Glutathione antioxidant complex and carotenoid composition in tissues of the bivalve mollusk <Emphasis Type="Italic">Anadara kagoshimensis</Emphasis> (Tokunaga, 1906)

The correlation of the state of glutathione complex composed of reduced glutathione (GSH), glutathione reductase (GR) activity and glutathione peroxidase (GP) and the qualitative composition of carotenoids was investigated in the bivalve mollusk Anadara kagoshimensis (Tokunaga, 1906). Using high-performance liquid chromatography, UV-Vis and mass spectra, 7 types of carotenoids (trans- and cis-pectenolon, alloxanthine, pectenol A, β-carotene, zeaxanthin and diatoxanthin) were identified in tissues of this species and their quantitative ratio was determined. A positive correlation (R ² > 0.9) was established between GSH and most carotenoid levels. A negative correlation was found for the GR–carotenoids (R ² > 0.75) and GP–pectenol A (R ² > 0.988) systems. The cause-and-effect relations of these regularities are discussed.     

Protective effect of propionyl-L-carnitine against ischaemia and reperfusion-damage

Summary Reperfusion of isolated rabbit heart after 60 min of ischaemia resulted in poor recovery of mechanical function, release of reduced (GSH) and oxidized glutathione (GSSG), reduction of tissue GSH/GSSG ratio and shift of cellular thiol redox state toward oxidation, suggesting the occurrence of oxidative stress. Pretreatment of the isolated heart with propionyl-L-carnitine (10^–7M) improved the functional recovery of the myocardium, reduced GSH and GSSG release and attenuated the accumulation of tissue GSSG. This effect was specific for propionyl-L-carnitine as L-carnitine and propionyl acid did not modify myocardial damage.     

Ozone detoxification in the apoplasm and symplasm of spinach,broad bean and beech leaves at ambient and elevated concentrations of ozone in air

Spinach (Spinacia oleracea L.), broad bean (Vicia faba L.) and beech (Fagus sylvatica L.) plants were exposed to ozone at concentrations often measured in air during the summer months (120–300 g·m^–3) and antioxidants were determined in the leaf tissue and in the aqueous phase of the cell wall, the apoplasm. Concentrations of both reduced ascorbate (AA) and its oxidized form, dehydroascorbate (DHA), showed the tendency to increase transiently in the apoplasm of spinach leaves 6–24 h after starting fumigation with ozone. In beech leaves, apoplasmic AA and DHA increased 3–7 d after beginning of treatment. At the very high concentration of 1600 g O₃·m^–3, an increase of apoplasmic AA was already measured after 1 d in beech leaves. Apparently, spinach and beech leaves respond to oxidative stress by increasing AA transport into the apoplasm and by accelerating DHA export. In contrast to these observations, DHA accumulated during 3 d of fumigation with only 120 g O₃·m^–3 in the apoplasm of broad bean leaves, while AA contents did not increase. After termination of fumigation, the extracellular redox state of ascorbate normalized within 1 d. Glutathione could not be detected in the apoplasm of any of the three leaf species. Intracellular AA changed its redox state in response to exposure to elevated concentrations of ozone. After 4–6 weeks of fumigation with 200–300 g O₃·m^–3 an increase of intracellular DHA was measured in beech leaves. At the same time, chlorophyll contents decreased and characteristic symptoms of ozone damage could be observed. However, no significant change in the redox state of apoplasmic ascorbate could be detected in beech leaves. Evidently, detoxification of ozone by apoplasmic AA was insufficient to protect the leaf tissue. Fumigation with a high ozone concentration (1600 g·m^–3) caused an appreciable increase in the cellular contents of the oxidized forms of ascorbate and glutathione in beech leaves. Whereas in spinach leaves intracellular antioxidant contents and redox states were not altered during fumigation with 120–240 g O₃·m^–3, in broad bean leaves the intracellular DHA concentration increased and intracellular ascorbate became more oxidized after fumigation of the plants with 120 g O₃·m^–3. Apparently, broad bean leaves are more sensitive to ozone than beech and spinach leaves.Abbreviations AA ascorbate, reduced form - DHA ascorbate, oxidized form (dehydroascorbate) - FW fresh weight - GSH glutathione, reduced form - GSSG glutathione, oxidized form - IWF intercellular washing fluid - V_air intercellular air space volume of leaves - V_apo apoplasmic water volume of leaves This work was supported within the Sonderforschungsbereich 251 of the University of Würzburg.     

Regulation of sulfate uptake and xylem loading of poplar roots (Populus tremula x P. alba)

Sulfate transport processes and its regulation were studied in roots of poplar trees (Populus tremula x P. alba). From the exponential increase in sulfate uptake with temperature an activation energy (E_a) of 9.0±0.8 kJ mol^–1 was calculated. In the concentration range 0.005–10 mM sulfate uptake showed biphasic Michaelis-Menten kinetics with a K_m of 3.2±3.4 M and a V_max of 49±11 nmol SO₄^2– g^–1 FW h^–1 for the high-affinity uptake system (phase 1) and a K_m of 1.33±0.41 mM and a V_max of 255±25 nmol SO₄^2– g^–1 FW h^–1 for the low-affinity system (phase 2). Xylem loading decreased linearly with temperature and remained unchanged within the sulfate concentration range studied. Regulation of sulfate uptake and xylem loading by O-acetyl serine (OAS), Cys, reduced glutathione (GSH), Met and S-methylmethionine (SMM) were tested by perfusion into the xylem sap with the pressure probe and by addition to the incubation medium. When added directly to the transport medium, Cys and GSH repressed, and OAS stimulated sulfate uptake; xylem loading was stimulated by Cys, repressed by GSH and only slightly affected by OAS. When perfused into the xylem, none of the compounds tested affected sulfate uptake of excised roots, but xylem loading was stimulated by SMM and OAS and repressed by Met. Apparently, the site of application strongly determined the effect of regulatory compounds of sulfate transport processes.     

Potential Role of Cerebral Glutathione in the Maintenance of Blood-Brain Barrier Integrity in Rat

Using the model of glutathione (GSH) depletion, possible role of GSH in the maintenance of blood-brain barrier (BBB) integrity was evaluated in rats. Administration (ip) of GSH depletors, diethyl maleate (DEM, 1–4 mmol/kg), phorone (2–3 mmol/kg) and 2-cyclohexene-1-one (CHX, 1 mmol/kg), to male adults was found to deplete brain and liver GSH and increase the BBB permeability to micromolecular tracers (sodium fluorescein and [¹⁴C]sucrose) in a dose-dependent manner at 2h. However, BBB permeability to macromolecular tracers such as horseradish peroxidase and Evan's blue remained unaltered. It was also shown that observed BBB permeability dysfunction was associated with brain GSH depletion. A lower magnitude of BBB increase in rat neonates, as compared to adults, indicated a possible bigger role of GSH in the BBB function of mature brain. The treatment with N-acetylcysteine, methionine and GSH provided a partial to full protection against DEM-induced brain (microvessel) GSH depletion and BBB dysfunction; however, the treatment with -tocopherol, ascorbic acid and turmeric were not effective. Our studies showed that cerebral GSH plays an important role in maintaining the functional BBB integrity.     

Soluble and microsomal glutatione S-transferase activities in pea seedlings (Pisum sativum L.)

Epicotyl and primary leaves of pea seedlings (Pisum sativum L., var. Alaska) were found to contain soluble and microsomal enzymes catalyzing the addition of glutathione to the olefinic double bond of cinnamic acid. Glutathione S-cinnamoyl transfer was also obtained with enzyme preparations from potato slices and cell suspension cultures of parsley and soybean.The pea transferases had pH-optima between pH 7.4 and 7.8 K_m-values were 0.1–0.4 mM and 1–4 mM for cinnamic acid and glutathione, respectively. V-values were between 2–15 nmol mg^-1 protein x min.Chromatography on Sephacryl S-200 indicated that the soluble pea glutathione S-cinnamoyl transferase activity existed in molecular weight forms of 37,000, 75,000, and 150,000. The glutathione-dependent cleavage of the herbicide fluorodifen was catalyzed by a different soluble enzyme activity which eluted in molecular weight positions of 47,000 and/or 82,000.The microsomal fraction from pea primary leaves also catalyzed the conjugation of the carcinogen benzo[]pyrene with glutathione.Abbreviations GSH glutathione - DDE 1,1-Dichloro-2,2-bis-(4-chlorophenyl)-ethylene - DDMU l-Chloro-2,2-bis-(4-chlorophenyl)-ethylene     

Import and Metabolism of Glutathione by Streptococcus mutans

Glutathione (γ-GluCysGly, GSH) is not found in most gram-positive bacteria, but some appear to synthesize it and others, including Streptococcus mutans ATCC 33402, import it from their growth medium. Import of oxidized glutathione (GSSG) by S. mutans 33402 in 7H9 medium was shown to require glucose and to occur with an apparent K_m of 18 ± 5 μM. GSSG, GSH, S-methylglutathione, and homocysteine-glutathione mixed disulfide (hCySSG) were imported at comparable rates (measured by depletion of substrate in the medium), as was the disulfide of γ-GluCys. In contrast, the disulfide of CysGly was not taken up at a measurable rate, indicating that the γ-Glu residue is important for efficient transport. During incubation with GSSG, little GSSG was detected in cells but GSH and γ-GluCys accumulated during the first 30 min and then declined. No significant intracellular accumulation of Cys or sulfide was found. Transient intracellular accumulation of d/l-homocysteine, as well as GSH and γ-GluCys, was observed during import of hCySSG. Although substantial levels of GSH were found in cells when S. mutans was grown on media containing glutathione, such GSH accumulation had no effect on the growth rate. However, the presence of cellular GSH did protect against growth inhibition by the thiol-oxidizing agent diamide. Import of glutathione by S. mutans ATCC 25175, which like strain 33402 does not synthesize glutathione, occurred at a rate comparable to that of strain 33402, but three species which appear to synthesize glutathione (S. agalactiae ATCC 12927, S. pyogenes ATCC 8668, and Enterococcus faecalis ATCC 29212) imported glutathione at negligible or markedly lower rates.Bacteria import peptides composed of two to eight residues by means of a number of different multiprotein uptake systems or permeases (14). Of the bacterial permeases, those of Escherichia coli, Lactococcus lactis, and Salmonella typhimurium are the best studied (6, 7). In these organisms, there are individual permeases that have high affinity for dipeptides, tripeptides, dipeptides and tripeptides, or oligopeptides. Among the bacterial peptide permeases (14), there seems to be no discrimination of the specific amino acids of the transported peptides. However, switching the stereochemistry of C_α from l to d or modifying the C-terminal carboxylate or N-terminal amine of transported peptides significantly reduces the rate of transport. One transport system which does seem to recognize peptide residue side chains has been reported to exist in Enterococcus faecalis; this system transports only peptides that possess an N-terminal Asp or Glu (13).In 1978, we reported that glutathione (γ-GluCysGly, GSH) is not synthesized by most gram-positive bacteria (4), apparent exceptions being Streptococcus agalactiae and L. lactis (previously Streptococcus lactis). However, some of the gram-positive bacteria appeared to acquire GSH by import of another form of GSH from the growth medium. Uptake of glutathione by Streptococcus mutans was later studied by Thomas (16), who found that total cellular thiol content, and radioactivity from labeled GSH or oxidized GSH (GSSG), increased with the same kinetics. A careful study of L. lactis subsp. cremoris by Wiederholt and Steele (17) established that strain Z8 efficiently accumulates GSH when grown in medium supplemented with GSH but is unable to synthesize it, whereas strain C2 can neither import nor synthesize GSH. Species of Peptostreptococcus and Fusobacterium have been shown to markedly increase their production of H₂S, apparently derived by import of glutathione from the growth medium (2). Finally, cellular accumulation of radioactivity from radiolabeled GSH or GSSG added to the incubation medium has been demonstrated in Streptococcus pneumoniae, and a mutant in which the apparent transport of glutathione is blocked has been found (9).In a recent report (10), we provided evidence for accumulation of GSH through transport and synthesis of GSH by streptococci and enterococci, but the occurrence of these processes appeared to be species dependent and even, for some species, strain dependent. Such strain dependence appears most variable for L. lactis, where different strains can synthesize GSH, accumulate GSH by import, or do neither (4, 17). In the present research, we expand on our studies of streptococci in order to gain insight into the nature of the glutathione species transported, the fate of the glutathione once it enters the cell, and the function of glutathione in the cell.     

Accumulation of ubiquitinated proteins in mouse neuronal cells induced by oxidative stress

Ubiquitin protein conjugates are commonly detected in neuronal brain inclusions of patients with neurodegenerative disorders. The failure to eliminate the ubiquitin-protein deposits in the degenerating neurons may result from changes in the activity of the ubiquitin/ATP-dependent proteolytic pathway. This proteolytic pathway plays a major role in the degradation of short lived, abnormal and denatured proteins. Cadmium is a potent cell poison and is known to affect the ubiquitin pathway and to cause oxidative stress. Increases in protein mixed-disulfides (Pr-SSG) and decreases in glutathione (GSH) are often used as markers of oxidative stress. To investigate the relationship between the ubiquitin pathway and cellular glutathione (GSH), we treated HT4 cells (a mouse neuronal cell line) and rat mesencephalic primary cultures with different concentrations of the heavy metal. We observed marked increases in Pr-SSG as well as decreases in GSH, after exposure of HT4 cells or primary mesencephalic cultures to Cd²⁺. Furthermore, our results show that Cd²⁺ induced the accumulation of ubiquitinated proteins. Detection was by Western blotting of total cell extracts probed with antibodies that recognize ubiquitin-protein conjugates. These results suggest that the ubiquitin-pathway is closely involved in the cell response to cadmium-mediated oxidative stress. Abbreviations: GSH – glutathione; GSSG – glutathione disulfide; Pr-SSG – protein mixed disulfides.     

Co-production of S-adenosyl-l-methionine and glutathione from spent brewer’s yeast cells

Both S-adenosyl-l-methionine (AdoMet) and glutathione (GSH) are important small molecules with pharmaceutical importance. The co-production of AdoMet and GSH using abundant spent brewer’s yeast cells from the beer industry and with l-methionine supplement was successfully realized. Experimental data showed that improvement of GSH productivity was accompanied by AdoMet accumulation. AdoMet productivity of 40–45 mg g⁻¹ (DCW) was successfully achieved and an additional 13–18 mg g⁻¹ (DCW) GSH was synthesized in spent brewer’s yeast cells.     

Pharmacological characterization of the N-methyl-d-aspartate (NMDA) receptor-channel in rodent and dog brain and rat spinal cord using [3H]MK-801 binding

The biochemical and pharmacological properties of [³H]MK-801 binding to the N-methyl-d-aspartate (NMDA) receptor-channel in homogenates of mouse, guinea pig and dog brain, dog cerebral cortex and rat spinal cord were determined using radioligand binding techniques. Specific [³H]MK-801 binding increased linearily with increasing tissue concentration and in general represented 80–93% of the total binding at 6–8 nM radioligand concentration. [³H]MK-801 interacted with brain and spinal homogenates with high affinity. The dissociation constants (K _d ) for all tissues studied were similar ranging between 7.9 and 11.9 nM, whereas the maximum number of binding sites (B_max) showed a wide, tissue-dependent range (0.1–6.75 pmol/mg protein). The rank order of tissue enrichment was found to be as follows: mouse brain>>dog cerebral cortex>>dog brain>> guinea pig brain>>rat spinal cord. Specific [³H]MK-801 binding in rodent and dog brain, dog cerebral cortex and rat spinal cord exhibited a similar pharmacological profile 9correlation coefficients=0.93–0.99). The rank order of potency of unlabelled compounds competing for [³H]MK-801 binding was: (+)MK-801>(–)MK-801>phencyclidine>(–)cyclazocine>>(+)cyclazocine ketamine>(+)N-allyl-N-normetazocine>(–)N-allyl-N-normetazocine>(–)pentazocine>(+)pentazocine. NMDA, Kainate, quisqualate and several other compounds failed to inhibit [³H]MK-801 binding at 100 M. In modulation studies conducted on extensively washed dog cortex membranes, Mg²⁺ ions stimulated [³H]MK-801 binding at 10 M-1 mM (EC₅₀=91.5 M) and then inhibited the binding from 1 mM to 10 mM (IC₅₀=3.1 mM). Glycine stimulated [³H]MK-801 binding at 30 nM-1 mM (EC₅₀=256 nM). In contrast, Zn²⁺ ions inhibited the binding of [³H]MK-801 binding site exhibited similar pharmacological and biochemical properties. These data appear to suggest that the pharmacological profile of the NMDA-receptor-channel is species and tissue independent.     

Determination of sulfite radical (SO 3 ⋅ −) reaction rate constants by means of competition kinetics

Summary The sulfite radical anion (SO ₃ ^–) was found to react rapidly with the flavonoid quercetin (k = 2.5 × 10⁸ dm³mol^–1 s^–1) and the carotenoids crocin (k = 1.0 × 10⁹ dm³mol^–1 s^1–) and crocetin (k = 1.5 × 10⁹ dm³mol^1– s^1–). The reactions can easily be monitored due to the strong absorptions of the substrates and, in the case of quercetin, the formation of a strongly absorbing transient species. Using these substances, we determined by means of competition kinetics rate constants of SO ₃ ^– reactions with nucleic acid components, polyunsaturated fatty acids, and glutathione.Abbreviations ABTS 2,2-azinobis(3-ethyl-6-benzothiazolinesulfonate) - cmc critical micellization concentration - GSH Glutathione - PUFA polyunsaturated fatty acids Preliminary results were presented at the Third Biennial Meeting of the Society for Free Radical Research in Düsseldorf in July, 1986     

Exogenous glutathione induces sister chromatid exchanges,clastogenicity and endoreduplication in V79-E Chinese hamster cells

Glutathione (GSH) dissolved in Eagle's MEM and added to cultures o of V79-E cells in concentrations between 2.5 × 10^–4 and 10^–3 moles/l for 1 h induces a dose-dependent cell cycle delay, sister chromatid exchanges and clastogenic damage. 7–8% of the metaphases showed endoreduplication at a recovery phase of 25 and 30 h after treatment with 10^–3 molesll GSH. Higher concentrations were lethal. The highest tolerated dose corresponds to the intracellular GSH level in V79-E cells. In the same range of concentrations, glutathione disulfide was inactive. Endoreduplication induction by GSH is G2-phase specific and endoreduplication metaphases show a reduced occurrence of single SCEs when extrapolated to the diploid complement. The adverse effects of GSH are independent of the presence of serum in the culture fluid but completely abolished when the treatment is performed in Hank's solution instead of MEM. The mechanism of genotoxicity of exogenous GSH is discussed but, at present, no pertinent explanation can be given.Abbreviations BUdR 5-bromodeoxyuridine - GSH glutathione - GSSG glutathione disulfide - SCE sister chromatid exchange     

Phytochelatin synthesis and cadmium localization in wild type of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

AlthoughArabidopsis thaliana is known as a model plant, in molecular studies, as well as heavy metal tolerance of higher plants, there have been no detailed studies of its cadmium accumulation, tolerance and cellular distribution in a wild type of this species. In hydroponic experiments the wild type of A. thaliana (L.) Heynh cv. Columbia plants grew at cadmium concentrations varying from 5 to 100 M with phytotoxicity symptoms depending on the concentration and time of application. The concentration of cadmium in roots and shoots increased from 0.28 and 0.08 mg g^–1 d.wt at 5 M Cd treatment after 7 days to 0.82 and 0.85 mg g^–1 d.wt at 100 M Cd treatment after 14 days, respectively. Most of the cadmium (69–88% of its total pool) was found in shoot. Cd application induced the biosynthesis of phytochelatins (PCs) in root and shoot tissues. Studies with buthionine sulfoximine [BSO, specific inhibitor of glutathione (GSH) synthesis] supported the presence of Cd–phytochelatin complexes and their role in Cd detoxification and tolerance in wild type of A. thaliana. Cellular distribution of cadmium was examined using energy-dispersive X-ray micro-analysis. Particularly interesting was the observation of cadmium localized in the root pericycle.     

Noncompetitive Activation of the Peroxidase-Catalyzed Oxidation of o-Phenylenediamine by Melamine

Peroxidase-catalyzed oxidation of o-phenylenediamine (PDA) is greatly activated with melamine (MA) in 15 mM phosphate–citrate buffer at pH 6.0–7.4 in a noncompetitive manner: k _cat and K _m increase in direct proportion to the MA concentration. An extent of the activation is quantitatively characterized with a coefficient (in M^–1), which essentially increases along with the rise in pH from 6.0 to 7.4. MA acts as a nucleophilic catalyst in the oxidation process: it most likely affects the peroxidase active site from the distal position of heme. MA noncompetitively inhibits the peroxidase oxidation of PDA at pH 4.3, since it completely loses its nucleophilic properties in acidic medium. A rapid, highly accurate, and simple analytical test system based on the kinetics of melamine-activated oxidation of PDA is proposed for the quantitative determination of melamine within the concentration range of 10^–4–10^–3 M. This test system uses the spectrophotometric determination of the PDA oxidation product at 455 nm.     

(h)GR,beans and drought stress

Reactive oxygen species (ROS) are commonly found in plants as natural by-products of the metabolism but their production is greatly enhanced under abiotic stresses. Particular metabolites and enzymes belonging to the ascorbate-glutathione cycle are able to scavenge these deleterious molecules and modulate the cellular redox-status. In the March issue of Journal of Plant Physiology, we have shown that drought stress induces a raise in glutathione reductase (GR) activity and gene expression that could be related to the intensity of the drought treatment and the drought susceptibility of the bean cultivar (cowpea and/or common bean). In the present addendum we show new data on GR specific activity during progressive drought stress and recovery of the drought-susceptible bean cultivar which can be related to the previously found dual-targeted GR gene expression. Furthermore, since in leguminous plants homoglutathione (hGSH) is generally the most abundant low molecular weight thiol form, we discuss on the occurrence of a (homo)glutathione reductase activity in beans.Key words: common bean, cowpea, drought stress, (homo)glutathione, (homo)glutathione reductase, legumes, Phaseolus vulgaris, recovery, Vigna unguiculataDrought stress is the most common form of abiotic stress and plants are likely to encounter periods of water shortage at least once in their lifecycle. One of the inevitable consequences of drought stress is enhanced reactive oxygen species (ROS) production which will imbalance the cellular redox-status. This shift in the steady-state cellular redox-status is currently believed to have an initial signaling effect, triggering adaptive/defense responses (reviewed in ref. ¹). However, in order to avoid oxidative stress, enhanced ROS production must be kept under tight control by the cellular antioxidant machinery. Glutathione reductase (GR; EC 1.6.4.2) is a major cellular antioxidant enzyme. It belongs to the ascorbate-glutathione cycle and it is ubiquitously found in all cellular compartments.² Using several bean plants (common bean, Phaseolus vulgaris and cowpea, Vigna unguiculata) as a model system to study drought responses and relate them to the degree of drought tolerance and/or susceptibility, we have shown that severe drought stress leads to an enhanced cellular GR activity related to the drought susceptibility of the cultivar.^3,4 Similar results have also been found in a wheat system composed of drought-tolerant and susceptible cultivars.⁵ Regarding the more susceptible cultivar of our bean system (P.v. Carioca) and under severe drought stress (S3, Ψ_w = −2.0 MPa; RWC = 50.9%), total leaf GR activity was raised to approximately 200% when compared to control plants (C, Ψ_w = −0.5 MPa; RWC = 95.3%) (Fig. 1). This could translate a higher degree of oxidative stress due to enhanced ROS production in drought-susceptible cultivars than in drought-tolerant ones. In fact it has been shown that at the cellular level these drought-susceptible bean plants suffer a higher degree of membrane integrity loss when compared to the drought-tolerant.^6–8 This can be related to enhanced ROS production since proteins and lipids of cellular membranes are main targets of ROS peroxidation.⁹Open in a separate windowFigure 1GR-specific activity and relative water content (RWC%) in common bean (Phaseolus vulgaris) ‘Carioca’ leaves. GR-specific activity and RWC were measured in control, severely drought stressed and on rewatered plants. Values are means ± s.d. of three to five independent measurements. GR activity was assayed by following the oxidation of NADPH (decrease in absorbance at 340 nm) and expressed in nmoles min⁻¹ mg⁻¹ protein. RWC was measured according to Weatherley.²⁴ Control plants (C), Ψ_w = −0.5 MPa; severely droughted plants (S3), Ψ_w = −2.0 MPa; 24 h rehydrated plant (24R), Ψ_w = −0.5 MPa; 48 h rehydrated plant (48R), Ψ_w = −0.5 MPa.Considering the responses to drought at the whole-plant level, susceptible and tolerant beans also differ. In fact, drought-tolerant bean cultivars present a water-saving strategy by precocious control of stomatal opening which allows for photosynthetic activity to proceed at lower leaf Ψ_w.^10–12 The maintenance of stomatal opening and photosynthetic activity during drought stress results in lower ROS production by photorespiration and/or the Mehler reaction as opposed to complete stomatal closure where inhibition of CO₂ fixation occurs.¹ Indeed, in the drought-tolerant cowpea cultivar, total GR activity was found constant throughout the progressive drought treatment.³After 24 h rewatering (24R, Ψ_w = −0.5 MPa; RWC = 88.1%), from a moderate water stress: Ψ_w = −1.5 MPa; RWC = 69.2%, GR activity in the drought-susceptible bean cultivar was further raised by ∼270% as compared to control (Fig. 1). This enhanced GR activity can be directly related to the upregulation of the dual-targeted form of the bean GR gene (PvGRdt) (targeted to both chloroplasts and mitochondria) observed on rewatering of this drought-susceptible cultivar.⁴ In fact a significant upregulation of PvGRdt was detected as soon as 6 h after rewatering and the high expression levels were maintained up to 24 h after rewatering to then decrease at 48 h after rewatering.⁴ Hence in the drought-susceptible cultivar it seems that the dual-targeted form is more responsive to drought and rewatering than the cytosolic form. The same pattern was also seen on the less tolerant cowpea cultivar.³ Enhanced dual-targeted GR expression (and GR activity) under drought could be related to enhanced ROS production at those particular cellular compartments (mitochondria and chloroplasts). In fact, under a PEG-induced water deficit, drought-susceptible bean plants showed a higher number of disorganized chloroplasts when compared to the drought-tolerant,⁷ indicating that these organelles experienced oxidative stress during the treatment.The GR enzyme is responsible for the reduction of glutathione disulfide (GSSG) to glutathione (GSH) using NADPH, and not only it keeps glutathione in the reduced state but it is also responsible for the maintenance of the cellular GSH:GSSG ratio.^13,14 Interestingly, in leguminous plants such as the present bean plants, homoglutathione (hGSH) replaces completely or in part, glutathione (GSH). Homoglutathione has been shown to be the most abundant tripeptide in common bean and pea (Pisum sativum),¹⁵ in soybean (Glycine max),¹⁶ and in Lotus japonicus.¹⁷ The synthesis of hGSH proceeds through two ATP-dependent steps, the first step being common with GSH synthesis, the second step adding a β-Alanine instead of a Glycine to form the tripeptide. In the case of cowpea, which was up to now considered to be a non hGSH producing legume,¹⁵ we have recently detected the presence of a homoglutathione synthetase (hGSHS) mRNA and activity (MH Cruz de Carvalho, J Brunet, A Lameta, Y Zuily-Fodil and D Contour-Ansel, unpublished data).Besides the chemical difference of the two thiol tripeptides, many of the roles ascribed to GSH are also performed by hGSH,^18,19 particularly the control of the cellular redox status and ROS scavenging.²⁰ However, the presence of hGSH questions on the occurrence of a homoglutathione reductase (hGR). A role for hGR as a detoxifying enzyme of the ascorbate-glutathione cycle has been suggested, using hGSSG (oxidized homoglutathione) instead of GSSG (oxidized glutathione), thus maintaining the cellular homoglutathione pool in the reduced state and acting as an antioxidative molecule in these plants.^21,22 It can hence be suggested that in beans and other leguminous plants where both thiols co-exist (hGSH and GSH), the (h)GR enzyme will act as either a GR or a hGR in accordance to the thiol utilized in the ascorbate-glutathione cycle.     

Modulation of glyceraldehyde-3-phosphate dehydrogenase inAnacystis nidulans by glutathione

Glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13; GAPDH) from the cyanobacteriumAnacystis nidulans was activated up to five-fold by reduced glutathione (GSH) in the physiological concentration range (0.1–2 mM GSH). Non-physiological reductants, like dithiothreitol (DTT) and -mercaptoethanol, also activated the enzyme. Oxidized glutathione (GSSG) had no effect on the cyanobacterial GAPDH but treatment with H₂O₂ led to a rapid, reversible deactivation of both untreated and GSH-treated enzyme preparations. GSH reversed the inhibition induced by H₂O₂. An oligomeric form of the enzyme (apparentM _r440,000) was dissociated by GSH into a lower-M _r, more active enzyme form (M _r200,000). The enzyme was shown to obey regular Michaelis-Menten kinetics. The activation of GAPDH by GSH was associated with a decrease inK _m and an increase inV _max values of the enzyme for 3-phosphoglycerate. GSH had virtually no effect on a GAPDH preparation isolated from corn chloroplasts and studied for comparison.Abbreviations GAPDH glyceraldehyde-3-phosphate dehydrogenase - GSH reduced glutathione - GSSG oxidized glutathione - DTT dithiothreitol     

Properties and physiological function of a glutathione reductase purified from spinach leaves by affinity chromatography

Glutathione reductase (EC 1.6.4.2) was purified from spinach (Spinacia oleracea L.) leaves by affinity chromatography on ADP-Sepharose. The purified enzyme has a specific activity of 246 enzyme units/mg protein and is homogeneous by the criterion of polyacrylamide gel electrophoresis on native and SDS-gels. The enzyme has a molecular weight of 145,000 and consists of two subunits of similar size. The pH optimum of spinach glutathione reductase is 8.5–9.0, which is related to the function it performs in the chloroplast stroma. It is specific for oxidised glutathione (GSSG) but shows a low activity with NADH as electron donor. The pH optimum for NADH-dependent GSSG reduction is lower than that for NADPH-dependent reduction. The enzyme has a low affinity for reduced glutathione (GSH) and for NADP⁺, but GSH-dependent NADP⁺ reduction is stimulated by addition of dithiothreitol. Spinach glutathione reductase is inhibited on incubation with reagents that react with thiol groups, or with heavymetal ions such as Zn²⁺. GSSG protects the enzyme against inhibition but NADPH does not. Pre-incubation of the enzyme with NADPH decreases its activity, so kinetic studies were performed in which the reaction was initiated by adding NADPH or enzyme. The Km for GSSG was approximately 200 M and that for NADPH was about 3 M. NADP⁺ inhibited the enzyme, assayed in the direction of GSSG reduction, competitively with respect to NADPH and non-competitively with respect to GSSG. In contrast, GSH inhibited non-competitively with respect to both NADPH and GSSG. Illuminated chloroplasts, or chloroplasts kept in the dark, contain equal activities of glutathione reductase. The kinetic properties of the enzyme (listed above) suggest that GSH/GSSG ratios in chloroplasts will be very high under both light and dark conditions. This prediction was confirmed experimentally. GSH or GSSG play no part in the light-induced activation of chloroplast fructose diphosphatase or NADP⁺-glyceraldehyde-3-phosphate dehydrogenase. We suggest that GSH helps to stabilise chloroplast enzymes and may also play a role in removing H₂O₂. Glucose-6-phosphate dehydrogenase activity may be required in chloroplasts in the dark in order to provide NADPH for glutathione reductase.Abbreviations GSH reduced form of the tripeptide glutathione - GSSG oxidised form of glutathione     

Light-dependent Reduction of Oxidized Glutathione by Ruptured Chloroplasts

Crude extracts of pea shoots (Pisum sativum) catalyzed oxidized glutathione (GSSG)-dependent oxidation of NADPH which was attributed to NADPH-specific glutathione reductase. The pH optimum was 8 and the K_m values for GSSG and NADPH were 23 μm and 4.9 μm, respectively. Reduced glutathione (GSH) inhibited the reaction. Crude extracts also catalyzed NADPH-dependent reduction of GSSG; the ratio of the rate of NADPH oxidized to GSH formed was 0.49. NADH and various substituted mono- and disulfides would not substitute for NADPH and GSSG respectively. Per mg of chlorophyll, enzyme activity of isolated chloroplasts was 69% of the activity of crude extracts.     

Glutathione depletion in lung cells by low-molecular-weight aldehydes

Use of oxygenates in gasoline in the United States may increase atmospheric levels of aldehydes. To assist in health assessments of inhalation exposure to aldehydes, we studied glutathione (GSH) depletion by low-molecular-weight n-alkanals and 2-alkenals, ubiquitous air pollutants, in adult rat lung (ARL) cells by laser cytometry. For each homologous series, the effective aldehyde concentration that depleted GSH by 50% (EC₅₀) in ARL cells correlates with published values for the median lethal dose of the chemicals and with Hammett/Taft electronic parameters, * for n-alkanals and _p* for 2-alkenals. n-Alkanals (EC₅₀, 110–400 mmol/L) were 1000 times less effective in depleting GSH than were 2-alkenals (EC₅₀, 2–180 mol/L), of which acrolein was the most potent. Ability of the 2-alkenals to deplete GSH follows the second-order rate constant for adduct formation. Ability of n-alkanals to deplete GSH follows chain length. Within a homologous series of low-molecular-weight aldehydes, structure–activity relationships are useful for predicting the toxicity of the aldehydes in vitro and in vivo.     

Disulfiram augments oxidative stress in rat brain following bilateral carotid artery occlusion

We examined the brain oxidative stress which accompanies 30 min of bilateral carotid artery ligation (BCAL) in terms of changes in brain levels of glutathione; reduced (GSH) and oxidized (GSSG) forms and the exacerbation of oxidative stress by disulfiram (DSF). These results indicate that BCAL alone decreases GSH content and limits glutathione reductase (GR) activity, and these changes were enhanced by DSF pretreatment. Similar observations were recorded with DSF alone. GR activity (74.3±4.0 µmol min^–1 mg^–1 tissue; p<0.001) and GSH content (1.23±0.06 µmol min^–1g^–1 tissue; p<0.001) was attenuated in rats subjected to synergistic effect of BCAL and DSF with a concomitant increase of GSSG (0.006±0.006 µmol min^–1 g^–1 tissue; p<0.001). Recovery of GSH/GSSG level and GR activity during reperfusion following 30 min BCAL was considerably delayed (96 h) in the BCAL and DSF group as compared to the recovery time of 24 h in the group subjected to BCAL-reperfusion alone. Perturbation of GSH/GSSG homeostasis as a result of BCAL was augmented by DSF. These findings clearly demonstrate central nervous system oxidative stress due to a BCAL-DSF synergistic effect. Based on the results obtained with this model, we conclude that DSF increases brain oxidative stress and this may be detrimental to alcoholics who might drink and develop an acetaldehyde-induced hypotension while taking DSF.