Isoforms Of Glutathione Reductase Research Articles

NOX-like ROS production by glutathione reductase

SummaryIn organisms from bacteria to mammals, NADPH oxidase (NOX) catalyzes the production of beneficial reactive oxygen species (ROS) such as superoxide (O2−). However, our previous research implicated glutathione reductase (GR), a canonical antioxidant enzyme, as a source of extracellular superoxide in the marine diatom Thalassiosira oceanica. Here, we expressed and characterized the two GR isoforms of T. oceanica. Both coupled the oxidation of NADPH, the native electron donor, to oxygen reduction, giving rise to superoxide in the absence of glutathione disulfide, the native electron acceptor. Superoxide production by ToGR1 exhibited similar kinetics as representative NOX enzymes, and inhibition assays agreed with prior organismal studies, supporting a physiological role. ToGR is similar to GR from human, yeast, and bacteria, suggesting that NOX-like ROS production by GR could be widespread. Yet unlike NOX, GR-mediated ROS production is independent of iron, which may provide an advantageous way of making ROS under micronutrient stress.

Toxic Effects of Imidazolium Ionic Liquids on the Green Seaweed Ulva lactuca: Oxidative Stress and DNA Damage

The green credentials of ionic liquids (ILs) are being increasingly questioned due to the growing evidence of their toxicity to aquatic ecosystems, although the mechanisms of toxicity are unknown. This study provides insights into the mechanism of toxicity and biological effects of 1-alkyl-3-methylimidazolium bromide [C(n)mim]Br (n = 4 to 16) on the marine macroalga Ulva lactuca. The cell viability of this alga during IL exposure was found to be negatively correlated to the chain length of the alkyl group. The IL ([C(12)mim]Br) exposure triggers the generation of reactive oxygen species (ROS viz. O(2)(•-), H(2)O(2), and OH(•)), damage of the membrane and DNA, and inhibition of antioxidant systems in the alga. The enhanced production of ROS and lipid peroxidation in the alga subjected to LC(50) concentration for 4 days was largely attributed to lipoxygenase (LOX) activity coupled with the induction of two LOX isoforms (~80 kDa and ~55 kDa). Pretreatment of the algal thallus with enzyme inhibitors such as diphenylene iodonium, sodium azide, cantharidin, and oxadiazoloquinoxalin-1-one, prior to [C(12)mim]Br exposure showed the regulation of ROS by the activation of membrane bound NADPH-oxidase and cytochrome oxidase. The IL exposure resulted in the accumulation of n-3 and n-6 fatty acids at 0.5 LC(50) concentration indicating the induction of desaturase enzymes. Furthermore, antioxidant enzyme activities such as superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase (GR) were enhanced by 1.3-2.0-fold, while glutathione peroxidase (GSH-Px) diminished, together with a higher regeneration rate of reduced ascorbate and glutathione. The isoforms of antioxidant enzymes, namely, Mn-SOD (~85 kDa), APX (~125 and 45 kDa), and GR (~135 kDa) regulated differentially to IL exposure. The comet assay performed for the first time for seaweeds revealed the significant induction of DNA damage (>50-70% increase in % tail DNA over control) in alga exposed to ≥ LC(50) concentration.

Ascorbate and glutathione metabolism in embryo axes and cotyledons of germinating lupine seeds

Changes in ascorbate and glutathione contents and the activities and isoenzyme patterns of enzymes of the ascorbate-glutathione cycle were investigated in embryo axes and cotyledons of germinating lupine (Lupinus luteus L.) seeds. Ascorbate content was not significantly affected over the initial 12 h of imbibition in embryo axes, but afterwards increased, with the most rapid accumulation coinciding with radicle emergence. A somewhat similar trend was observed for glutathione with significant increase in embryo axes shortly before radicle protrusion followed by decline in the next hours. In cotyledons the ascorbate pool rose gradually during germination but the amount of glutathione showed fluctuations during a whole germination period. The activity of ascorbate peroxidase (APX) rose progressively in embryo axes, while activities of dehydroascorbate reductase (DHAR) and glutathione reductase (GR) showed transient increase during germination. New isoforms of APX and GR were synthesized, suggesting that they play a relevant role during germination. All analyzed enzymes were already present in dry seeds which allowed them to be active immediately after imbibition.

Changes in the pattern of antioxidant enzymes in wheat exposed to water deficit and rewatering

Antioxidant defenses in two wheat cultivars differing in sensitivity to dehydration (YouJian (YJ-24) more sensitive than LongChun (LC-20) were analyzed during water deficit and rewatering. Resistant cultivar (LC-20) showed a higher relative water content than the sensitive cultivar (YJ-24) during the whole period of water withholding. In order to analyze the changes of antioxidant enzymes, native PAGE analysis of protein extract were performed. Wheat leaves had two isoforms of Mn-superoxide dismutase (SOD), two isoforms of Cu/Zn-SOD and one of Fe-SOD. Three catalase (CAT) isoforms were identified in the leaves of wheat. The activities of SOD and CAT isoforms were increased in two cultivars under water deficit. The intensities of SOD and CAT isoforms were slightly lower in LC-20 and increased continuously in YJ-24 after rewatering. Peroxidase (POD) isoforms were significantly increased during the whole dehydration-rehydration period. Three ascorbate peroxidase (APX) isoforms were present in gel. APX-1 and APX-3 were enhanced during water deficit and decreased during rewatering in LC-20. In YJ-24 only the activities of APX-2 were increased under water deficit. Seven isoforms of glutathione reductase (GR) were detected in the native gel. Activities of most of GR isoforms were higher in tolerant (LC-20) than in sensitive cultivar (YJ-24). Different isoforms of GR in two wheat cultivars behaved differently under water deficit and rewatering. These results collectively suggest that water deficit activates the SOD, CAT and ascorbate-glutathione cycle in wheat leaves. The response of enzyme isoforms to drought is not the same for all isoforms of antioxidant enzymes in two wheat cultivars.

Investigations on the antioxidative defence responses to NaCl stress in a mangrove, Bruguiera parviflora: Differential regulations of isoforms of some antioxidative enzymes

Two-month-old healthy seedlings of a true mangrove, Bruguiera parviflora, raised from propagules in normal nursery conditions were subjected to varying concentrations of NaCl for 45 d under hydroponic culture conditions to investigate the defence potentials of antioxidative enzymes against NaCl stress imposed oxidative stress. Changes in the activities of the antioxidative enzymes catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (POX), glutathione reductase (GR) and superoxide dismutase (SOD) were assayed in leaves to monitor the temporal regulation. Among the oxidative stress triggered chemicals, the level of H2O2 was significantly increased while total ascorbate and total glutathione content decreased. The ratio of reduced to oxidized glutathiones, however, increased due to decreased levels of oxidized glutathione in the leaf tissue. Among the five antioxidative enzymes monitored, the APX, POX, GR and SOD specific activities were significantly enhanced at high concentration (400 mM NaCl), while the catalase activities declined, suggesting both up and downregulations of antioxidative enzymes occurred due to NaCl imposed osmotic and ionic stress. Analysis of the stress induced alterations in the isoforms of CAT, APX, POX, GR and SOD revealed differential regulations of the isoforms of these enzymes. In B. parviflora one isoform of each of Mn-SOD and Cu/Zn-SOD while three isoforms of Fe-SOD were observed by activity staining gel. Of these, only Mn-SOD and Fe-SOD2 content was preferentially elevated by NaCl treatment, whereas isoforms of Cu/Zn-SOD, Fe-SOD1 and Fe-SOD3 remained unchanged. Similarly, out of the six isoforms of POX, the POX-1,-2,-3 and -6 were enhanced due to salt stress but the levels of POX-4 and -5 remained same as in control plants suggesting preferential upregulation of selective POX isoforms. Activity staining gel revealed only one prominent band of APX and this band increased with increased salt concentration. Similarly, two isoforms of GR (GR1 and GR2) were visualized on activity staining gel and both these isoforms increased upon salt stress. In this mangrove four CAT-isoforms were identified, among which the prominent CAT-2 isoform level was maximally reduced again suggesting differential downregulation of CAT isoforms by NaCl stress. The results presented in this communication are the first report on the resolutions of isoforms APX, POX and GR out of five antioxidative enzymes studied in the leaf tissue of a true mangrove. The differential changes in the levels of the isoforms due to NaCl stress may be useful as markers for recognizing salt tolerance in mangroves. Further, detailed analysis of the isoforms of these antioxidative enzymes is required for using the various isoforms as salt stress markers. Our results indicate that the overproduction of H2O2 by NaCl treatment functions as a signal of salt stress and causes upregulation of APX, POX, GR and deactivations of CAT in B. parviflora. The concentrations of malondialdehyde, a product of lipid peroxidation and lipoxygenase activity remained unchanged in leaves treated with different concentrations of NaCl, which again suggests that the elevated levels of the antioxidant enzymes protect the plants against the activated oxygen species thus avoiding lipid peroxidation during salt stress.

Defense potentials to NaCl in a mangrove, Bruguiera parviflora: Differential changes of isoforms of some antioxidative enzymes

In order to assess the role of the antioxidative defense system against salt treatment, the activities of some antioxidative enzymes and levels of antioxidants were monitored in a true mangrove, Bruguiera parviflora, subjected to varying levels of NaCl under hydroponic culture. In the leaves of B. parviflora, salt treatment preferentially enhanced the content of H2O2 as well as the activity of ascorbate peroxidase (APX), guaiacol peroxidase (GPX), glutathione reductase (GR), and superoxide dismutase (SOD), whereas it induced the decrease of total ascorbate and glutathione (GSH+GSSG) content as well as catalase (CAT) activity. Analysis of isoforms of antioxidative enzymes by native PAGE and activity staining revealed that leaves of B. parviflora had one isoform each of Mn-SOD and Cu/Zn-SOD and three isoforms of Fe-SOD. Expression of Mn-SOD and Fe-SOD-2 was preferentially elevated by NaCl. Similarly, out of the six isoforms of GPX, the GPX-1, 2, 3 and 6 were enhanced by salt treatment but the levels of GPX-4 and -5 changed minimally as compared to those of a control. Activity staining gel revealed only one prominent isoform of APX and two isoforms of GR (GR-1 and GR-2), all of these isoforms increased upon salt exposure. Four CAT-isoforms were identified, among which the prominent CAT-2 isoform level was maximally reduced, suggesting differential down regulation of CAT isoforms by NaCl. The concentrations of malondialdehyde (MDA), a product of lipid peroxidation, remained unchanged in leaves of the plant treated with different concentrations of NaCl. This suggests that plants are protected against activated oxygen species by the elevated levels of certain antioxidative enzymes, thus avoiding lipid peroxidation during salt exposure. The differential changes in the levels of the isoforms due to NaCl treatment may be useful as markers for recognizing salt tolerance in mangroves.

Biochemical and Molecular Characterisation of Wheat Chloroplastic Glutathione Reductase

Wheat leaves contain two charge/mass-separable isoforms of glutathione reductase (GR, EC 1.6.4.2), one chloroplastic and the other probably cytosolic. The chloroplastic GR was purified to homogeneity, and its biochemical and molecular characterisation showed features very similar to the other plant GRs. In its native conformation the enzyme is composed by two subunits of 56 kDa and an associated polypeptide of 32 kDa, with an overall molecular mass of approximately 150 kDa. Optimum activity was observed at pH 8.00 and with an ionic strength between 60 to 100 mM. GR activity is highly sensitive to temperature changes, exhibiting an exponential increase up to 45 °C. It showed a high affinity for oxidised glutathione and an intermediate affinity for NADPH at pH 8.0. Inhibition tests with thiol and histidine modifiers demonstrated that -SH groups and histidine residues are essential for the catalytic properties of the enzyme. T study the origin of GR isoforms, the number of GR gene copies and the number and size of GR transcripts were determined. Southern analyses showed that wheat GR isoforms are encoded by multiple gene copies. However, a single size transcript of approximately 1.4 kb was observed, suggesting that different GR isoforms could be generated by post-transcriptional and/or post-translational modifications.

The inductive responses of the antioxidant enzymes by salt stress in the rice ( Oryza sativa L.)

Summary To access contributions of inductive responses of the antioxidant enzymes in the resistance to salt stress, activities of the enzymes were determined in the rice ( Oryza sativa L. cv. Dongjin) plant. In the leaves of the rice plant, salt stress preferentially enhanced the content of H 2 O 2 as well as the activities of the superoxide dismutase (SOD), ascorbate peroxidase (APX), and peroxidase specific to guaiacol, whereas it induced the decrease of catalase activity. On the other hand, salt stress had little effect on the activity levels of glutathione reductase (GR). In order to analyze the changes of antioxidant enzyme isoforms against salt stress, plant extracts were subjected to native PAGE. Leaves of the rice plant had two isoforms of Mn-SOD and five isoforms of Cu/Zn-SOD. Fe-SOD isoform was not observed in the activity gels. Expression of Cu/Zn-1, -2, and Mn-SOD-2 isoforms was preferentially enhanced by salt stress. Seven APX isoforms were presented in the leaves of the rice plants. The intensities of APX-4 to -7 were enhanced by salt stress, whereas those of APX-1 to -3 were minimally in changed response to salt stress. There were seven GR isoforms in the leaves of rice plants. Levels of activity for most GR isoforms did not change in the stressed plants compared to the control plants. On the other hand, the levels of activity for most antioxidant enzymes changed little in the roots of stressed plants compared to the control plants. These results collectively suggest that SOD leads to the overproduction of hydrogen peroxide in the leaves of rice plants subjected to salt stress: The overproduction of hydrogen peroxide functions as the signal of salt stress, which induces the induction of specific APX isoforms but not specific GR isoforms under catalase deactivation.

The ascorbate-glutathione cycle in the cytosolic and chloroplastic fractions of drought-tolerant and drought-sensitive poplars

Summary To determine whether a drought-tolerant Populus euramericana clone (Dorskamp) exhibits a more efficient reactive oxygen species (ROS)-scavenging system than a drought-sensitive one (Luisa Avanzo), we determined the activity of two enzymes of the ascorbate-glutathione cycle. Because ROS were mainly generated in illuminated chloroplasts, cytosolic and chloroplastic ascorbate peroxidase (AP) and glutathione reductase (GR) were followed in seedlings exposed for 12 h to control or 100 mmol/L mannitol (corresponding to -0.224 MPa). Whatever the treatment, the activities of plastidial ascorbate peroxidase (AP) and glutathione reductase (GR) were lower than those of cytosolic fractions. Mannitol treatment did not affect plastidial GR activity of Dorskamp, but increased cytosolic AP activity. Although ROS were not produced in 12 h stressed Luisa Avanzo, decreases in cytosolic AP and GR activities and increases in plastidial AP activity occurred. Changes in apparent Km values of the two enzymes were observed in both subcellular compartments of each stressed clone. When AP and GR of both clones were incubated in the presence of mannitol in vitro, the percent of inhibition of plastidial AP and GR was the highest in Luisa Avanzo. Whatever the clones, the GR isoforms of cytosolic and plastidial fractions were differently responsive to ROS-generated in vitro and to inhibitors of catalase and AP. The consequences of the efficiency of the ascorbate-glutathione cycle in each subcellular compartment and of the differential reactivity of AP and GR towards mannitol and ROS are discussed for each clone with regard to their drought responses.

Chilling stress-induced changes of antioxidant enzymes in the leaves of cucumber: in gel enzyme activity assays

To investigate the antioxidant defense system, chilling stress-induced changes of antioxidant enzymes were examined in the leaves of cucumber ( Cucumis sativus L.). Chilling stress preferentially enhanced the activities of the superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR) and peroxidase specific to guaiacol, whereas it induced the decrease of catalase activity. In order to analyze the changes of antioxidant enzyme isoforms against chilling stress, foliar extracts were subjected to native PAGE. Leaves of cucumber had four isoforms of Mn-SOD and two isoforms of Cu/Zn-SOD. Fe-SOD isoform was not observed in this plant. Expression of Cu/Zn-SOD and Mn-SOD was preferentially enhanced by chilling stress. Expression of Mn-SOD-2 and -4 was enhanced after 48 h of the poststress period. Five APX isoforms were presented in the leaves of cucumber. The intensities of APX-4 and -5 were enhanced by chilling stress, whereas that of APX-3 was significantly increased in the poststress periods after chilling stress. Gel stained for GR activity revealed six isoforms in the plant. Activation levels for most of GR isoforms were higher in the stressed-plants than the control and poststressed-plants, but that of GR-1 isoform was significantly higher in the poststressed-plants than chilling stressed-plants. These results collectively suggest that chilling stress activates the enzymes of an SOD/ascorbate-glutathione cycle under catalase deactivation in the leaves of cucumber, but the response timing of enzyme isoforms against various environmental stresses is not the same for all isoforms of antioxidant enzymes.

Cloning and characterisation of glutathione reductase cDNAs and identification of two genes encoding the tobacco enzyme.

We have isolated 4 cDNA clones (GRT1-4) encoding glutathione reductase (GR) from a tobacco (Nicotiana tabacum L.) leaf cDNA library. The cDNAs were almost identical: GRT1, GRT3 and GRT4 represented the same gene, differing only in that GRT4 contained an intron within the C-terminal part of the coding sequence. Failure to splice out this intron resulted in a substitution of the final 13 amino acids of the deduced amino acid sequence. A second gene was represented by GRT2. Southern blots indicated that there were two related GR genes in tobacco. The presence of multiple isoforms of GR in tobacco may be explained in part by the expression of a small gene family. In addition, alternative isoforms may result from translation of different mRNAs derived from the same gene by intron skipping during the splicing of nascent GR mRNAs.

Occurrence of two isoforms of glutathione reductase in the green alga Chlamydomonas reinhardtii

Two isoforms (isoenzymes) of glutathione reductase (NADPH: oxidized glutathione oxidoreductase, EC 1.6.4.2; GR) were clearly resolved when enzyme preparations partially purified from the unicellular alga Chlamydomonas reinhardtii were subjected to column chromatofocusing in the pH range from 8 to 4. One isoform (GR I) exhibited an almost electroneutral isoelectric point (pI, 6.9–7.1) and the other (GR II) was a very acidic protein (pI, 4.7–4.9). Both GRs are, however, homodimeric flavoproteins with similar molecular masses of approx. 127 kDa. Cross-reaction with an antibody against the cyanobacterial GR allowed determination of their subunit molecular masses by Western blotting after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, a value of 66 kDa being estimated in both cases. The two algal GR isoforms showed similar K m values for the oxidized form of glutathione (approx. 50 μM). However, the K m values for NADPH were different, being 7 μM and 28 μM for GR I and GR II, respectively. The two isoforms also differed in their optimum pH. Thus, whereas GR I showed a clear maximum at neutral pH, GR II exhibited a broader optimum around pH 8.5 and was more active in the alkaline range. The relative contribution of the two isoforms to the total activity in enzyme preparations of cells disrupted by two different methods indicates that GR I should be a cytoplasmic isoform and GR II a plastidic isoform. The physiological roles of the GR isoenzymes found in Chlamydomonas are discussed and some of their properties compared with those of GRs isolated from other photosynthetic organisms.

Seasonal Variation in the Antioxidant System of Eastern White Pine Needles

Antioxidant metabolites in eastern white pine (Pinus strobus L.) needles increased two- to fourfold from the summer to the winter season. Antioxidant enzymes in needle tissue increased between 2- and 122-fold during this same period. These seasonal changes were determined by monitoring ascorbate and glutathione concentrations and the activity of ascorbate peroxidase, glutathione reductase (GR), and superoxide dismutase. Levels of antioxidant metabolites and enzymes were observed always to be lowest during the summer, or active growing season, and highest during the winter, or dormant season. These data correlated well with the thermal kinetic window for purified GR obtained from summer needles. The minimum, apparent K(m,NADPH) for two isoforms of GR (GR(A) and GR(B)) occurred at 5 and 10 degrees C, respectively. The upper limit of the thermal kinetic window (200% of the minimum K(m)) for GR(A) and GR(B) was 20 and 25 degrees C, respectively, indicating that needle temperatures exceeding 25 degrees C may result in impairment of antioxidant metabolism. The needle content and kinetic properties of GR, the increased activities of other enzymes, and the high substrate concentrations observed during the winter are consistent with the protective function this pathway may provide against photooxidative, winter injury.

Purification, Characterization, and Immunological Properties for Two Isoforms of Glutathione Reductase from Eastern White Pine Needles

Glutathione reductase (EC 1.6.4.2) was purified from Eastern white pine (Pinus strobus L.) needles. The purification steps included affinity chromatography using 2', 5'-ADP-Sepharose, FPLC-anion-exchange, FPLC-hydrophobic interaction, and FPLC-gel filtration. Separation of proteins by FPLC-anion-exchange resulted in the recovery of two distinct isoforms of glutathione reductase (GR(A) and GR(B)). Purified GR(A) had a specific activity of 1.81 microkatals per milligram of protein and GR(B) had a specific activity of 6.08 microkatals per milligram of protein. GR(A) accounted for 17% of the total units of glutathione reductase recovered after anion-exchange separation and GR(B) accounted for 83%. The native molecular mass for GR(A) was 103 to 104 kilodaltons and for GR(B) was 88 to 95 kilodaltons. Both isoforms of glutathione reductase were dimers composed of identical subunit molecular masses which were 53 to 54 kilodaltons for GR(A) and 57 kilodaltons for GR(B). The pH optimum for GR(A) was 7.25 to 7.75 and for GR(B) was 7.25. At 25 degrees C the K(m) for GSSG was 15.3 and 39.8 micromolar for GR(A) and GR(B), respectively. For NADPH, the K(m) was 3.7 and 8.8 micromolar for GR(A) and GR(B), respectively. Antibody produced from purified GR(B) was reactive with both native and denatured GR(B), but was cross-reactive with only native GR(A).