Scavenger Enzyme Activities in Subcellular Fractions of White Clover (Trifolium repens L.) under PEG-induced Water Stress

Scavenger enzyme activities in subcellular fractions under polyethylene glycol (PEG)-induced water stress in white clover (Trifolium repens L.) were studied. Water stress decreased ascorbic acid (AA) content and catalase (CAT) activity and increased the contents of hydrogen peroxide (H₂O₂), thiobarbituric acid reactive substances (TBARS) (measure of lipid peroxidation), and activities of superoxide dismutase (SOD), its various isozymes, ascorbate peroxidase (APOX), and glutathione reductase (GR) in cellular cytosol, chloroplasts, mitochondria, and peroxisomes of Trifolium repens leaves. In both the PEG-treated plants and the control, chloroplastic fractions showed the highest total SOD, APOX, and GR activities, followed by mitochondrial fractions in the case of total SOD and GR activities, whereas cytosolic fractions had the second greatest APOX activity. However, CAT activity was the highest in peroxisomes, followed by the cytosol, mitochondria, and chloroplasts in decreasing order. Although Mn-SOD activity was highest in mitochondrial fractions, residual activity was also observed in cytosolic fractions. Cu/Zn-SOD and Fe-SOD were observed in all subcellular fractions; however, the activities were the highest in chloroplastic fractions for both isoforms. Total Cu/Zn-SOD activity, the sum of activities observed in all fractions, was higher than other SOD isoforms. These results suggest that cytosolic and chloroplastic APOX, chloroplastic and mitochondrial GR, mitochondrial Mn-SOD, cytosolic and chloroplastic Cu/Zn-SOD, and chloroplastic Fe-SOD are the major scavenger enzymes, whereas cellular CAT may play a minor role in scavenging of O₂⁻ and H₂O₂ produced under PEG-induced water stress in Trifolium repens.     

Increased levels of peroxisomal active oxygen-related enzymes in copper-tolerant pea plants

The effect in vivo of high nutrient levels of copper (240 micromolar) on the activity of different metalloenzymes containing Cu, Mn, Fe, and Zn, distributed in chloroplasts, peroxisomes, and mitochondria, was studied in leaves of two varieties of Pisum sativum L. plants with different sensitivity to copper. The metalloenzymes studied were: cytochrome c oxidase, Mn-superoxide dismutase (Mn-SOD) and Cu,Zn-superoxide dismutase I (Cu,Zn-SOD I), for mitochondria; catalase and Mn-SOD, for peroxisomes; and isozyme Cu,Zn-SOD II for chloroplasts. The activity of mitochondrial SOD isozymes (Mn-SOD and Cu,Zn-SOD I) was very similar in Cu-tolerant and Cu-sensitive plants, whereas cytochrome c oxidase was lower in Cu-sensitive plants. Chloroplastid Cu,Zn-SOD activity was the same in the two plant varieties. In contrast, the peroxisomal Mn-SOD activity was considerably higher in Cu-tolerant than in Cu-sensitive plants, and the activity of catalase was also increased in peroxisomes of Cu-tolerant plants. The higher activities of these peroxisomal active oxygen-related enzymes in Cu-tolerant plants suggest the involvement of reactive oxygen intermediates (O₂⁻, OH) in the mechanism of Cu lethality, and also imply a function for peroxisomal Mn-SOD in the molecular mechanisms of plant tolerance to Cu in Pisum sativum L.     

Dynamic Regulation of Ero1α and Peroxiredoxin 4 Localization in the Secretory Pathway

In the early secretory compartment (ESC), a network of chaperones and enzymes assists oxidative folding of nascent proteins. Ero1 flavoproteins oxidize protein disulfide isomerase (PDI), generating H₂O₂ as a byproduct. Peroxiredoxin 4 (Prx4) can utilize luminal H₂O₂ to oxidize PDI, thus favoring oxidative folding while limiting oxidative stress. Interestingly, neither ER oxidase contains known ER retention signal(s), raising the question of how cells prevent their secretion. Here we show that the two proteins share similar intracellular localization mechanisms. Their secretion is prevented by sequential interactions with PDI and ERp44, two resident proteins of the ESC-bearing KDEL-like motifs. PDI binds preferentially Ero1α, whereas ERp44 equally retains Ero1α and Prx4. The different binding properties of Ero1α and Prx4 increase the robustness of ER redox homeostasis.     

T-LAK Cell-originated Protein Kinase (TOPK) Phosphorylation of Prx1 at Ser-32 Prevents UVB-induced Apoptosis in RPMI7951 Melanoma Cells through the Regulation of Prx1 Peroxidase Activity

Protein kinases are potential targets for the prevention and control of UV-induced skin cancer. T-cell-originated protein kinase (TOPK) is highly expressed in skin cancer cells, but its specific function is still unknown. We investigated the role of TOPK in UVB-induced apoptosis in RPMI7951 human melanoma cells. Liquid chromatography-tandem mass spectrometry analysis was used to identify proteins that bind with TOPK. Immunofluorescence, Western blot, and flow cytometry were used to assess the effect of UVB on TOPK, peroxiredoxin 1 (Prx1), and apoptosis in RPMI7951 cells. TOPK binds with Prx1 and its phosphorylation of Prx1 at Ser-32 is important for regulation of H₂O₂-mediated signal transduction. Analysis of the CD spectra of Prx1 and mutant Prx1 (S32A) proteins showed that the secondary structure of Prx1 was significantly altered by phosphorylation of Prx1 at Ser-32. UVB irradiation induced phosphorylation of TOPK in RPMI7951 human melanoma cells and phosphorylated TOPK co-localized with Prx1 in the nucleus. UVB induced the peroxidase activity of Prx1 in vitro and ex vivo. Following treatment with UVB, H₂O₂ levels and apoptosis were increased in RPMI7951 cells stably expressing TOPK siRNA or stably mutant Prx1 (S32A). Phosphorylation of Prx1 (Ser-32) by TOPK prevents UVB-induced apoptosis in RPMI7951 melanoma cells through regulation of Prx1 peroxidase activity and blockade of intracellular H₂O₂ accumulation.     

Some Enzymes of Hydrogen Peroxide Metabolism in Leaves and Root Nodules of Medicago sativa

Leaves and nodules (bacteroids and cytosol) of alfalfa (Medicago sativa L. cv Aragon) plants inoculated with Rhizobium meliloti strain 102F51 have been analyzed for the presence of the enzymes superoxide dismutase (SOD, EC 1.15.1.1), catalase (EC 1.11.1.6), and peroxidase (EC 1.11.1.7). All three fractions investigated (leaves, bacteroids, and nodular cytosol) show Cu,Zn-SOD activity. Besides, the bacteroids and cytosol of nodules possess CN⁻-insensitive SOD activities. Studies of SOD inactivation with H₂O₂ indicate that, very likely, a Mn-SOD is present in the bacteroids, and suggest that the cytosol contain both Mn-SOD and Fe-SOD. Bacteroids show high catalase activity but lack peroxidase. By contrast, the nodule cytosol exhibits an elevated peroxidase activity as compared with the foliar tissue; this activity was completely inhibited by 50 to 100 micromolar KCN. The significantly lower contents of H₂O₂ and malondialdehyde (a product of lipid peroxidation) in nodules with respect to those in leaves reveal that the above-mentioned bacteroid and cytosol enzymes act in an efficient and combined manner to preserve integrity of nodule cell membranes and to keep leghemoglobin active.     

鉴别超氧化物歧化酶类型的定位染色法

介绍一种鉴别SOD类型的方法─—聚丙烯酰胺凝胶电泳的定位染色法. 由于不同类型的SOD对抑制剂的表现各异, 电泳后的凝胶经不同的抑制剂处理, 染色, 结果展示在凝胶上, CuZn-SOD酶带在H₂O₂或CN^-的作用下消失, Mn-SOD在CHCl₃-CH₃OH作用下消失, Fe-SOD在H₂O₂或. CHCl₃-CH₃CH₂OH作用下失活, 从酶带消失或存活的情况, 可以判断SOD的类型.     

Moderate intermittent hypoxia/hyperoxia: implication for correction of mitochondrial dysfunction

The purpose of this study was to appreciate the acute hypoxia-induced mitochondrial oxidative damage development and the role of adaptation to hypoxia/hyperoxia (H/H) in correction of mitochondrial dysfunction. It was demonstrated that long-term sessions of moderate H/H [5 cycles of 5 min hypoxia (10% O₂ in N₂) alternated with 5 min hyperoxia (30% O₂ in N₂) daily for two weeks]_attenuated basal and Fe²⁺/ascorbate-induced lipid peroxidation (LPO) as well as production of carbonyl proteins and H₂O₂ in liver mitochondria of rats exposed to acute severe hypoxia (7% O₂ in N₂, 60 min) in comparison with untreated animals. It was shown that H/H increases the activity of glutathione peroxidase (GPx), reduces hyperactivation of Mn-SOD, and decreases Cu,Zn-SOD activity as compared with untreated rats. It has been suggested that the induction of Mn-SOD protein expression and the coordinated action of Mn-SOD and GPx could be the mechanisms underlying protective effects of H/H, which promote the correction of the acute hypoxia-induced mitochondrial dysfunction. The increase in Mn-SOD protein synthesis without changes in Mn-SOD mRNA level under H/H pretreatment indicates that the Mn-SOD activity is most likely dependent on its posttranslational modification or on the redox state of liver mitochondria.     

Functional Characterization of Peroxiredoxins from the Human Protozoan Parasite Giardia intestinalis

The microaerophilic protozoan parasite Giardia intestinalis, causative of one of the most common human intestinal diseases worldwide, infects the mucosa of the proximal small intestine, where it has to cope with O₂ and nitric oxide (NO). Elucidating the antioxidant defense system of this pathogen lacking catalase and other conventional antioxidant enzymes is thus important to unveil novel potential drug targets. Enzymes metabolizing O₂, NO and superoxide anion (O₂ ^−•) have been recently reported for Giardia, but it is yet unknown how the parasite copes with H₂O₂ and peroxynitrite (ONOO⁻). Giardia encodes two yet uncharacterized 2-cys peroxiredoxins (Prxs), GiPrx1a and GiPrx1b. Peroxiredoxins are peroxidases implicated in virulence and drug resistance in several parasitic protozoa, able to protect from nitroxidative stress and repair oxidatively damaged molecules. GiPrx1a and a truncated form of GiPrx1b (deltaGiPrx1b) were expressed in Escherichia coli, purified and functionally characterized. Both Prxs effectively metabolize H₂O₂ and alkyl-hydroperoxides (cumyl- and tert-butyl-hydroperoxide) in the presence of NADPH and E. coli thioredoxin reductase/thioredoxin as the reducing system. Stopped-flow experiments show that both proteins in the reduced state react with ONOO⁻ rapidly (k = 4×10⁵ M⁻¹ s⁻¹ and 2×10⁵ M⁻¹ s⁻¹ at 4°C, for GiPrx1a and deltaGiPrx1b, respectively). Consistent with a protective role against oxidative stress, expression of GiPrx1a (but not deltaGiPrx1b) is induced in parasitic cells exposed to air O₂ for 24 h. Based on these results, GiPrx1a and deltaGiPrx1b are suggested to play an important role in the antioxidant defense of Giardia, possibly contributing to pathogenesis.     

Glutathionylation of the Active Site Cysteines of Peroxiredoxin 2 and Recycling by Glutaredoxin

Peroxiredoxin 2 (Prx2) is a thiol protein that functions as an antioxidant, regulator of cellular peroxide concentrations, and sensor of redox signals. Its redox cycle is widely accepted to involve oxidation by a peroxide and reduction by thioredoxin/thioredoxin reductase. Interactions of Prx2 with other thiols are not well characterized. Here we show that the active site Cys residues of Prx2 form stable mixed disulfides with glutathione (GSH). Glutathionylation was reversed by glutaredoxin 1 (Grx1), and GSH plus Grx1 was able to support the peroxidase activity of Prx2. Prx2 became glutathionylated when its disulfide was incubated with GSH and when the reduced protein was treated with H₂O₂ and GSH. The latter reaction occurred via the sulfenic acid, which reacted sufficiently rapidly (k = 500 m⁻¹ s⁻¹) for physiological concentrations of GSH to inhibit Prx disulfide formation and protect against hyperoxidation to the sulfinic acid. Glutathionylated Prx2 was detected in erythrocytes from Grx1 knock-out mice after peroxide challenge. We conclude that Prx2 glutathionylation is a favorable reaction that can occur in cells under oxidative stress and may have a role in redox signaling. GSH/Grx1 provide an alternative mechanism to thioredoxin and thioredoxin reductase for Prx2 recycling.     

Pretreatment with interferon-γ protects microglia from oxidative stress via up-regulation of Mn-SOD

Microglial cells, resident macrophage-like immune cells in the brain, are exposed to intense oxidative stress under various pathophysiological conditions. For self-defense against oxidative injuries, microglial cells must be equipped with antioxidative mechanisms. In this study, we investigated the regulation of antioxidant enzyme systems in microglial cells by interferon-γ (IFN-γ) and found that pretreatment with IFN-γ for 20 h protected microglial cells from the toxicity of various reactive species such as hydrogen peroxide (H₂O₂), superoxide anion, 4-hydroxy-2(E)-nonenal, and peroxynitrite. The cytoprotective effect of IFN-γ pretreatment was abolished by the protein synthesis inhibitor cycloheximide. In addition, treatment of microglial cells with both IFN-γ and H₂O₂ together did not protect them from the H₂O₂-evoked toxicity. These results imply that protein synthesis is required for the protection by IFN-γ. Among various antioxidant enzymes such as manganese or copper/zinc superoxide dismutase (Mn-SOD or Cu/Zn-SOD), catalase, and glutathione peroxidase (GPx), only Mn-SOD was up-regulated in IFN-γ-pretreated microglial cells. Transfection with siRNA of Mn-SOD abolished both up-regulation of Mn-SOD expression and protection from H₂O₂ toxicity by IFN-γ pretreatment. Furthermore, whereas the activities of Mn-SOD and catalase were up-regulated by IFN-γ pretreatment, those of Cu/Zn-SOD and GPx were not. These results indicate that IFN-γ pretreatment protects microglial cells from oxidative stress via selective up-regulation of the level of Mn-SOD and activity of Mn-SOD and catalase.     

T cell activation induces CuZn superoxide dismutase (SOD)-1 intracellular re-localization,production and secretion

Reactive oxygen species (ROS) behave as second messengers in signal transduction for a series of receptor/ligand interactions. A major regulatory role is played by hydrogen peroxide (H₂O₂), more stable and able to freely diffuse through cell membranes. Copper–zinc superoxide dismutase (CuZn-SOD)-1 is a cytosolic enzyme involved in scavenging oxygen radicals to H₂O₂ and molecular oxygen, thus representing a major cytosolic source of peroxides. Previous studies suggested that superoxide anion and H₂O₂ generation are involved in T cell receptor (TCR)-dependent signaling. Here, we describe that antigen-dependent activation of human T lymphocytes significantly increased extracellular SOD-1 levels in lymphocyte cultures. This effect was accompanied by the synthesis of SOD-1-specific mRNA and by the induction of microvesicle SOD-1 secretion. It is of note that SOD-1 increased its concentration specifically in T cell population, while no significant changes were observed in the “non-T” cell counterpart. Moreover, confocal microscopy showed that antigen-dependent activation was able to modify SOD-1 intracellular localization in T cells. Indeed, was observed a clear SOD-1 recruitment by TCR clusters. The ROS scavenger N-acetylcysteine (NAC) inhibited this phenomenon. Further studies are needed to define whether SOD-1-dependent superoxide/peroxide balance is relevant for regulation of T cell activation, as well as in the functional cross talk between immune effectors.     

Molecular Basis for the Resistance of Human Mitochondrial 2-Cys Peroxiredoxin 3 to Hyperoxidation

Peroxiredoxins (Prxs) detoxify peroxides and modulate H₂O₂-mediated cell signaling in normal and numerous pathophysiological contexts. The typical 2-Cys subclass of Prxs (human Prx1–4) utilizes a Cys sulfenic acid (Cys-SOH) intermediate and disulfide bond formation across two subunits during catalysis. During oxidative stress, however, the Cys-SOH moiety can react with H₂O₂ to form Cys sulfinic acid (Cys-SO₂H), resulting in inactivation. The propensity to hyperoxidize varies greatly among human Prxs. Mitochondrial Prx3 is the most resistant to inactivation, but the molecular basis for this property is unknown. A panel of chimeras and Cys variants of Prx2 and Prx3 were treated with H₂O₂ and analyzed by rapid chemical quench and time-resolved electrospray ionization-TOF mass spectrometry. The latter utilized an on-line rapid-mixing setup to collect data on the low seconds time scale. These approaches enabled the first direct observation of the Cys-SOH intermediate and a putative Cys sulfenamide (Cys-SN) for Prx2 and Prx3 during catalysis. The substitution of C-terminal residues in Prx3, residues adjacent to the resolving Cys residue, resulted in a Prx2-like protein with increased sensitivity to hyperoxidation and decreased ability to form the intermolecular disulfide bond between subunits. The corresponding Prx2 chimera became more resistant to hyperoxidation. Taken together, the results of this study support that the kinetics of the Cys-SOH intermediate is key to determine the probability of hyperoxidation or disulfide formation. Given the oxidizing environment of the mitochondrion, it makes sense that Prx3 would favor disulfide bond formation as a protection mechanism against hyperoxidation and inactivation.     

Endogenous Superoxide Dismutase Levels Regulate Iron-Dependent Hydroxyl Radical Formation in Escherichia coli Exposed to Hydrogen Peroxide

Aerobic organisms contain antioxidant enzymes, such as superoxide dismutase (SOD) and catalase, to protect them from both direct and indirect effects of reactive oxygen species, such as O₂^·− and H₂O₂. Previous work by others has shown that Escherichia coli mutants lacking SOD not only are more susceptible to DNA damage and killing by H₂O₂ but also contain larger pools of intracellular free iron. The present study investigated if SOD-deficient E. coli cells are exposed to increased levels of hydroxyl radical (^·OH) as a consequence of the reaction of H₂O₂ with this increased iron pool. When the parental E. coli strain AB1157 was exposed to H₂O₂ in the presence of an α-(4-pyridyl-1-oxide)-N-tert-butyl-nitrone (4-POBN)–ethanol spin-trapping system, the 4-POBN–^·CH(CH₃)OH spin adduct was detectable by electron paramagnetic resonance (EPR) spectroscopy, indicating ^·OH production. When the isogenic E. coli mutant JI132, lacking both Fe- and Mn-containing SODs, was exposed to H₂O₂ in a similar manner, the magnitude of ^·OH spin trapped was significantly greater than with the control strain. Preincubation of the bacteria with the iron chelator deferoxamine markedly inhibited the magnitude of ^·OH spin trapped. Exogenous SOD failed to inhibit ^·OH formation, indicating the need for intracellular SOD. Redox-active iron, defined as EPR-detectable ascorbyl radical, was greater in the SOD-deficient strain than in the control strain. These studies (i) extend recent data from others demonstrating increased levels of iron in E. coli SOD mutants and (ii) support the hypothesis that a resulting increase in ^·OH formation generated by Fenton chemistry is responsible for the observed enhancement of DNA damage and the increased susceptibility to H₂O₂-mediated killing seen in these mutants lacking SOD.     

The Extracellular A-loop of Dual Oxidases Affects the Specificity of Reactive Oxygen Species Release

NADPH oxidase (Nox) family proteins produce superoxide (O₂^⨪) directly by transferring an electron to molecular oxygen. Dual oxidases (Duoxes) also produce an O₂^⨪ intermediate, although the final species secreted by mature Duoxes is H₂O₂, suggesting that intramolecular O₂^⨪ dismutation or other mechanisms contribute to H₂O₂ release. We explored the structural determinants affecting reactive oxygen species formation by Duox enzymes. Duox2 showed O₂^⨪ leakage when mismatched with Duox activator 1 (DuoxA1). Duox2 released O₂^⨪ even in correctly matched combinations, including Duox2 + DuoxA2 and Duox2 + N-terminally tagged DuoxA2 regardless of the type or number of tags. Conversely, Duox1 did not release O₂^⨪ in any combination. Chimeric Duox2 possessing the A-loop of Duox1 showed no O₂^⨪ leakage; chimeric Duox1 possessing the A-loop of Duox2 released O₂^⨪. Moreover, Duox2 proteins possessing the A-loops of Nox1 or Nox5 co-expressed with DuoxA2 showed enhanced O₂^⨪ release, and Duox1 proteins possessing the A-loops of Nox1 or Nox5 co-expressed with DuoxA1 acquired O₂^⨪ leakage. Although we identified Duox1 A-loop residues (His¹⁰⁷¹, His¹⁰⁷², and Gly¹⁰⁷⁴) important for reducing O₂^⨪ release, mutations of these residues to those of Duox2 failed to convert Duox1 to an O₂^⨪-releasing enzyme. Using immunoprecipitation and endoglycosidase H sensitivity assays, we found that the A-loop of Duoxes binds to DuoxA N termini, creating more stable, mature Duox-DuoxA complexes. In conclusion, the A-loops of both Duoxes support H₂O₂ production through interaction with corresponding activators, but complex formation between the Duox1 A-loop and DuoxA1 results in tighter control of H₂O₂ release by the enzyme complex.     

Nicotinamide Nucleotide Transhydrogenase (Nnt) Links the Substrate Requirement in Brain Mitochondria for Hydrogen Peroxide Removal to the Thioredoxin/Peroxiredoxin (Trx/Prx) System

Mitochondrial reactive oxygen species are implicated in the etiology of multiple neurodegenerative diseases, including Parkinson disease. Mitochondria are known to be net producers of ROS, but recently we have shown that brain mitochondria can consume mitochondrial hydrogen peroxide (H₂O₂) in a respiration-dependent manner predominantly by the thioredoxin/peroxiredoxin system. Here, we sought to determine the mechanism linking mitochondrial respiration with H₂O₂ catabolism in brain mitochondria and dopaminergic cells. We hypothesized that nicotinamide nucleotide transhydrogenase (Nnt), which utilizes the proton gradient to generate NADPH from NADH and NADP⁺, provides the link between mitochondrial respiration and H₂O₂ detoxification through the thioredoxin/peroxiredoxin system. Pharmacological inhibition of Nnt in isolated brain mitochondria significantly decreased their ability to consume H₂O₂ in the presence, but not absence, of respiration substrates. Nnt inhibition in liver mitochondria, which do not require substrates to detoxify H₂O₂, had no effect. Pharmacological inhibition or lentiviral knockdown of Nnt in N27 dopaminergic cells (a) decreased H₂O₂ catabolism, (b) decreased NADPH and increased NADP⁺ levels, and (c) decreased basal, spare, and maximal mitochondrial oxygen consumption rates. Nnt-deficient cells possessed higher levels of oxidized mitochondrial Prx, which rendered them more susceptible to steady-state increases in H₂O₂ and cell death following exposure to subtoxic levels of paraquat. These data implicate Nnt as the critical link between the metabolic and H₂O₂ antioxidant function in brain mitochondria and suggests Nnt as a potential therapeutic target to improve the redox balance in conditions of oxidative stress associated with neurodegenerative diseases.     

Subcellular localization and stress responses of superoxide dismutase isoforms from leaves in the C3-CAM intermediate halophyte Mesembryanthemum crystallinum L.

BSA, bovine serum albumin
CAM, Crassulacean acid metabolism
DTT, dithiothreitol
EDTA, ethylenediaminetetraacetic acid
FPLCfast protein liquid chromatography
HEPES, N-(2-hydroxyethyl)piperazine-?-(ethanesulphonic acid)
ME, β-mercaptoethanol
NBT, nitro blue tetrazolium
PAGE, polyacrylamide gel electrophoresis
SDS, sodium dodecyl sulphate
SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis
Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39)
SOD, superoxide dismutase (EC 1.15.1.1)
TEMED, N,N,?,?-tetramethylethylenediamine
Tris, Tris (hydroxymethyl) aminomethane
Tricine, N-Tris(hydroxymethyl)methylglycine

Treatment of Mesembryanthemum crystallinum for several days with 0·4 kmol m^–3 NaCl in the root medium, in parallel to an increase of the cell sap osmolarity enhances activity of important antioxidative enzymes, such as superoxide dismutases (SODs). M. crystallinum is equipped with three SOD isoforms. These isoforms were identified as Mn-, Fe-, and Cu/Zn-SODs, respectively. Mn-SOD was found in the mitochondrial fraction, Fe-SOD in the chloroplast fraction, and Cu/Zn-SOD is probably localized in the cytosol. The Fe-SOD found in M. crystallinum is the first iron-containing SOD enzyme to be characterized in the plant family Aizoaceae. Salt treatment increases the activity of this isoform earlier than the other SODs. Molecular masses of SOD isoforms were determined as 82, 48 and 34 kDa for Mn-, Fe-, Cu/Zn-SODs, respectively. Native Mn-SOD seems to be a tetramer, while Fe-SOD and Cu/Zn-SOD are dimers. All SOD isoforms show high thermal stability. Mn-SOD is active even after short heating at 90 °C and Fe-SOD at 70 °C. Moreover, high concentrations of β-mercaptoethanol used as a reducing agent did not destroy the function of all isoforms. With the salinity treatment in M. crystallinum, Crassulacean acid metabolism (CAM) is induced. Build-up of large stationary O₂ concentrations in the leaf air spaces is associated with the photosynthetic CO₂ reduction behind closed stomata in phase III of CAM. This illustrates why M. crystallinum may require higher antioxidative activities under NaCl stress and also explains earlier findings that CAM plants are more resistant than C₃ plants to environmental stress as imposed by, for example, SO₂ and O₃.     

Utilizing Natural and Engineered Peroxiredoxins As Intracellular Peroxide Reporters

It is increasingly apparent that nature evolved peroxiredoxins not only as H₂O₂ scavengers but also as highly sensitive H₂O₂ sensors and signal transducers. Here we ask whether the H₂O₂ sensing role of Prx can be exploited to develop probes that allow to monitor intracellular H₂O₂ levels with unprecedented sensitivity. Indeed, simple gel shift assays visualizing the oxidation of endogenous 2-Cys peroxiredoxins have already been used to detect subtle changes in intracellular H₂O₂ concentration. The challenge however is to create a genetically encoded probe that offers real-time measurements of H₂O₂ levels in intact cells via the Prx oxidation state. We discuss potential design strategies for Prx-based probes based on either the redox-sensitive fluorophore roGFP or the conformation-sensitive fluorophore cpYFP. Furthermore, we outline the structural and chemical complexities which need to be addressed when using Prx as a sensing moiety for H₂O₂ probes. We suggest experimental strategies to investigate the influence of these complexities on probe behavior. In doing so, we hope to stimulate the development of Prx-based probes which may spearhead the further study of cellular H₂O₂ homeostasis and Prx signaling.     

Involvement of reactive oxygen species during early stages of ectomycorrhiza establishment between <Emphasis Type="Italic">Castanea sativa</Emphasis> and <Emphasis Type="Italic">Pisolithus tinctorius</Emphasis>

Evidence for the participation of reactive oxygen species (ROS) and antioxidant systems in ectomycorrhizal (ECM) establishment is lacking. In this paper, we evaluated ROS production and the activities of superoxide dismutase (SOD) and catalase (CAT) during the early contact of the ECM fungus Pisolithus tinctorius with the roots of Castanea sativa (chestnut tree). Roots were placed in contact with P. tinctorius mycelia, and ROS production was evaluated by determining the levels of H₂O₂ and O₂ ^·− during the early stages of fungal contact. Three peaks of H₂O₂ production were detected, the first two coinciding with O₂ ^·− bursts. The first H₂O₂ production peak coincided with an increase in SOD activity, whereas CAT activity seemed to be implicated in H₂O₂ scavenging. P. tinctorius growth was evaluated in the presence of P. tinctorius-elicited C. sativa crude extracts prepared during the early stages of fungal contact. Differential hyphal growth that matched the H₂O₂ production profile with a delay was detected. The result suggests that during the early stages of ECM establishment, H₂O₂ results from an inhibition of ROS-scavenging enzymes and plays a role in signalling during symbiotic establishment.     

Unusual location and characterization of Cu/Zn-containing superoxide dismutase from filamentous fungus Humicola lutea

The present study aims to provide new information about the unusual location of Cu/Zn-superoxide dismutase (Cu/Zn-SOD) in lower eukaryotes such as filamentous fungi. Humicola lutea, a high producer of SOD was used as a model system. Subcellular fractions [cytosol, mitochondrial matrix, and intermembrane space (IMS)] were isolated and tested for purity using activity measurements of typical marker enzymes. Evidence, based on electrophoretic mobility, sensitivity to KCN and H₂O₂ and immunoblot analysis supports the existence of Cu/Zn-SOD in mitochondrial IMS, and the Mn-SOD in the matrix. Enzyme activity is almost equally partitioned between both the compartments, thus suggesting that the intermembrane space could be one of the major sites of exposure to superoxide anion radicals. The mitochondrial Cu/Zn-SOD was purified and compared with the previously published cytosolic enzyme. They have identical molecular mass, cyanide- and H₂O₂-sensitivity, N-terminal amino acid sequence, glycosylation sites and carbohydrate composition. The H. lutea mitochondrial Cu/Zn-SOD is the first identified naturally glycosylated enzyme, isolated from IMS. These findings suggest that the same Cu/Zn-SOD exists in both the mitochondrial IMS and cytosol. Ekaterina Krumova and Alexander Dolashki equally contributed to this work.