The dynamics of cysteine, glutathione and their disulphides in astrocyte culture medium

Glutathione (GSH) plays an important neuroprotective role, and its synthesis depends on the amount of available cysteine (CSH) in the cells. Various kinds of evidence suggest that astrocytes can provide CSH or GSH to neurons, but the delivery mechanism of the thiol-compounds has not been elucidated. In this study, the dynamics of CSH, GSH and their disulphides in astrocyte culture medium were investigated by following the time-course of concentration changes and by computer simulation and curve fitting to experimental data using a mathematical model. The model consists of seven reactions and three transports, which are grouped into four categories: autoxidation of thiols into disulphides, thiol-disulphide exchange and reactions of thiols with medium components, as well as the cellular influx and efflux of thiols and disulphides. The obtained results are interpreted that cystine (CSSC) after entering astrocyte is reduced to CSH, most of which is released to medium and autoxidized to CSSC. The efflux of GSH was estimated to be considerably slower than that of CSH, and most of the excreted GSH is converted to cysteine-glutathione disulphide principally through the thiol-disulphide exchange. The results seem to indicate that astrocytes provide neurons mainly with CSH, rather than GSH, as the antioxidant material for neuroprotection.     

Redox state of low-molecular-weight thiols and disulphides during somatic embryogenesis of salt-treated suspension cultures of Dactylis glomerata L

The tripeptide antioxidant γ-L-glutamyl-L-cysteinyl-glycine, or glutathione (GSH), serves a central role in ROS scavenging and oxidative signalling. Here, GSH, glutathione disulphide (GSSG), and other low-molecular-weight (LMW) thiols and their corresponding disulphides were studied in embryogenic suspension cultures of Dactylis glomerata L. subjected to moderate (0.085 M NaCl) or severe (0.17 M NaCl) salt stress. Total glutathione (GSH + GSSG) concentrations and redox state were associated with growth and development in control cultures and in moderately salt-stressed cultures and were affected by severe salt stress. The redox state of the cystine (CySS)/2 cysteine (Cys) redox couple was also affected by developmental stage and salt stress. The glutathione half-cell reduction potential (E(GSSG/2 GSH)) increased with the duration of culturing and peaked when somatic embryos were formed, as did the half-cell reduction potential of the CySS/2 Cys redox couple (E(CySS/2 Cys)). The most noticeable relationship between cellular redox state and developmental state was found when all LMW thiols and disulphides present were mathematically combined into a 'thiol-disulphide redox environment' (E(thiol-disulphide)), whereby reducing conditions accompanied proliferation, resulting in the formation of pro-embryogenic masses (PEMs), and oxidizing conditions accompanied differentiation, resulting in the formation of somatic embryos. The comparatively high contribution of E(CySS/2 Cys) to E(thiol-disulphide) in cultures exposed to severe salt stress suggests that Cys and CySS may be important intracellular redox regulators with a potential role in stress signalling.     

Some properties of mitochondrial glutathione.

1. The presence of GSH in rat liver mitochondria is confirmed. GSH diffuses from the suspended particles in the presence of phosphate but respiratory inhibitors inhibit the diffusion. 2. GSH is oxidised in situ by oxidants including t-butyl hydroperoxide. The products formed include GSSG and GSS-protein mixed disulphides. The oxidation occurs at lower oxidant concentrations if phosphate or oxaloacetate are also present. Respiratory inhibitors abolish their effect. 3. With phosphate, the GSSG produced is found chiefly outside the mitochondria whereas with oxaloacetate, it is found chiefly inside. 4. The GSSG formed by the oxidation is reduced by Krebs-cycle acids with the exception of the ketoacids. Exogenous GSSG is reduced by these substrates only after lysis. Intact particles, however, catalyse the reduction of GSSG by either NADH2 or NADPH2.     

The mechanism of disulphide reduction by mitochondria

1. Cystamine was reduced to the corresponding thiol by rat liver mitochondria, even in the presence of rotenone or antimycin A. 2. The reduction of disulphides was stimulated by the accumulation of NADH or by the addition of NADH to osmotically ;shocked' mitochondria. 3. Energy made available by oxidative phosphorylation was not essential for the reduction of disulphides. 4. Cystamine was not reduced during the oxidation of NADH by ultrasonically treated particles, which had lost their capacity for oxidation of alpha-oxo acids. 5. In intact mitochondria, arsenite and other inhibitors of vicinal dithiols caused a decrease in the capacity for reduction of disulphides concomitantly with an inhibition of the oxidation of alpha-oxo acids. 6. Isolated lipoamide dehydrogenase reduced cystamine at the expense of NADH, provided that lipoic acid was also present. 7. It is concluded that in mitochondria the reduction of cystamine and related disulphides is probably brought about by interaction with reduced lipoic acid, generated by the alpha-oxo acid dehydrogenase complexes during the oxidation of alpha-oxo acids or by reaction of lipoamide dehydrogenase with NADH.     

Useful agents for the study of glutathione metabolism in erythrocytes. Organic hydroperoxides

t-Butyl hydroperoxide and cumene hydroperoxide, both known to be substrates for glutathione peroxidase, were used to oxidize erythrocyte GSH. Addition of concentrations of hydroperoxides equimolar with respect to GSH in the erythrocytes or whole blood quantitatively oxidizes GSH in the erythrocytes with a half-time of 4.5s at 37 degrees C and about three times as long at 4 degrees C. In the presence of glucose, normal erythrocytes regenerate all the GSH in about 25min. However, glucose 6-phosphate dehydrogenase-deficient erythrocytes failed to regenerate GSH. Treatment of erythrocytes with hydroperoxides does not affect erythrocyte survival in rabbits. Oxidation of erythrocyte GSH with equimolar concentrations of hydroperoxides does not lead to formation of mixed disulphides of haemoglobin and GSH. The hydroperoxides do not affect erythrocyte glycolytic and hexose monophosphate-shunt-pathway enzymes. Previous studies on transport of GSSG from erythrocytes were confirmed by using t-butyl hydroperoxide to oxidize erythrocyte GSH.     

Studies on the fungitoxicity of captan

The cellular distribution of ³⁵S after incubating labelled captan with Neurospora crassa conidia has been determined. Nearly all the ³⁵S in the spores is bound to the water-soluble and protein fractions. Thin-layer chromatography of the hot-water extract of spores has shown that ³⁵S occurs largely in oxidized glutathione (GSSG) and in a product tentatively identified as a thiazolidine derivative of glutathione. It is suggested that this derivative, which only forms above pH 6·5, is produced by reaction between glutathione (GSH) and the thiocarbonyl chloride liberated on the decomposition of captan. Captan toxicity could not be completely reversed by pretreatment with thiols and disulphides capable of penetrating the cell membrane, confirming the previous hypothesis that fungitoxicity is due to irreversible changes following the oxidation of the protein thiols to disulphides.     

Changes in low-molecular-weight thiol-disulphide redox couples are part of bread wheat seed germination and early seedling growth

The tripeptide antioxidant glutathione (γ-l-glutamyl-l-cysteinyl-glycine; GSH) essentially contributes to thiol-disulphide conversions, which are involved in the control of seed development, germination, and seedling establishment. However, the relative contribution of GSH metabolism in different seed structures is not fully understood. We studied the GSH/glutathione disulphide (GSSG) redox couple and associated low-molecular-weight (LMW) thiols and disulphides related to GSH metabolism in bread wheat (Triticum aestivum L.) seeds, focussing on redox changes in the embryo and endosperm during germination. In dry seeds, GSH was the predominant LMW thiol and, 15?h after the onset of imbibition, embryos of non-germinated seeds contained 12 times more LMW thiols than the endosperm. In germinated seeds, the embryo contained 17 and 11 times more LMW thiols than the endosperm after 15 and 48?h, respectively. This resulted in the embryo having significantly more reducing half-cell reduction potentials of GSH/GSSG and cysteine (Cys)/cystine (CySS) redox couples (E_GSSG/2GSH and E_CySS/2Cys, respectively). Upon seed germination and early seedling growth, Cys and CySS concentrations significantly increased in both embryo and endosperm, progressively contributing to the cellular LMW thiol-disulphide redox environment (E_{thiol-disulphide}). The changes in E_CySS/2Cys could be related to the mobilisation of storage proteins in the endosperm during early seedling growth. We suggest that E_GSSG/2GSH and E_CySS/2Cys can be used as markers of the physiological and developmental stage of embryo and endosperm. We also present a model of interaction between LMW thiols and disulphides with hydrogen peroxide (H₂O₂) in redox regulation of bread wheat germination and early seedling growth.     

Chemical reactivity of some isothiazolone biocides

Chemical reactions between the isothiazolone biocides, N-methylisothiazol-3-one (MIT), benzisothiazol-3-one (BIT) and 5-chloro-N-methylisothiazol-3-one (CMIT) with cysteine have been investigated by u.v. and NMR spectroscopy. At physiological pH all three agents interacted oxidatively with thiols to form disulphides. Further interaction with thiols caused the release of cystine and formation of a reduced, ring-opened form of the biocide (mercaptoacrylamide). In an analogous fashion to the initial reaction the mercaptoacrylamide reacted with another molecule of biocide to give biocide dimers. NMR spectral studies indicated that for CMIT the mercaptoacrylamide form is capable of tautomerization to a highly reactive thio-acyl chloride. Formation of mercaptoacrylamide was in all cases highly pH-dependent. Alcohol dehydrogenase was insensitive to all three agents but was highly sensitive to CMIT when co-administered with dithiothreitol. Capacity to form a thioacyl chloride from the mercaptoacrylamide is suggested to account for much of this enhanced activity. Stopped-flow spectroscopic studies showed rates of reaction with glutathione (GSH) to directly parallel antimicrobial activity. Additionally, CMIT was able to react directly with both ionization states of GSH (pH 7-10) whilst BIT and MIT appeared only to interact when the glutamyl-nitrogen of GSH was charged (pH 8.5).     

Redox state of low-molecular-weight thiols and disulphides during somatic embryogenesis of salt-treated suspension cultures of Dactylis glomerata L.

Abstract

The tripeptide antioxidant γ-L-glutamyl-L-cysteinyl-glycine, or glutathione (GSH), serves a central role in ROS scavenging and oxidative signalling. Here, GSH, glutathione disulphide (GSSG), and other low-molecular-weight (LMW) thiols and their corresponding disulphides were studied in embryogenic suspension cultures of Dactylis glomerata L. subjected to moderate (0.085 M NaCl) or severe (0.17 M NaCl) salt stress. Total glutathione (GSH + GSSG) concentrations and redox state were associated with growth and development in control cultures and in moderately salt-stressed cultures and were affected by severe salt stress. The redox state of the cystine (CySS)/2 cysteine (Cys) redox couple was also affected by developmental stage and salt stress. The glutathione half-cell reduction potential (E_{GSSG/2 GSH}) increased with the duration of culturing and peaked when somatic embryos were formed, as did the half-cell reduction potential of the CySS/2 Cys redox couple (E_{CySS/2 Cys}). The most noticeable relationship between cellular redox state and developmental state was found when all LMW thiols and disulphides present were mathematically combined into a ‘thiol–disulphide redox environment’ (E_{thiol–disulphide}), whereby reducing conditions accompanied proliferation, resulting in the formation of pro-embryogenic masses (PEMs), and oxidizing conditions accompanied differentiation, resulting in the formation of somatic embryos. The comparatively high contribution of E_{CySS/2 Cys} to E_{thiol–disulphide} in cultures exposed to severe salt stress suggests that Cys and CySS may be important intracellular redox regulators with a potential role in stress signalling.     

Investigation of the transport of intact glutathione in human and rat type II pneumocytes

The aim of the study was to investigate whether there is transmembrane transport of intact glutathione ([³H]-GSH, 0.1 μCi) in rat and human type II pneumocytes (T2P), and if this transport might be dependent on the redox state of the extracellular fluid. The T2P were pretreated with acivicin (250 μM) to inhibit γ-glutamyl-transferase activity and with L-buthionine-[SR]-sulfoximine (1 mM) to inhibit intracellular GSH synthesis. After 48 h in culture, initial GSH influx rate was 0.70 ± 0.20 nmol/min/mg protein (37°C) and 0.35 ± 0.04 nmol/min/mg protein (4°C) during the first 5 min in rat T2P. In human T2P, the initial GSH influx rate was 0.36 ± 0.30 nmol/min/mg protein (37°C) and 0.32 ± 0.06 nmol/min/mg protein (4°C) during the first 10 min. Thereafter no further influx was found. The influx of 1 mM GSH in freshly isolated rat and human T2P in suspension was 2.3 ± 0.3 and 1.2 ± 0.3 nmol/mg protein after 15 min at 37°C, and 2.8 ± 0.2 and 1.0 ± 0.3 nmol/mg protein at 4°C, respectively. When GSH influx was studied at different concentrations between 0 and 40 mM, a linear increase without saturation or difference between 37°C and 4°C was found. Preexposure to ouabain had no effect on GSH influx. Efflux of GSH was stimulated and influx inhibited by preexposure of the cells to reduced thiols, while disulphides inhibited efflux and favoured inward uptake. Thus, in human and rat T2P a GSH-carrier exists which operates as an effluxer. At GSH concentrations in the physiological range no uptake is seen, but some uptake can be observed at GSH concentrations above normal physiological levels. The uptake appears to be energy-independent and non-saturable. Efflux of GSH is stimulated and influx inhibited by reduced thiols, while disulphides inhibit the efflux and favour inward uptake. GSH uptake in T2P thus may depend on concentration gradients and driving forces, such as the redox state of the extracellular fluid.     

Investigation of the transport of intact glutathione in human and rat type II pneumocytes

The aim of the study was to investigate whether there is transmembrane transport of intact glutathione ([³H]-GSH, 0.1 μCi) in rat and human type II pneumocytes (T2P), and if this transport might be dependent on the redox state of the extracellular fluid. The T2P were pretreated with acivicin (250 μM) to inhibit γ-glutamyl-transferase activity and with L-buthionine-[SR]-sulfoximine (1 mM) to inhibit intracellular GSH synthesis. After 48 h in culture, initial GSH influx rate was 0.70 ± 0.20 nmol/min/mg protein (37°C) and 0.35 ± 0.04 nmol/min/mg protein (4°C) during the first 5 min in rat T2P. In human T2P, the initial GSH influx rate was 0.36 ± 0.30 nmol/min/mg protein (37°C) and 0.32 ± 0.06 nmol/min/mg protein (4°C) during the first 10 min. Thereafter no further influx was found. The influx of 1 mM GSH in freshly isolated rat and human T2P in suspension was 2.3 ± 0.3 and 1.2 ± 0.3 nmol/mg protein after 15 min at 37°C, and 2.8 ± 0.2 and 1.0 ± 0.3 nmol/mg protein at 4°C, respectively. When GSH influx was studied at different concentrations between 0 and 40 mM, a linear increase without saturation or difference between 37°C and 4°C was found. Preexposure to ouabain had no effect on GSH influx. Efflux of GSH was stimulated and influx inhibited by preexposure of the cells to reduced thiols, while disulphides inhibited efflux and favoured inward uptake. Thus, in human and rat T2P a GSH-carrier exists which operates as an effluxer. At GSH concentrations in the physiological range no uptake is seen, but some uptake can be observed at GSH concentrations above normal physiological levels. The uptake appears to be energy-independent and non-saturable. Efflux of GSH is stimulated and influx inhibited by reduced thiols, while disulphides inhibit the efflux and favour inward uptake. GSH uptake in T2P thus may depend on concentration gradients and driving forces, such as the redox state of the extracellular fluid.     

The formation of mixed disulphides in rat lung following paraquat administration Correlation with changes in intermediary metabolism

We have investigated the hypothesis that the formation of mixed disulphides between protein sulphydryl and glutathione may be responsible for controlling the activity of the pentose phosphate pathway and fatty acid synthesis in rat lung. Using lung slices, taken from rats 2 h after dosing with a range of concentrations (5–80 mg/kg) of the pulmonary toxin paraquat, the pentose phosphate pathway was found to be stimulated in direct proportion to a reduction in fatty acid synthesis. These effects were also linearly related to an increase in mixed (total) disulphide levels in the lung. This was quantitatively similar to an increase in mixed (glutathione) disulphides, although non-protein sulphydryl and oxidised levels remained normal. Thus, an early biochemical event in the mechanism of paraquat toxicity in the lung involves an increased formation of mixed (glutathione) disulphides and simulatneous regulation of pentose phosphate pathway activity and fatty acid synthesis. These data support the concept that the formation of mixed disulphides of protein and glutathione is a mechanism for maintaining NADPH levels despite the ‘redox’ stress caused by the cyclical and NADPH dependent reduction and reoxidation of paraquat.     

Heme Inhibition of [delta]-Aminolevulinic Acid Synthesis Is Enhanced by Glutathione in Cell-Free Extracts of Chlorella

In plants, algae, and many bacteria, the heme and chlorophyll precursor, [delta]-aminolevulinic acid (ALA), is synthesized from glutamate in a reaction involving a glutamyl-tRNA intermediate and requiring ATP and NADPH as cofactors. In particulate-free extracts of algae and chloroplasts, ALA synthesis is inhibited by heme. Inclusion of 1.0 mM glutathione (GSH) in an enzyme and tRNA extract, derived from the green alga Chlorella vulgaris, lowered the concentration of heme required for 50% inhibition approximately 10-fold. The effect of GSH could not be duplicated with other reduced sulfhydryl compounds, including mercaptoethanol, dithiothreitol, and cysteine, or with imidazole or bovine serum albumin, which bind to heme and dissociate heme dimers. Absorption spectroscopy indicated that heme was fully reduced in incubation medium containing dithiothreitol, and addition of GSH did not alter the heme reduction state. Oxidized GSH was as effective in enhancing heme inhibition as the reduced form. Co-protoporphyrin IX inhibited ALA synthesis nearly as effectively as heme, and 1.0 mM GSH lowered the concentration required for 50% inhibition approximately 10-fold. Because GSH did not influence the reduction state of heme in the incubation medium, and because GSH could not be replaced by other reduced sulfhydryl compounds or ascorbate, the effect of GSH cannot be explained by action as a sulfhydryl protectant or heme reductant. Preincubation of enzyme extract with GSH, followed by rapid gel filtration, could not substitute for inclusion of GSH with heme during the reaction. The results suggest that GSH must specifically interact with the enzyme extract in the presence of the inhibitor to enhance the inhibition.     

GSH depletion, protein S-glutathionylation and mitochondrial transmembrane potential hyperpolarization are early events in initiation of cell death induced by a mixture of isothiazolinones in HL60 cells

We recently described that brief exposure of HL60 cells to a mixture of 5-chloro-2-methyl-4-isothiazolin-3-one (CMI) and 2-methyl-4-isothiazolin-3-one (MI) induces apoptosis at low concentrations (0.001-0.01%) and necrosis at higher concentrations (0.05-0.1%). In this study, we show that glutathione (GSH) depletion, reactive oxygen species generation, hyperpolarization of mitochondrial transmembrane potential (DeltaPsim) and formation of protein-GSH mixed disulphides (S-glutathionylation) are early molecular events that precede the induction of cell death by CMI/MI. When the cells exhibit common signs of apoptosis, they show activation of caspase-9, reduction of DeltaPsim and, more importantly, decreased protein S-glutathionylation. In contrast, necrosis is associated with severe mitochondrial damage and maximal protein S-glutathionylation. CMI/MI-induced cytotoxicity is also accompanied by decreased activity of GSH-related enzymes. Pre-incubation with L-buthionine-(S,R)-sulfoximine (BSO) clearly switches the mode of cell death from apoptosis to necrosis at 0.01% CMI/MI. Collectively, these results demonstrate that CMI/MI alters the redox status of HL60 cells, and the extent and kinetics of GSH depletion and S-glutathionylation appear to determine whether cells undergo apoptosis or necrosis. We hypothesize that S-glutathionylation of certain thiol groups accompanied by GSH depletion plays a critical role in the molecular mechanism of CMI/MI cytotoxicity.     

Generation of PM2 DNA breaks in the course of reduction of chromium(VI) by glutathione

The carcinogen chromate is efficiently taken up and reduced to chromium(III) compounds by various biological systems. To test the possible DNA damage induced in the course of chromium(VI) reduction, we used a combination of chromate with the reductant glutathione (GSH) as well as a green complex of chromium(V), which is formed in the reaction of chromate with GSH. The combination of chromate and glutathione was found to cause single-strand breaks in supercoiled circular DNA of the bacteriophage PM2. The green chromium(V) complex Na4(GSH)4Cr(V).8H2O, prepared from chromate and glutathione, also cleaved supercoiled PM2 DNA. No DNA-degrading effects were observed with either chromate or the final product of the reaction with GSH, a purple anionic chromium(III) GSH complex. The nature of the buffering agents revealed a strong influence on the extent of DNA strand breaks produced by chromate and GSH. A variation of the GSH concentration in the reaction with chromate and PM2 DNA, performed in sodium phosphate-buffered solutions showed an initial increase in the number of strand breaks at GSH concentrations up to 1 mM followed by a decline at higher GSH concentrations. Since neither chromate, when administered individually, nor the final product of chromium(VI) reduction, the purple chromium(III) GSH complex, produced any detectable DNA cleavage, the critical steps leading to DNA strand breaks occur in the course of the conversion of chromium(VI) to chromium(III) by GSH, the most abundant intracellular low molecular thiol. Moreover, the demonstration that DNA cleavage is induced in the presence of the chromium(V) complex identifies chromium(V) as the oxidation state of the metal, which is involved in the steps leading to DNA-damaging effects of chromate.     

Kinetics of inhibition of aldehyde dehydrogenase isoenzymes of rat liver by fluorine-containing disulfides

New disulphides synthesized on the basis of dithiocarboxylic acid derivatives and heterocyclic thiols containing the fluorine atoms were studied as applied to inhibit aldehyde dehydrogenase (ALDH) isozymes of the rat liver mitochondria. The most effective rat liver inhibitors of ALDH isozymes were revealed. Inhibition of the rat liver isozymes by disulphides I, II, IV, VI-VIII and fluorinated pyridine disulphide was found to be irreversible. The values of isozyme inactivation rate constants are reported. The ALDH inhibition by disulphides I, IV, VI-VIII was competitive both for the cofactor and for the substrate of the reaction. The protective effect of the NAD+ against ALDH I and II inactivation by disulfiram and disulphides I, IV, VI-VIII and X is shown. NADP+ protects isozyme II against inactivation by disulfiram and also disulphides I, VI-VIII.     

Mechanism of the reaction catalyzed by dehydroascorbate reductase from spinach chloroplasts.

Dehydroascorbate reductase (DHAR) reduces dehydroascorbate (DHA) to ascorbate with glutathione (GSH) as the electron donor. We analyzed the reaction mechanism of spinach chloroplast DHAR, which had a much higher reaction specificity for DHA than animal enzymes, using a recombinant enzyme expressed in Escherichia coli. Kinetic analysis suggested that the reaction proceeded by a bi-uni-uni-uni-ping-pong mechanism, in which binding of DHA to the free, reduced form of the enzyme was followed by binding of GSH. The Km value for DHA and the summed Km value for GSH were determined to be 53 +/- 12 micro m and 2.2 +/- 1.0 mm, respectively, with a turnover rate of 490 +/- 40 s-1. Incubation of 10 microm DHAR with 1 mm DHA and 10 microm GSH resulted in stable binding of GSH to the enzyme. Bound GSH was released upon reduction of the GSH-enzyme adduct by 2-mercaptoethanol, suggesting that the adduct is a reaction intermediate. Site-directed mutagenesis indicated that C23 in DHAR is indispensable for the reduction of DHA. The mechanism of catalysis of spinach chloroplast DHAR is proposed.     

Role of glutathione reductase system in disulfiram conversion to diethyldithiocarbamate.

Experiments were carried out to establish the role of glutathione reductase (GR), if any, in the metabolic conversion of disulfiram (DS) to diethyldithiocarbamate (DDC). It was observed that, under standard assay conditions, whereas DS was incorporated as a substrate instead of oxidised glutathione (GSSG), the enzymes from both human liver extract and yeast sources failed to reduce the parent compound, implying that glutathione reductase perse do not reduce disulfiram. However, the incorporation of disulfiram into an assay system comprising of GSSG, NADPH and reductase resulted in DS reduction to DDC. Further, the observation, that the GR assay system devoid of either GSSG or NADPH was found to lack DS reducing ability, implies that GSH as a reaction product of GR system is responsible for the reduction of DS to DDC. The results of in-vitro experiments indicated that GSH perse could reduce DS to DDC nonenzymatically, with a stoichiometric relationship of 2:1. Thus it is inferred that GR perse do not reduce DS, whereas GSH, as an intermediary metabolite of GR system, brings about non-enzymatic reduction of DS via a sulfhydral group exchange reaction.     

Actin S-glutathionylation: evidence against a thiol-disulphide exchange mechanism

Many proteins, including actin, are targets for S-glutathionylation, the reversible formation of mixed disulphides between protein cysteinyl thiol groups and glutathione (GSH) that can be induced in cells by oxidative stress. Proposed mechanisms of protein S-glutathionylation follow mainly two distinct pathways. One route involves the initial oxidative modification of a reduced protein thiol to an activated protein, which may then react with GSH to the mixed disulphide. The second route involves the oxidative modification of GSH to an activated form such as glutathione disulphide (GSSG), which may then react with a reduced protein thiol, yielding the corresponding protein mixed disulphide. We show here that physiological levels of GSSG induce a little extent of actin S-glutathionylation. Instead, actin with the exposed cysteine thiol activated by diamide or 5,5'-dithiobis(2-nitrobenzoic acid) reacts with physiological levels of GSH, incorporating about 0.7 mol GSH/mol protein. Differently, an extremely high concentration of GSSG induces an increased level of S-glutathionylation that causes a 50% inhibition in actin polymerization not reversed by dithiotreitol. In mammalian cells, GSH is present in millimolar concentrations and is in about 100-fold excess over GSSG. The high concentration of GSSG required for obtaining a significant actin S-glutathionylation as well as attendant irreversible changes in protein functions make unlikely that actin may be S-glutathionylated by a thiol-disulphide exchange mechanism within the cell.     

Bacterial conjugation: a two-step mechanism for DNA transport

Ten years ago it was thought that disulphide bond formation in prokaryotes occurred spontaneously. Now two pathways involved in disulphide bond formation have been well characterized, the oxidative pathway, which is responsible for the formation of disulphides, and the isomerization pathway, which shuffles incorrectly formed disulphides. Disulphide bonds are donated directly to unfolded polypeptides by the DsbA protein; DsbA is reoxidized by DsbB. DsbB generates disulphides de novo from oxidized quinones. These quinones are reoxidized by the electron transport chain, showing that disulphide bond formation is actually driven by electron transport. Disulphide isomerization requires that incorrect disulphides be attacked using a reduced catalyst, followed by the redonation of the disulphide, allowing alternative disulphide pairing. Two isomerases exist in Escherichia coli, DsbC and DsbG. The membrane protein DsbD maintains these disulphide isomerases in their reduced and thereby active form. DsbD is kept reduced by cytosolic thioredoxin in an NADPH-dependent reaction.