The novel disulfide reductase bis-gamma-glutamylcystine reductase and dihydrolipoamide dehydrogenase from Halobacterium halobium: purification by immobilized-metal-ion affinity chromatography and properties of the enzymes.

An NADPH-specific disulfide reductase that is active with bis-gamma-glutamylcystine has been purified 1,900-fold from Halobacterium halobium to yield a homogeneous preparation of the enzyme. Purification of this novel reductase, designated bis-gamma-glutamylcystine reductase (GCR), and purification of halobacterial dihydrolipoamide dehydrogenase (DLD) were accomplished with the aid of immobilized-metal-ion affinity chromatography in high-salt buffers. Chromatography of GCR on immobilized Cu2+ resin in buffer containing 1.23 M (NH4)2SO4 and on immobilized Ni2+ resin in buffer containing 4.0 M NaCl together effected a 120-fold increase in purity. Native GCR was found to be a dimeric flavoprotein of Mr 122,000 and to be more stable to heat when in buffer of very high ionic strength. DLD was chromatographed on columns of immobilized Cu2+ resin in buffer containing NaCl and in buffer containing (NH4)2SO4, the elution of DLD differing markedly in the two buffers. Purified DLD was found to be a heat-stable, dimeric flavoprotein of Mr 120,000 and to be very specific for NAD. The utility of immobilized-metal-ion affinity chromatography for the purification of halobacterial enzymes and the likely cellular function of GCR are discussed.     

Glutathione is required for growth and prespore cell differentiation in Dictyostelium

Glutathione (GSH) is the most abundant non-protein thiol in eukaryotic cells and acts as reducing equivalent in many cellular processes. We investigated the role of glutathione in Dictyostelium development by disruption of gamma-glutamylcysteine synthetase (GCS), an essential enzyme in glutathione biosynthesis. GCS-null strain showed glutathione auxotrophy and could not grow in medium containing other thiol compounds. The developmental progress of GCS-null strain was determined by GSH concentration contained in preincubated media before development. GCS-null strain preincubated with 0.2 mM GSH was arrested at mound stage or formed bent stalk-like structure during development. GCS-null strain preincubated with more than 0.5 mM GSH formed fruiting body with spores, but spore viability was significantly reduced. In GCS-null strain precultured with 0.2 mM GSH, prestalk-specific gene expression was delayed, while prespore-specific gene and spore-specific gene expressions were not detected. In addition, GCS-null strain precultured with 0.2 mM GSH showed prestalk tendency and extended G1 phase of cell cycle. Since G1 phase cells at starvation differentiate into prestalk cells, developmental defect of GCS-null strain precultured with 0.2 mM GSH may result from altered cell cycle. These results suggest that glutathione itself is essential for growth and differentiation to prespore in Dictyostelium.     

FAD analogues as prosthetic groups of human glutathione reductase. Properties of the modified enzyme species and comparisons with the active site structure

Human glutathione reductase (NADPH + GSSG + H+ in equilibrium with NADP+ + 2 GSH) is a suitable enzyme for correlating spectroscopic properties and chemical reactivities of protein-bound FAD analogues with structural data. FAD, the prosthetic group of the enzyme, was replaced by FAD analogues, which were modified at the positions 8, 1, 2, 4, 5 and 6, respectively, of the isoalloxazine ring. When compared with a value of 100% for native glutathione reductase, the specific activities of most enzyme species ranged from 40% to 17%, in the order of the prosthetic groups 8-mercapto-FAD greater than 8-azido-FAD = 8-F-FAD = 8-C1-FAD greater than 4-thio-FAD = 1-deaza-FAD greater than 2-thio-FAD. The enzymic activities indicate a correct orientation of the bound analogues. The enzyme species containing 5-deaza-FAD and 6-OH-FAD, respectively, had no more glutathione reductase activity than the FAD-free apoenzyme. 5-Deaza-FAD X glutathione reductase was crystallized for X-ray diffraction analysis. Detailed studies were focussed on position 8 of the flavin. 8-Cl-FAD X glutathione reductase and 8-F-FAD X glutathione reductase reacted only poorly with HS- to give 8-mercapto-FAD X glutathione reductase, which suggests that the region around Val61 hinders the halogen anion from leaving the tetrahedral intermediate. Other experiments showed that position 8 is accessible to certain solvent-borne reagents. 8-Mercapto-FAD X glutathione reductase, for instance, reacted readily and stoichiometrically with the thiol reagent methylmethanethiosulfonate. 8-Mercapto-FAD X glutathione reductase does not exhibit a long wavelength charge transfer absorption band upon reduction, as it is the case for the 2-electron-reduced FAD-containing enzyme. This behaviour indicates that the charge transfer interaction between flavin and the thiolate of Cys63 in the native enzyme is not per se essential for catalysis. The absorption spectrum of the blue anionic 8-mercapto-FAD bound to glutathione reductase suggests that the protein concurs to the stabilization of a negative charge in the pyrimidine subnucleus. In light of the protein structure this effect is attributed to the dipole moment of alpha-helix 338-354 which starts out close to the N(1)/C(2)/O(2 alpha) region of the flavin. 1-Deaza-FAD binds as tightly as FAD to the apoenzyme. The resulting holoenzyme was found to be enzymically active but structurally unstable. In this respect 1-deaza-FAD . glutathione reductase mimics the properties of the enzyme species found in inborn glutathione reductase deficiency.(ABSTRACT TRUNCATED AT 400 WORDS)     

Elevated glutathione biosynthetic capacity in the chloroplasts of transgenic tobacco plants paradoxically causes increased oxidative stress

Glutathione (GSH), a major antioxidant in most aerobic organisms, is perceived to be particularly important in plant chloroplasts because it helps to protect the photosynthetic apparatus from oxidative damage. In transgenic tobacco plants overexpressing a chloroplast-targeted gamma-glutamylcysteine synthetase (gamma-ECS), foliar levels of GSH were raised threefold. Paradoxically, increased GSH biosynthetic capacity in the chloroplast resulted in greatly enhanced oxidative stress, which was manifested as light intensity-dependent chlorosis or necrosis. This phenotype was associated with foliar pools of both GSH and gamma-glutamylcysteine (the immediate precursor to GSH) being in a more oxidized state. Further manipulations of both the content and redox state of the foliar thiol pools were achieved using hybrid transgenic plants with enhanced glutathione synthetase or glutathione reductase activity in addition to elevated levels of gamma-ECS. Given the results of these experiments, we suggest that gamma-ECS-transformed plants suffered continuous oxidative damage caused by a failure of the redox-sensing process in the chloroplast.     

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.     

Coordinate induction of glutathione biosynthesis and glutathione-metabolizing enzymes is correlated with salt tolerance in tomato

The acclimation of reduced glutathione (GSH) biosynthesis and GSH-utilizing enzymes to salt stress was studied in two tomato species that differ in stress tolerance. Salt increased GSH content and GSH:GSSG (oxidized glutathione) ratio in oxidative stress-tolerant Lycopersicon pennellii (Lpa) but not in Lycopersicon esculentum (Lem). These changes were associated with salt-induced upregulation of gamma-glutamylcysteine synthetase protein, an effect which was prevented by preincubation with buthionine sulfoximine. Salt treatment induced glutathione peroxidase and glutathione-S-transferase but not glutathione reductase activities in Lpa. These results suggest a mechanism of coordinate upregulation of synthesis and metabolism of GSH in Lpa, that is absent from Lem.     

Thiol-disulfide exchanges modulate aldo–keto reductase family 1 member B10 activity and sensitivity to inhibitors

The reversible thiol/disulfide exchange is an important regulatory mechanism of protein enzymatic activity. Many protein enzymes are susceptible to S-thiolation induced by reactive oxygen species (ROS); and the glutathione (GSH) and free amino acid cysteine (Cys) are critical cellular thiol anti-oxidants, protecting proteins from irreversible oxidative damage. In this study, we found that aldo–keto reductase family 1 member B10 (AKR1B10) contains 4 Cys residues, i.e., Cys45, Cys187, Cys200, and Cys299. Exposing AKR1B10 to ROS mixtures resulted in significant decrease of its free sulfhydryl groups, up to 40–50% in the presence of physiological thiol cysteine at 0.5 or 1.0 mM; and accordingly, AKR1B10 enzymatic activity was reversibly decreased, in parallel with the oxidation of the sulfhydryl groups. ROS-induced thiolation also affected the sensitivity of AKR1B10 to inhibitors EBPC, epalrestat, and statil. Together our results showed for the first time that AKR1B10's enzymatic activity and inhibitor sensitivity are modulated by thiol/disulfide exchanges.     

Evolution of antioxidant mechanisms: Thiol-Dependent peroxidases and thioltransferase among procaryotes

Summary Glutathione peroxidase and glutathione S-transferase both utilize glutathione (GSH) to destroy organic hydroperoxides, and these enzymes are thought to serve an antioxidant function in mammalian cells by catalyzing the destruction of lipid hydroperoxides. Only two groups of procaryotes, the purple bacteria and the cyanobacteria, produce GSH, and we show in the present work that representatives from these two groups (Escherichia coli, Beneckea alginolytica, Rhodospirillum rubrum, Chromatium vinosum, andAnabaena sp. strain 7119) lack significant glutathione peroxidase and glutathione S-transferase activities. This finding, coupled with the general absence of polyunsaturated fatty acids in procaryotes, suggests that GSH-dependent peroxidases evolved in eucaryotes in response to the need to protect against polyunsaturated fatty acid oxidation. A second antioxidant function of GSH is mediated by glutathione thiol-transferase, which catalyzes the reduction of various cellular disulfides by GSH. Two of the five GSH-producing bacteria studied (E. coli andB. alginolytica) produced higher levels of glutathione thiol-transferase than found in rat liver, whereas the activity was absent in the other three species studied. The halobacteria produced γ-glutamylcysteine rather than GSH, and assays for γ-glutamylcysteine-dependent enzymes demonstrated an absence of peroxidase and S-transferase activities but the presence of significant thioltransferase activity. Based upon these results it appears that GSH and γ-glutamylcysteine do not function in bactera as antioxidants directed against organic hydroperoxides but do play a significant, although not universal, role in main-taining disulfides in a reduced state. The function of GSH in the photosynthetic bacteria, aside from providing a form of cysteine resistant toward autoxidation, remains a puzzle, as none of the GSH-dependent enzymes tested other than glutathione reductase were present in these organisms.     

24-Epibrassinolide maintains elevated redox state of AsA and GSH in radish (Raphanus sativus L.) seedlings under zinc stress

Exogenous-applied 24-epibrassinolide (EBR) increased the seedling growth of radish (Raphanus sativus L.) in terms of seedling length, fresh weight and dry weight both in zinc (Zn²⁺)-stressed and unstressed conditions. Moreover, EBR lowered the Zn²⁺ uptake and bioaccumulation. Increased oxidation of ascorbate (AsA) and glutathione (GSH) pools to dehydroascorbate and glutathione disulfide respectively was observed in Zn²⁺-stressed seedlings, a clear indication of oxidative stress. However, exogenous application of EBR to stressed seedlings inhibited the oxidation of ascorbate and glutathione, maintaining redox molecules in reduced form. Under Zn²⁺ stress, enzymatic activities of ascorbate–glutathione cycle such as ascorbate peroxidase, monodehydroascorbate reductase increased but the dehydroascorbate reductase, glutathione reductase decreased. Zn²⁺ stress induced the gamma-glutamylcysteine synthetase, and glutathione-s-transferase activities in radish seedlings were further enhanced with EBR application. Zn²⁺ toxicity decreased the thiol content but, EBR supplementation resulted in restoration of thiol pool. The results of present study clearly demonstrated that external application of EBR modulates the AsA and GSH redox status to combat the oxidative stress of Zn²⁺ in seedlings via the AsA–GSH cycle and glutathione metabolism as an antioxidant defense system.     

Purification and properties of the glutathione reductase of Chromatium vinosum.

The Chromatium vinosum glutathione reductase [NAD(P)H: glutathione disulfide oxidoreductase, EC 1.6.4.2] was purified to apparent homogeneity. The enzyme was found to require reduced nicotinamide adenine dinucleotide (NADH) as a reductant and to be specific for oxidized glutathione (GSSG). The polypeptide molecular weight in sodium dodecyl sulfate was found to be 52,000. Incubation of enzyme with NADH in the absence of GSSG resulted in a significant loss in activity. The enzyme was stimulated by phosphate and sulfate ion, but was inhibited by chloride ion, heavy metals, and sulfhydryl reagents. Adenylate nucleotides were inhibitory, and the data suggested that they were acting as competitive inhibitors of flavin adenine dinucleotide (FAD). The Km values of 7 X 10-3 for GSSG and 6 X 10-5 M for NADH were the highest reported of any previously investigated glutathione reductase. The order of addition of components markedly affected the response of the enzyme to FAD. A requirement for FAD (Km 5.2 X 10-7 M) was seen if the enzyme was incubated with NADH prior to GSSG addition, whereas no FAD was required if the order was reversed.     

Allosteric and non-allosteric forms of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase: differential inhibition of activity by adenosine 2'-monophospho-5'-diphosphoribose

Adenosine 2'-monophospho-5'-diphosphoribose (P-ADP-Rib) is a structural analog of NADPH which was reported to competitively inhibit (Kiapp = 21.7 microM) solubilized rat liver 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (Tanazawa, K., and A. Endo. 1979. Eur. J. Biochem. 98: 195-201). However, microsomal HMG-CoA reductase, which at low thiol concentrations exhibits allosteric properties, is only poorly inhibited by P-ADP-Rib (Kiapp = 550 microM at 4.5 mM GSH). Gradual shift of the microsomal reductase towards a non-allosteric form by increasing glutathione (GSH) concentrations resulted in a higher inhibition by P-ADP-Rib. Under these conditions, Ki values for P-ADP-Rib were 165 microM and 53 microM at 9 mM and 27 mM GSH, respectively. The largest change in the degree of inhibition by P-ADP-Rib was observed within the 10 mM range of GSH. By contrast, freeze-thaw solubilized HMG-CoA reductase, which does not display allosteric properties, is readily inhibited by P-ADP-Rib, even when assayed at a low concentration of GSH (Kiapp = 50 microM at 4.5 mM GSH). Assaying the solubilized reductase in the presence of increased thiol concentration results in a minor decrease in the apparent Ki for P-ADP-Rib (22 microM at 27 mM GSH). Microsomal HMG-CoA reductase is allosterically activated by various nucleotides. When activated by NADH, the enzyme is effectively inhibited by P-ADP-Rib even at a 4.5-mM GSH concentration (Kiapp = 175 microM in the presence of 300 microM NADH).(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties and utility of the peculiar mixed disulfide in the bacterial glutathione transferase B1-1

Bacterial glutathione transferases appear to represent an evolutionary link between the thiol:disulfide oxidoreductase and glutathione transferase superfamilies. In particular, the observation of a mixed disulfide in the active site of Proteus mirabilis glutathione transferase B1-1 is a feature that links the two families. This peculiar mixed disulfide between Cys10 and one GSH molecule has been studied by means of ESR spectroscopy, stopped-flow kinetic analysis, radiochemistry, and site-directed mutagenesis. This disulfide can be reduced by dithiothreitol but even a thousand molar excess of GSH is poorly effective due to an unfavorable equilibrium constant of the redox reaction (K(eq) = 2 x 10(-4)). Although Cys10 is partially buried in the crystal structure, in solution it reacts with several thiol reagents at a higher or comparable rate than that shown by the free cysteine. Kinetics of the reaction of Cys10 with 4,4'-dithiodipyridine at variable pH values is consistent with a pK(a) of 8.0 +/- 0.1 for this residue, a value about 1 unit lower than that of the free cysteine. The 4,4'-dithiodipyridine-modified enzyme reacts with GSH in a two-step mechanism involving a fast precomplex formation, followed by a slower chemical step. The natural Cys10-GSH mixed disulfide exchanges rapidly with free [3H]GSH in a futile redox cycle in which the bound GSH is continuously replaced by the external GSH. Our data suggest that the active site of the bacterial enzyme has intermediate properties between those of the recently evolved glutathione transferases and those of the thiol:disulfide oxidoreductase superfamily.     

Endogenous defenses against the cytotoxicity of hydrogen peroxide in cultured rat hepatocytes

The catalase activity of cultured rat hepatocytes was inhibited by 90% pretreatment with 20 mM aminotriazole without effect on the activities of glutathione peroxidase or glutathione reductase, or on the viability of the cells over the subsequent 24 h. Glutathione reductase was inhibited by 85% by pretreatment with 300 microM 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) without effect on glutathione peroxidase, catalase, or on viability. Both pretreatments sensitized the hepatocytes to the cytotoxicity of H2O2 generated either by glucose oxidase (0.05-0.5 units/ml) or by the autoxidation of the one-electron-reduced state of menadione (50-250 microM). Aminotriazole pretreatment had no effect on the GSH content of the hepatocytes. BCNU reduced GSH levels by 50%. Depletion of GSH levels to less than 20% of control by treatment with diethyl maleate, however, did not sensitize the cells to either glucose oxidase or menadione, indicating that the effect of BCNU is related to inhibition of the GSH-GSSG redox cycle rather than to the depletion of GSH. With glucose oxidase, most of the cell killing in hepatocytes pretreated with either aminotriazole or BCNU occurred between 1 and 3 h. The antioxidant diphenylphenylenediamine (DPPD) had no effect on viability at 3 h. Catalase added to the culture medium 1 h after the addition of glucose oxidase prevented the cell killing measured at 3 h. The sulfhydryl reagents dithiothreitol (200 microM), N-acetyl-L-cysteine (4 mM), and alpha-mercaptopropionyl-L-glycine (2.5 mM) prevented the cell killing with exogenous H2O2 in hepatocytes sensitized by the inhibition of catalase or glutathione reductase. With menadione, there was no killing of nonpretreated hepatocytes at 1 h, and DPPD did not prevent the cell death after 3 h. Aminotriazole pretreatment enhanced the cell killing at 3 h but not at 1 h, and DPPD was not protective. Catalase added to the medium at 1 h inhibited the cell death measured at 3 h. In contrast, menadione killed hepatocytes pretreated with BCNU within 1 h. DPPD prevented cell death at 1 h, and there was evidence of lipid peroxidation in the accumulation of malondialdehyde in the culture medium. Catalase added with menadione did not prevent the cell killing at 1 h but did prevent it at 3 h. These data indicate that catalase and the GSH-GSSG cycle are active in the defense of hepatocytes against the toxicity of H2O2.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Nitric oxide modulates antioxidant defense and the methylglyoxal detoxification system and reduces salinity-induced damage of wheat seedlings

The present study investigates the possible regulatory role of exogenous nitric oxide (NO) in antioxidant defense and methylglyoxal (MG) detoxification systems of wheat seedlings exposed to salt stress (150 and 300 mM NaCl, 4 days). Seedlings were pre-treated for 24 h with 1 mM sodium nitroprusside, a NO donor, and then subjected to salt stress. The ascorbate (AsA) content decreased significantly with increased salt stress. The amount of reduced glutathione (GSH) and glutathione disulfide (GSSG) and the GSH/GSSG ratio increased with an increase in the level of salt stress. The glutathione S-transferase (GST) activity increased significantly with severe salt stress (300 mM). The ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), catalase (CAT) and glutathione peroxidase (GPX) activities did not show significant changes in response to salt stress. The glutathione reductase (GR), glyoxalase I (Gly I), and glyoxalase II (Gly II) activities decreased upon the imposition of salt stress, especially at 300 mM NaCl, with a concomitant increase in the H₂O₂ and lipid peroxidation levels. Exogenous NO pre-treatment of the seedlings had little influence on the non-enzymatic and enzymatic components compared to the seedlings of the untreated control. Further investigation revealed that NO pre-treatment had a synergistic effect; that is, the pre-treatment increased the AsA and GSH content and the GSH/GSSG ratio, as well as the activities of MDHAR, DHAR, GR, GST, GPX, Gly I, and Gly II in most of the seedlings subjected to salt stress. These results suggest that the exogenous application of NO rendered the plants more tolerant to salinity-induced oxidative damage by enhancing their antioxidant defense and MG detoxification systems.     

20 S proteasome from Saccharomyces cerevisiae is responsive to redox modifications and is S-glutathionylated

The 20 S proteasome core purified from Saccharomyces cerevisiae is inhibited by reduced glutathione (GSH), cysteine (Cys), or the GSH precursor gamma-glutamylcysteine. Chymotrypsin-like activity was more affected by GSH than trypsin-like activity, whereas the peptidylglutamyl-hydrolyzing activity (caspase-like) was not inhibited by GSH. Cys-sulfenic acid formation in the 20 S core was demonstrated by spectral characterization of the Cys-S(O)-4-nitrobenzo-2-oxa-1,3-diazole adduct, indicating that 20 S proteasome Cys residues might react with reduced sulfhydryls (GSH, Cys, and gamma-glutamylcysteine) through the oxidized Cys-sulfenic acid form. S-Glutahionylation of the 20 S core was demonstrated in vitro by GSH-biotin incorporation and by decreased alkylation with monobromobimane. Compounds such as N-ethylmaleimide (-S-sulfhydril H alkylating), dimedone (-SO sulfenic acid H reactant), or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (either -SH or -SOH reactant) highly inhibited proteasomal chymotrypsin-like activity. In vivo experiments revealed that 20 S proteasome extracted from H(2)O(2)-treated cells showed decreased chymotrypsin-like activity accompanied by S-glutathionylation as demonstrated by GSH release from the 20 S core after reduction with NaBH(4). Moreover, cells pretreated with H(2)O(2) showed decreased reductive capacity assessed by determination of the GSH/oxidized glutathione ratio and increased protein carbonyl levels. The present results indicate that at the physiological level the yeast 20 S proteasome is regulated by its sulfhydryl content, thereby coupling intracellular redox signaling to proteasome-mediated proteolysis.     

Cysteine does not repress adenosine-5′-phosphosulfate reductase through its conversion to either sulfate or glutathione

Cysteine (Cys) represses the activity of several key regulatory enzymes in the plant sulfate assimilatory pathway. However, it is not clear whether this effect arises from Cys itself or through its conversion to either sulfate or glutathione (GSH). Therefore, we examined this phenomenon by analyzing the activity of adenosine-5′-phosphosulfate (APS) reductase. Both APS reductase (AR) activity and mRNA levels were decreased by treatingArabidopsis thaliana roots with 1 mM Cys. The intracellular sulfate concentration was not affected, whereas enzymatic activity and, to some extent, the mRNA level, declined. Cys treatment in sulfur-starved plants also diminished both parameters. However, this response to Cys was more efficient than when plants were treated with an equal amount of sulfate. When Cys was removed from both Cys-and sulfate-fed plants, AR activity was recovered; the same removal of sulfate was not so effective. Moreover, buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, did not influence the repression of AR by Cys. Finally the AR enzyme was inhibited by cysteinein vitro. These results indicate that Cys represses AR by inhibiting mRNA expression and by directly repressing enzymatic activity, rather than through its conversion to either sulfate or GSH.     

Selenium-Induced Up-Regulation of the Antioxidant Defense and Methylglyoxal Detoxification System Reduces Salinity-Induced Damage in Rapeseed Seedlings

The present study investigates the regulatory role of exogenous selenium (Se) in the antioxidant defense and methylglyoxal (MG) detoxification systems in rapeseed seedlings exposed to salt stress. Twelve-day-old seedlings, grown in Petri dishes, were supplemented with selenium (25 μM Na₂SeO₄) and salt (100 and 200 mM NaCl) separately and in combination, and further grown for 48 h. The ascorbate (AsA) content of the seedlings decreased significantly with increased salt stress. The amount of reduced glutathione (GSH) and glutathione disulfide (GSSG) increased with an increase in the level of salt stress, while the GSH/GSSG ratio decreased. In addition, the ascorbate peroxidase (APX) and glutathione S-transferase (GST) activity increased significantly with increased salt concentration (both at 100 and 200 mM NaCl), while glutathione peroxidase (GPX) activity increased only at moderate salt stress (100 mM NaCl). Glutathione reductase (GR) activity remained unchanged at 100 mM NaCl, while it was decreased under severe (200 mM NaCl) salt stress. Monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), catalase (CAT), glyoxalase I (Gly I), and glyoxalase II (Gly II) activities decreased upon the imposition of salt stress, whereas a sharp decrease of these activities was observed under severe salt stress (200 mM NaCl). Concomitant increases in the levels of H₂O₂ and lipid peroxidation (MDA) were also measured. Exogenous Se treatment alone had little effect on the non-enzymatic and enzymatic components. However, further investigation revealed that Se treatment had a synergistic effect: in salt-stressed seedlings, it increased the AsA and GSH contents; GSH/GSSG ratio; and the activities of APX, MDHAR, DHAR, GR, GST, GPX, CAT, Gly I, and Gly II. As a result, addition of Se in salt-stressed seedlings led to a reduction in the levels of H₂O₂ and MDA as compared to salt stress alone. These results suggest that the exogenous application of Se rendered the plants more tolerant to salt stress-induced oxidative damage by enhancing their antioxidant defense and MG detoxification systems.     

Regulation of sulphate assimilation by glutathione in poplars (Populus tremula x P. alba) of wild type and overexpressing gamma-glutamylcysteine synthetase in the cytosol

Glutathione (GSH) is the major low molecular weight thiol in plants with different functions in stress defence and the transport and storage of sulphur. Its synthesis is dependent on the supply of its constituent amino acids cysteine, glutamate, and glycine. GSH is a feedback inhibitor of the sulphate assimilation pathway, the primary source of cysteine synthesis. Sulphate assimilation has been analysed in transgenic poplars (Populus tremula x P. alba) overexpressing gamma-glutamylcysteine synthetase, the key enzyme of GSH synthesis, and the results compared with the effects of exogenously added GSH. Although foliar GSH levels were 3-4-fold increased in the transgenic plants, the activities of enzymes of sulphate assimilation, namely ATP sulphurylase, adenosine 5'-phosphosulphate reductase (APR), sulphite reductase, serine acetyltransferase, and O-acetylserine (thiol)lyase were not affected in three transgenic lines compared with the wild type. Also the mRNA levels of these enzymes were not altered by the increased GSH levels. By contrast, an increase in GSH content due to exogenously supplied GSH resulted in a strong reduction in APR activity and mRNA accumulation. This feedback regulation was reverted by simultaneous addition of O-acetylserine (OAS). However, OAS measurements revealed that OAS cannot be the only signal responsible for the lack of feedback regulation of APR by GSH in the transgenic poplars.