equilibrium constant for the reaction CoASH + GSSG in equilibrium CoASSG + GSH was found to be 3.1 at pH 8.0. These equilibrium constants for thiol/disulfide exchange are such that modulation of phosphofructokinase activity by thiol/disulfide exchange in vivo is feasible. The ability of the thiol/disulfide ratio in vivo to modulate the activity of the fructose 6-phosphate/fructose 1,6-diphosphate futile cycle is discussed. The possibility is considered that modulation of the thiol/disulfide ratio in vivo may serve as a "third messenger" in response to cAMP levels, and that the activity of key enzymes of glycolysis/gluconeogenesis may be regulated in response to changing thiol/disulfide ratios.     

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.     

Thiol/disulfide exchange between rabbit muscle phosphofructokinase and glutathione. Kinetics and thermodynamics of enzyme oxidation

Reversible thiol/disulfide exchange equilibria between rabbit muscle phosphofructokinase and glutathione redox buffers results in a dependence of the activity of the enzyme on the thiol to disulfide ratio of the redox buffer (Gilbert, H. F. (1982) J. Biol. Chem. 257, 12086-12091). The transition between fully reduced (active) and fully oxidized (inactive) enzyme is half complete at a [GSH]/[GSSG] ratio of 6.5 +/- 1 at pH 8.0 and 5.6 +/- 0.9 at pH 7.2. In the presence of excess GSSG approximately 40-50% of the activity is lost in a rapid process (k = 110 M-1 min-1), while the remaining activity is lost more slowly (k = 1.9 M-1 min-1). Two equivalents of radiolabeled glutathione are incorporated covalently, one coincident with each phase of inactivation. The most rapidly oxidized sulfhydryl group is also the most rapidly reduced by GSH in the reverse reaction (k = 150 M-1 min-1). Reduction of a more slowly reacting protein-glutathione mixed disulfide is required to regenerate the original activity (k = 0.33 M-1 min-1). The thiol/disulfide oxidation equilibrium constant (Kox) for the most rapidly oxidized sulfhydryl group is estimated to be 0.7 while that for the more slowly oxidized group is 6.1. The sulfhydryl group which is more easily oxidized kinetically is the more thermodynamically resistant to oxidation. The magnitude of the equilibrium constants for these reversible oxidations would suggest that the oxidation state (and activity) of phosphofructokinase would not be significantly affected by typical metabolic changes in the glutathione oxidation state in vivo.     

The reaction of choline acetyltransferase with sulfhydryl reagents. Methoxycarbonyl-CoA disulfide as an active site-directed reagent.

The reaction of choline acetyltransferase with methoxycarbonyl alkyl disulfides leads to a progressive loss in enzyme activity as the size of the alkyl group increases from methyl to n-butyl. Reaction with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) or methoxycarbonyl coenzyme A (CoA) disulfide, leads to a total loss of enzyme activity. DTNB inactivation is biphasic (k1 = approximately 9 x 10(2) M-1 s-1, k2 = approximately 6 x 10(1) M-1 s-1) with the slow phase being diminished by acetyl-CoA. Methoxycarbonyl-CoA disulfide inactivation is also biphasic (k1 = approximately 2.1 x 10(3) M-1 s-1, k2 = approximately 6 x 10(1) M-1 s-1), with the rapid phase being diminished in the presence of acetyl-CoA. Inactivation by methoxycarbonyl methyl disulfide, ethyl disulfide, or hydroxyethyl disulfide, or by methyl methanethiosulfonate is not biphasic. Pretreatment of the enzyme with methyl methanethiosulfonate, which leads to a 25% loss in enzyme activity, abolishes the fast phase of DTNB inactivation, the slow phase of methoxycarbonyl-CoA disulfide inactivation, and any further inactivation by methoxycarbonyl ethyl disulfide. These results are interpreted to suggest that choline acetyltransferase contains two classes of reactive sulfhydryl groups, neither of which are required for enzyme activity.     

Glutathione,glutathione disulfide,and S-glutathionylated proteins in cell cultures

The analysis of the global thiol–disulfide redox status in tissues and cells is a challenging task since thiols and disulfides can undergo artificial oxido-reductions during sample manipulation. Because of this, the measured values, in particular for disulfides, can have a significant bias. Whereas this methodological problem has already been addressed in samples of red blood cells and solid tissues, a reliable method to measure thiols and disulfides in cell cultures has not been previously reported.Here, we demonstrate that the major artifact occurring during thiol and disulfide analysis in cultured cells is represented by glutathione disulfide (GSSG) and S-glutathionylated proteins (PSSG) overestimation, due to artificial oxidation of glutathione (GSH) during sample manipulation, and that this methodological problem can be solved by the addition of N-ethylmaleimide (NEM) immediately after culture medium removal. Basal levels of GSSG and PSSG in different lines of cultured cells were 3–5 and 10–20 folds higher, respectively, when the cells were processed without NEM. NEM pre-treatment also prevented the artificial reduction of disulfides that occurs during the pre-analytical phase when cells are exposed to an oxidant stimulus. In fact, in the absence of NEM, after medium removal, GSH, GSSG and PSSG levels restored their initial values within 15–30 min, due to the activity of reductases and the lack of the oxidant. The newly developed protocol was used to measure the thiol–disulfide redox status in 16 different line cells routinely used for biomedical research both under basal conditions and after treatment with disulfiram, a thiol-specific oxidant (0–200 μM concentration range).Our data indicate that, in most cell lines, treatment with disulfiram affected the levels of GSH and GSSG only at the highest concentration. On the other hand, PSSG levels increased significantly also at the lower concentrations of the drug, and the rise was remarkable (from 100 to 1000 folds at 200 μM concentration) and dose-dependent for almost all the cell lines. These data support the suitability of the analysis of PSSG in cultured cells as a biomarker of oxidative stress.     

Membrane thiol-disulfide status in glucose-6-phosphate dehydrogenase deficient red cells. Relationship to cellular glutathione

The behavior of glucose-6-phosphate dehydrogenase (G6PD)-deficient red cell membrane proteins upon treatment with diamide, the thiol-oxidizing agent (Kosower, N.S. et al. (1969) Biochem. Biophys. Res. Commun. 37, 593–596), was studied with the aid of monobromobimane, a fluorescent labeling agent (Kosower, N.S., Kosower, E.M., Newton, G.L. and Ranney, H.M. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 3382–3386) convenient for following membrane thiol group status. In diamide-treated G6PD-deficient red cells (and in glucose deprived normal cells), glutathione (GSH) is oxidized to glutathione disulfide (GSSG). When cellular GSH is absent, membrane protein thiols are oxidized with the formation of intrachain and interchain disulfides. Differences in sensitivity to oxidation are found among membrane thiols. In diamidetreated normal red cells, GSH is regenerated in the presence of glucose and membrane disulfides reduced. In G6PD-deficient cells, GSSG is not reduced, and the oxidative damage (disulfide formation) in the membrane not repaired. Reduction of membrane disulfides does occur after the addition of GSH to these membranes. A direct link between the thiol status of the cell membrane and cellular GSH is thereby established. GSH serves as a reductant of membrane protein disulfides, in addition to averting membrane thiol oxidation.     

Study on human erythrocyte thioltransferase: comparative characterization with bovine enzyme and its physiological role under oxidative stress.

Thioltransferase, an enzyme which catalyzes the thiol/disulfide exchange reaction in the presence of GSH, was purified to homogeneity on 15% SDS-PAGE from human (36,000-fold purification) and bovine (23,000-fold) erythrocyte hemolysates. These enzymes had similar properties in their monomeric structures (M(r) = 11,000) and broad specificities for substrates ranging from low-molecular disulfides (S-sulfocysteine, cystamine, and cystine) to protein disulfides (trypsin and insulin). They were highly sensitive to SH-reagents (monoiodoacetic acid and mercuric chloride), but were protected from inactivation by the presence of disulfides (GSSG, cystamine, and cystine). Phosphofructokinase and pyruvate kinase that had been inactivated by disulfides were reactivated effectively by the addition of thioltransferase with GSH. In addition, disulfides in membrane proteins of human erythrocytes that have been oxidatively damaged by diamide treatment were reduced to the SH-free form more effectively by incubation with thioltransferase.     

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.     

The effects of mevinolin on the thiol/disulfide exchange between 3-hydroxy-3-methyglutaryl-coenzyme A reductase and glutathione

The feeding of mevinolin plus cholestyramine to rats results in the production of a form of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR-CM) having thiol/disulfide redox properties different from those of 3-hydroxy-3-methylglutaryl-CoA reductase isolated from animals which had been given only cholestyramine (HMGR-C). The second-order rate constant for the inactivation of HMGR-CM by GSSG is 7-fold slower than for HMGR-C, while the second-order rate constant for the reactivation of oxidized enzyme by GSH is 100-fold slower. However, in the presence of saturating concentrations of both substrates, the rate constants for thiol/disulfide exchange are similar for both forms of the enzyme. HMGR-CM behaves as if a protein-glutathione mixed disulfide having a Kox of 27 +/- 4 is formed at equilibrium. In contrast, HMGR-C has previously been shown to form a protein-protein disulfide (Cappel, R. E., and Gilbert, H. F. (1988) J. Biol. Chem. 263, 12204-12212). Both forms of the enzyme are more difficult to oxidize thermodynamically in the presence of saturating levels of both substrates. For HMGR-CM, NADPH alone has no effect on the equilibrium constant for oxidation, but hydroxymethylglutaryl-CoA alone makes the enzyme approximately twice as difficult to oxidize. Under physiological conditions, HMGR-CM is thermodynamically more difficult to oxidize than HMGR-C. HMGR-C can be converted to HMGR-CM by in vitro treatment with mevinolinate. A direct or indirect interaction of mevinolin with HMGR-C results in some persistent, as yet undefined, structural alteration which inhibits the formation of a protein-SS-protein disulfide upon oxidation by glutathione disulfide.     

In vitro inhibition of topoisomerase IIα by reduced glutathione

In most cells, the major intracellular redox buffer is glutathione (GSH) and its disulfide-oxidized (GSSG) form. The GSH/GSSG system maintains the intracellular redox balance and the essential thiol status of proteins by thiol disulfide exchange. Topoisomerases are thiol proteins and are a target of thiol-reactive substances. In this study, the inhibitory effect of physiological concentration of GSH and GSSG on topoisomerase IIα activity in vitro was investigated. GSH (0-10 mM) inhibited topoisomerase IIα in a concentration-dependent manner while GSSG (1-100 μM) had no significant effect. These findings suggest that the GSH/GSSG system could have a potential in vivo role in regulating topoisomerase IIα activity.     

Significance of Thiol-Disulfide Exchange in Resting Stages of Plant Development

Abstract: Desiccation tolerance is a fundamental principle for resting stages of plant development which include the dormancy of seeds and the quiescent stages of resurrection plants. To prevent the deleterious effects of cellular desiccation, a complex interplay of several adaption mechanisms is required. The ability to cope with free radicals, the formation of which is well documented in desiccated tissues, is one of these basic requirements. Detoxification of free radicals by several antioxidants and scavenging enzymes include reactions of reduced glutathione (GSH) resulting in the formation of glutathione disulfide (GSSG). In free radical processing pathways GSSG is considered to be immediately reduced back to GSH by the action of glutathione reductase (EC 1.6.4.2.). However, in desiccated tissues GSSG accumulates. Protein-glutathione mixed disulfides (PSSG) are also reported to increase in plants under drought leading to the hypothesis that glutathione protects protein thiol groups from auto-oxidation. The irreversible formation of intramolecular disulfides resulting in denaturation of proteins would be one of the primary sites of desiccation injury. We suggest that PSSG is formed by the reaction of GSSG with high molecular weight thiols and introduce a thiol-disulfide cycle that involves reduction/oxidation processes of glutathione and protein thiol groups during the dehydration/rehydration processes in desiccation tolerant tissues.     

Redox state of glutathione in human plasma

Thiol and disulfide forms of glutathione (GSH) and cysteine (Cys) were measured in plasma from 24 healthy individuals aged 25-35 and redox potential values (E(h)) for thiol/disulfide couples were calculated using the Nernst equation. Although the concentration of GSH (2.8 +/- 0.9 microM) was much greater than that of GSSG (0.14 +/- 0.04 microM), the redox potential of the GSSG/2GSH pool (-137 +/- 9 mV) was considerably more oxidized than values for tissues and cultured cells (-185 to -258 mV). This indicates that a rapid oxidation of GSH occurs upon release into plasma. The difference in values between individuals was remarkably small, suggesting that the rates of reduction and oxidation in the plasma are closely balanced to maintain this redox potential. The redox potential for the Cys and cystine (CySS) pool (-80 +/- 9 mV) was 57 mV more oxidized, showing that the GSSG/2GSH and the CySS/2Cys pools are not in redox equilibrium in the plasma. Potentials for thiol/disulfide couples involving CysGly were intermediate between the values for these couples. Regression analyses showed that the redox potentials for the different thiol/disulfide couples within individuals were correlated, with the E(h) for CySS-mono-Gly/(Cys. CysGly) providing the best correlation with other low molecular weight pools as well as protein disulfides of GSH, CysGly and Cys. These results suggest that E(h) values for GSSG/2GSH and CySS-mono-Gly/(Cys. CysGly) may provide useful means to quantitatively express the oxidant/antioxidant balance in clinical and epidemiologic studies.     

Thiol/disulfide exchange in the thioredoxin-catalyzed reductive activation of spinach chloroplast fructose-1,6-bisphosphatase. Kinetics and thermodynamics

Two kinetically and thermodynamically distinct thiol/disulfide redox changes are observed during the reversible thioredoxin fb-catalyzed reduction and oxidation of spinach chloroplast fructose-1,6-bisphosphatase by dithiothreitol. The two processes, which occur at different rates and with different equilibrium constants, can be observed independently in either the reduction (activation) or oxidation (inactivation) direction by assaying the enzyme activity at different magnesium and fructose-1,6-bisphosphate concentrations. The two processes, in both the reduction and oxidation directions, are kinetically zero-order in dithiothreitol concentration and first-order in thioredoxin fb concentration. The rate-limiting step in both directions is the reaction of fructose-1,6-bisphosphatase with thioredoxin. The more kinetically and thermodynamically favored reduction of fructose-1,6-bisphosphatase lowers the apparent Km for fructose-1,6-bisphosphate while the less favorable process lowers the Km for magnesium. Both of the thiol/disulfide redox changes reach equilibrium in redox buffers consisting of different ratios of reduced to oxidized dithiothreitol (Ered + DTTox in equilibrium Eox + DTTred). The equilibrium constants (Kox) are 0.12 +/- 0.02 and 0.39 +/- 0.08 for the fast and slow reduction processes at pH 8.0. The equilibrium constants for oxidation of the enzyme by glutathione disulfide (Ered + GSSG in equilibrium Eox + 2 GSH) can be estimated to be approximately 2400 and 7800 M, respectively. Thermodynamically the fructose-1,6-bisphosphatase/thioredoxin fb system is extremely sensitive to oxidation, comparable to disulfide bond formation in extracellular proteins.     

Reversible inactivation of tyrosine aminotransferase from guinea pig liver by thiol and disulfide compounds.

Tyrosine aminotransferase from guinea pig liver is strongly inactivated by a variety of natural thiols and disulfides. L-cysteine was used as a model compound in the study of inactivation. Inactivation is due to the disulfide produced by spontaneous oxidation of thiol during incubation. Binding studies with [³⁵S]-cysteine revealed simultaneous incorporation of [³⁵S] into tyrosine aminotransferase and loss of enzyme activity. The reversibility demonstrates that the inactivation is the result of the formation of mixed disulfide between the disulfide and the sulfhydryl group of tyrosine aminotransferase. Some features of the enzyme active site are showed by the inactivation reaction.     

Thioltransferase in human red blood cells: kinetics and equilibrium

Thioltransferase from human red blood cells (HRBC TTase), coupled to GSSG reductase, catalyzed glutathione (GSH)-dependent reduction of prototype substrates hydroxyethyl disulfide (HEDS) and sodium S-sulfocysteine as well as of other homo- and heterodisulfides, including the protein mixed disulfide albumin-S-S-cysteine. Whereas apparent KM values for the substrates varied over more than a 20-fold range, the Vmax values agreed quite closely, usually within less than a factor of 2, suggesting that initial interaction of oxidized substrate with enzyme is not rate determining. HRBC TTase was inactivated by iodoacetamide (IAA), and this was prevented by pretreatment with disulfides. The pH dependence of IAA inactivation gave a remarkably low apparent pKa of 3.5, which was independent of ionic strength (0.05-2 M). At pH 6, one radiolabeled carboxyamidomethyl moiety was bound to the enzyme after treatment with [14C]IAA. This unusual thiol reactivity suggests that the active-site cysteine moiety of the TTase may be involved in a hydrogen bond with a carboxylate moiety. In contrast, the pH dependence for GSH-dependent TTase catalysis of disulfide reduction displayed an inflection point near pH 8.0, also suggesting that the initial reaction of oxidized substrate with the active-site thiol is not involved in rate determination. Two substrate kinetic studies of HRBC TTase and rat liver TTase (e.g., [GSH] and [HEDS] varied independently) gave patterns of intersecting lines on double-reciprocal plots (1/v vs 1/S), indicating a sequential mechanism for the TTase reactions, rather than a ping-pong mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxidation of extracellular cysteine/cystine redox state in bleomycin-induced lung fibrosis

Several lines of evidence indicate that depletion of glutathione (GSH), a critical thiol antioxidant, is associated with the pathogenesis of idiopathic pulmonary fibrosis (IPF). However, GSH synthesis depends on the amino acid cysteine (Cys), and relatively little is known about the regulation of Cys in fibrosis. Cys and its disulfide, cystine (CySS), constitute the most abundant low-molecular weight thiol/disulfide redox couple in the plasma, and the Cys/CySS redox state (E(h) Cys/CySS) is oxidized in association with age and smoking, known risk factors for IPF. Furthermore, oxidized E(h) Cys/CySS in the culture media of lung fibroblasts stimulates proliferation and expression of transitional matrix components. The present study was undertaken to determine whether bleomycin-induced lung fibrosis is associated with a decrease in Cys and/or an oxidation of the Cys/CySS redox state and to determine whether these changes were associated with changes in E(h) GSH/glutathione disulfide (GSSG). We observed distinct effects on plasma GSH and Cys redox systems during the progression of bleomycin-induced lung injury. Plasma E(h) GSH/GSSG was selectively oxidized during the proinflammatory phase, whereas oxidation of E(h) Cys/CySS occurred at the fibrotic phase. In the epithelial lining fluid, oxidation of E(h) Cys/CySS was due to decreased food intake. Thus the data show that decreased precursor availability and enhanced oxidation of Cys each contribute to the oxidation of extracellular Cys/CySS redox state in bleomycin-induced lung fibrosis.     

Interaction of menadione (2-methyl-1,4-naphthoquinone) with glutathione

The interaction of menadione with reduced glutathione (GSH) led to a removal of menadione and formation of menadione-GSH conjugate and glutathione disulfide (GSSG). The changes in thiol level were essentially biphasic with an initial rapid decrease in GSH and appearance of GSSG (less than 1 min) followed by secondary less pronounced changes. The interaction of menadione and GSH caused an oxygen uptake and both superoxide anion radical and hydrogen peroxide were produced during the reaction, the amount dependent on the GSH/menadione ratio. Catalase did not protect against the initial decrease in GSH level but markedly inhibited the secondary changes while superoxide dismutase had little effect. These results suggest that the initial changes in thiol level are the result in part of a redox reaction between menadione and GSH as well as conjugate formation, whilst the secondary changes reflect conjugate formation and the activity of other oxidants such as hydrogen peroxide. The potential biological significance of this reaction was investigated using hepatocytes depleted of reduced pyridine nucleotides and thus not able to perform enzyme-catalyzed reduction of menadione. In these cells menadione induced GSSG formation at a rate similar to that observed in control cells. This suggests that quinone-induced oxidative challenge caused by the chemical interactions of a quinone and glutathione may have biological relevance.     

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.     

Histamine chloramine reactivity with thiol compounds, ascorbate, and methionine and with intracellular glutathione

Histamine is stored in granules of mast cells and basophils and released by inflammatory mediators. It has the potential to intercept some of the HOCl generated by the neutrophil enzyme, myeloperoxidase, to produce histamine chloramine. We have measured rate constants for reactions of histamine chloramine with methionine, ascorbate, and GSH at pH 7.4, of 91 M(-1)s(-1), 195 M(-1)s(-1), and 721 M(-1)s(-1), respectively. With low molecular weight thiols, the reaction was with the thiolate and rates increased exponentially with decreasing thiol group pK(a). Comparing rate constants for different chloramines reacting with ascorbate or a particular thiol anion, these were higher when there was less negative charge in the vicinity of the chloramine group. Histamine chloramine was the most reactive among biologically relevant chloramines. Consumption of histamine chloramine and oxidation of intracellular GSH were examined for human fibroblasts. At nontoxic doses, GSH loss over 10 min was slightly greater than that with HOCl, but the cellular uptake of histamine chloramine was 5-10-fold less. With histamine chloramine, GSSG was a minor product and most of the GSH was converted to mixed disulfides with proteins. HOCl gave a different profile of GSH oxidation products, with significantly less GSSG and mixed disulfide formation. There was irreversible oxidation and losses to the medium, as observed with HOCl and other cell types. Thus, histamine chloramine shows high preference for thiols both in isolation and in cells, and in this respect is more selective than HOCl.