Superoxide dismutase and hydrogen peroxide formation in Campylobacter sputorum subspecies bubulus

Cell-free extracts of Campylobacter sputorum subspecies bubulus contained superoxide dismutase. The enzyme was located in the cytoplasmic fraction and insensitive to cyanide. After centrifuging a cell-free extract at 144000 x g for 1.5 h the total activity in the supernatant fraction was threefold higher than in the crude cell-free extract. The pellet fraction thus obtained was shown to have a lowering effect on superoxide dismutase activities from different sources in the assay method used here. C. sputorum responded to a raised oxygen tension in the culture by an increase in the superoxide dismutase activity. The ability to produce superoxide anion radicals (O₂ ^-·) during oxidation of formate and lactate was demonstrated. Furthermore C. sputorum was found to produce H₂O₂ while oxidizing formate. In experiments in which the reduction of cytochrome c by formate was followed, step-wise kinetics were observed. One of the steady states then obtained was attributed to the oxidizing action of H₂O₂, because it was abolished by the addition of catalase and lengthened by H₂O₂ added in addition to H₂O₂ formed as a product of formate oxidation. An overall reaction for formate oxidation by C. sputorum is discussed.Abbreviations O₂ ^-· superoxide anion radical - NBT p-nitro blue tetrazolium chloride - ABTS 2,2-azino-di-[3-ethylbenzthiazoline sulfonate (6)] - TL-medium tryptose-lactate medium     

Cotton-fiber germin-like protein. II: Immunolocalization,purification, and functional analysis

Cotton (Gossypium hirsutum L.) contains a germin-like protein (GLP), GhGLP1, that shows tissue-specific accumulation in fiber. The fiber GLP is an oligomeric, glycosylated protein with a subunit size of approximately 25.5 kDa. Accumulation of GhGLP1 occurs during the period of fiber elongation [4–14 days post-anthesis (DPA)]. During early phases of fiber development (2–4 DPA), GhGLP1 localizes to cytoplasmic vesicles as shown by confocal immunofluorescent microscopy. In slightly older fibers (7–10 DPA), GhGLP1 localizes to the apoplast. In other plants, germins and GLPs have been reported to have enzymatic activities including oxalate oxidase (OxO), superoxide dismutase, and ADP-glucose pyrophosphatase. Cotton fiber extracts did not contain OxO activity, nor did intact fibers stain for OxO activity. A four-step purification protocol involving ammonium sulfate precipitation of a 1.0 M NaCl extract, ion-exchange chromatography on DEAE-Trisacryl M, lectin-affinity chromatography, and gel filtration chromatography resulted in electrophoretically pure GhGLP1. While 1.0 M NaCl extracts from 10–14 DPA fiber contained superoxide dismutase and phosphodiesterase activities, GhGLP1 could be separated from both enzyme activities by the purification protocol. Although a GLP accumulates in the cotton fiber apoplast during cell elongation, the function of this protein in fiber growth and development remains unknown.Abbreviations ABP Auxin binding protein - AGPPase ADP-Glucose pyrophosphatase/phosphodiesterase - bis-PNPP Bis-p-nitrophenol phosphate - ConA Concanavalin A - DOA Day of anthesis - DPA Days post-anthesis - GLP Germin-like protein - Mn-SOD Manganese superoxide dismutase - OxO Oxalate oxidase - PBS Phosphate-buffered saline     

Anin vitro effect of soil organic matter fractions and synthetic humic acids on the generation of superoxide radicals

Summary Humic acid, and the acid-extracted residue obtained from it, stimulated the production of superoxide radicals (O₂ ^.–) generated in the xanthine-xanthine oxidase system. Several synthetic humic acids, prepared by the oxidation of simple phenolic substances, also stimulated the production of O₂ ^.– but the degree of stimulation depended on the initial phenol. Fulvic acid and water-extractable soil organic matter were less effective in stimulating O₂ ^.– production than was humic acid. The activity of superoxide dismutase, an enzyme which destroys O₂ ^.–, was also enhanced by HA. In contrast, fulvic acid and water-extractable soil organic matter had little effect on the activity of the dismutase.     

Lethality of hydrogen peroxide in wild type and superoxide dismutase mutants of Escherichia coli. (A hypothesis on the mechanism of H2O2-induced inactivation of Escherichia coli)

The toxicity of H₂O₂ in Escherichia coli wild type and superoxide dismutase mutants was investigated under different experimental conditions. Cells were either grown aerobically, and then treated in M9 salts or K medium, or grown anoxically, and then treated in K medium. Results have demonstrated that the wild type and superoxide dismutase mutants display a markedly different sensitivity to both modes of lethality produced by H₂O₂ (i.e. mode one killing, which is produced by concentrations of H₂O₂ lower than 5 mM, and mode two killing which results from the insult generated by concentrations of H₂O₂ higher than 10 mM). Although the data obtained do not clarify the molecular basis of H₂O₂ toxicity and/or do not explain the specific function of superoxide ions in H₂O₂-induced bacterial inactivation, they certainly demonstrate that the latter species plays a key role in both modes of H₂O₂ lethality. A mechanism of H₂O₂ toxicity in E. coli is proposed, involving the action of a hypothetical enzyme which should work as an O₂^-• generating system. This enzyme should be active at low concentrations of H₂O₂ (<5 mM) and high concentrations of the oxidant (>5 mM) should inactivate the same enzyme. Superoxide ions would then be produced and result in mode one lethality. The resistance at intermediate H₂O₂ concentrations may be dependent on the inactivation of such enzyme with no superoxide ions being produced at levels of H₂O₂ in the range 5–10 mM. Mode two killing could be produced by the hydroxyl radical in concert with superoxide ions, chemically produced via the reaction of high concentrations of H₂O₂ (>10 mM) with hydroxyl radicals. The rate of hydroxyl radical production may be increased by the higher availability of Fe²⁺ since superoxide ions may also reduce trivalent iron to the divalent form.     

Growth of Chlorella sorokiniana in the presence of sulfite elevates cell content of superoxide dismutase and imparts resistance towards paraquat

1. Growth of Chlorella sorokiniana in the presence of 7.5 mM sulfite, which halved the growth rate while doubling the superoxide dismutase (SOD; EC 1.15.1.1) content per cell, rendered the cells resistant to the toxic effects of 30 M paraquat. 2. While increasing total SOD content, sulfite increased the relative amount of the H₂O₂-resistant manganese-containing SOD. 3. It appears that O₂ ^– may be involved in mediating the toxicity of SO₂ in this green alga.Abbreviations SOD superoxide, dismutase - FeSOD ironcontaining superoxide dismutase - MnSOD manganese-containing superoxide dismutase     

Lipid peroxidation and superoxide dismutase activity in relation to photoinhibition induced by chilling in moderate light

The effect of a chilling stress, at a moderate photon flux density for a few hours, on the peroxidation of membrane lipids and on superoxide dismutase (SOD) activity was compared in leaf slices of chilling-sensitive and chilling-insensitive plants. The aim was to determine if susceptibility to chill-temperature photoinhibition could be related to either damage to membrane lipids by superoxide and-or a decrease in activity of chloroplast SOD. Plants used were Nerium oleander L., grown at 45° C, and Cucumis sativus L., both susceptible to chill-temperature photoinhibition, and N. oleander, grown at 20° C and Spinacia oleracea L., both insensitive to chill-temperature photoinhibition. Lipid peroxidation was assessed by measuring the concentration of malondialdehyde (MDA). Leaf slices from all plants showed a basal level of MDA which decreased by about 15% when the leaf slices were chilled in the light. The level of MDA was not increased by the addition of either KHCO₃ or methyl viologen during chilling but it was increased, up to threefold, by the addition of Rose Bengal, which produces singlet oxygen. Chloroplast SOD activity was assessed in leaf extracts as the cyanide-sensitive production of H₂O₂ in a system which produced superoxide. Activity of SOD was similar in all the plants and was altered little by chilling. The results show that for the plants tested, chilling at a moderate photon flux density for 5 h does not increase the susceptibility of cell membranes to peroxidative damage nor does it decrease the activity of SOD. It was concluded that the susceptibility of chilling-sensitive plants to chill-temperature photoinhibition cannot be explained on the basis of differences in the vulnerability of membrane lipids to damage by superoxide or differences in SOD activity.Abbreviations Chl chlorophyll - MDA malondialdehyde - MV methyl viologen - O ₂ ^- superoxide - 20°-oleander Nerium oleander grown at 20° C - 45°-oleander N. oleander grown at 45° C - PFD photon flux density - SOD superoxide dismutase Deceased     

Evidence that high-dose<Emphasis Type="Italic">L</Emphasis>-arginine may be inappropriate for use by diabetic patients as a prophylactic blocker of methylglyoxal glycation

Previous reports have suggested that high-doseL-arginine could be used in diabetic patients as a prophylactic blocker for the initial glycation reaction of proteins by methylglyoxal (MG), a reactive dicarbonyl compound of glucose metabolism. Here, we present several lines of evidence to substantiate that this prophylactic intervention may be inappropriate and should be used with care. First, we demonstrated that when various concentrations ofL-arginine (2.0–8.0 mM) were added to a fixed concentration of MG (1.56 µM) in a buffered lucigenin solution, dose-dependent generation of superoxide anion (O ₂ ^– )-mediated ultraweak chemiluminescence (uwCL) occurs. The suppression of uwCL generation by exogenously added superoxide dismutase further substantiated that the interaction between MG andL-arginine generated O ₂ ^– . This phenomenon can also be demonstrated in a serum-based system. Furthermore, when a fixed concentration ofL-arginine (8.0 mM) was added exogenously to a group of sera obtained from either diabetic patients (n=10) or their matched nondiabetic controls (n=10), a marked discrepancy in the generation of O ₂ ^– -mediated uwCL could be demonstrated (12,534 ± 3,147 vs. 950 ± 350 counts; p<0.001). Taken together, this evidence demonstrates that the appropriateness of using high-doseL-arginine for prophylactic measures in diabetic patients may be questioned, because the inhibition of the glycation reaction between MG and proteins by high-doseL-arginine unexpectedly produces plethoric O ₂ ^– as a by-product, which may subsequently aggravate the preexisting oxidative stress status of diabetic patients.     

Determination of the isotope distribution in malate-14C with fumarase-negative lactic acid bacteria

No fumarase activity could be found in whole cells or in cell-free crude extracts from Leuconostoc mesenteroides or Lactobacillus curvatus. The degradation of l-malate-4-¹⁴C by these organisms yielded more than 95% of the label as ¹⁴CO₂. It is therefore recommended that these organisms, rather than Lactobacillus plantarum, should be used in the determination of isotope distribution in l-malate-¹⁴C, since L. plantarum exhibits a significant fumarase activity and thus randomizes malate prior to the decarboxylation of this substance by the malolactic enzyme.     

Escherichia coli Hmp, an “oxygen-binding flavohaemoprotein”, produces superoxide anion and self-destructs

Escherichia coli Hmp is a homologue of Ralstonia eutropha FHP, the first reported bacterial flavohaemoglobin, and functions in NO detoxification. Photolysis of CO-ligated Hmp in the presence of oxygen gave a photodissociable oxy species with k_on 2.82×10⁷ M^–1 s^–1 and k_off 4.49×10³ s^–1. The dissociation constant of the primary O₂ compound was 160 M (25°C, pH 7.0). In order to detect superoxide formation, ferric horseradish peroxidase was used. Hmp formed the oxy compound within milliseconds, followed by formation of compound III, arising from superoxide formation. The rate of superoxide formation was independent of oxygen concentration between 0.05 and 0.7 mM oxygen, suggesting a K_m <0.05 mM. During prolonged oxidation of NADH, the spectral signals of Hmp decayed and iron was released in a process prevented by superoxide dismutase or catalase. NADH oxidation by purified Hmp was characterised by progressive slowing of oxygen uptake. Inclusion of NO, superoxide dismutase or catalase during NADH oxidation partially protected oxygen uptake, consistent with the formation, in the absence of NO, of reactive oxygen species that inhibit Hmp function. The results are discussed in relation to the tight control exerted on Hmp synthesis in vivo.This paper is dedicated to Professor Dr Hans G. Schlegel, on the occasion of his 80th birthday.     

Transgenic cucumber fruits that produce elevated level of an anti-aging superoxide dismutase

Superoxide dismutase (SOD) plays an important role in cellular defense against oxidative stress in aerobic organisms. To generate cucumber (Cucumis sativus L.) fruits producing high yields of SOD for an anti-aging cosmetic material as a plant bioreactor, the CuZnSOD cDNA (mSOD1) from cassava was introduced into cucumber fruits by Agrobacterium-mediated transformation using the ascorbate oxidase promoter with high expression in fruits. The bialaphos-resistant shoots were selected on medium containing MS basal salts, 2 mg l^–1 BA, 0.1 mg l^–1 IAA, 300 mg l^–1 claforan, and 2 mg l^–1 bialaphos. After 6 weeks of culture on the selection medium, the shoots were transferred to MS medium containing 1 mg l^–1 IAA, 300 mg l^–1 claforan, 2 mg l^–1 bialaphos to induce roots. Southern blot analysis confirmed that the mSOD1 gene was properly integrated into the nuclear genomes of three cucumber plants tested. The mSOD1 gene was highly expressed in the transgenic cucumber fruits, whereas it was expressed at a low level in the transgenic leaves. The SOD specific activity (units/mg protein) in transgenic fruits was approximately 3 times higher than in those of non-transgenic plants.     

Lysosomal enzyme leakage during the hypoxanthine/xanthine oxidase reaction

Impairment of lysosomal stability due to reactive oxygen species generated during the oxidation of hypoxanthine by xanthine oxidase was studied in rat liver lysosomes isolated in a discontinuous Nycodenz gradient. Production of O ₂ ^⋅ and H₂O₂ during the hypoxanthine/xanthine oxidase reaction occurred for at least 5 min, while lysosomal damage, indicated by the release of N-acetyl-β-glucosaminidase, occurred within 30 s, there being no further damage to these organelles thereafter. The extent of lysosomal enzyme release increased with increasing xanthine oxidase concentration. Superoxide dismutase and catalase did not prevent lysosomal damage during the hypoxanthine/xanthine oxidase reaction. Lysosomes reduced xanthine oxidase activity, as assessed in terms of O₂ consumption, only slightly but substantially inhibited in a competitive manner the O ₂ ^⋅ -mediated reduction of cytochrome c. This inhibition was almost completely reversed by potassium cyanide, thus pointing to the presence of a cyanide-sensitive Superoxide dismutase in the lysosomal fraction. However, potassium cyanide did not affect the hypoxanthine/xanthine oxidase-mediated lysosomal damage, thus suggesting an inability of the lysosomal superoxide dismutase to protect the organelles. Negligible malondialdehyde formation was observed in the lysosomes either during the hypoxanthine/xanthine oxidase reaction or with different selective experimental approaches known to produce lipid peroxidation in other organelles such as microsomes and mitochondria. These results are interpreted in terms of a possible lysosomal membrane permeability to O ₂ ^⋅ causing organelle impairment by a process that, though leading to enzyme-marker leakage, does not involve lipid peroxidation.     

Negative air ions as a source of superoxide

The physico-chemical characteristics and possible formation mechanisms of negative air ions are considered. It was found that the products of oxygen and nitrogen negative ionization reduce ferricytochromec and nitroblue tetrazolium, and that these reactions were inhibited by superoxide dismutase. The interaction of negatively ionized oxygen with water led to hydrogen peroxide accumulation, which was inhibited by tetranitromethane or catalase. Nitrogen ionization under these conditions caused the formation of the hydrated electron e _aq ^— and the superoxide anion O ₂ ^— . The data obtained indicate that the biological activity of negative air ions may be dependent on superoxide. The generation of reactive oxygen ions in the gas phase and also at a gas/water interface is described. A scheme for superoxide production under oxygen and nitrogen ionization is proposed.     

Age-related increases in superoxide dismutase activity and thiobarbituric acid-reactive substances: Effect of bio-catalyzer in aged rat brain

This study describes, using electron spin resonance spectrometry/spin trapping technique, the increase superoxide dismutase (SOD) activity in the mitochondrial and cytosolic fraction of the cortex, midbrain, pons-medulla oblongata and cerebellum, and in thiobarbituric acid-reactive substances (TBARS) in the cortex, cerebellum and hippocampus of the aged rats. The results show that corresponding to the increased life span and improved physical conditions observed after peroral long-term treatment with Bio-catalyzer, a commercial natural fermented health food supplement marketed in Japan and in the Philippines and earlier reported to be a hydroxyl radical scavenger with weaker scavenging activity on superoxide radical (O ₂ ^– ), SOD which is involved in the metabolic degradation of O ₂ ^– was further increased, whereas TBARS decreased. These findings suggest that the increased SOD activity in the brain as a defense mechanism against age-related accumulation of reactive oxygen species, in particular superoxide radicals, was enhanced with Biocatalyzer treatment while age-related peroxidation of neuronal membrane, as measured by TBARS, was decreased.     

Protective systems against active oxygen species in spinach: response to cold acclimation in excess light

Spinach (Spinacia oleracea L.) plants were acclimated to 1° C or maintained at 18° C under the same light regime (260–300 mol photons·m^–2·s^–1). The cold acclimation led to several metabolic and biochemical changes that apparently include improved protection of the photosynthetic apparatus against active oxygen species. In particular, cold-acclimated leaves exhibited a considerably higher ascorbate content and significantly increased activities of superoxide dismutase, ascorbate peroxidase, and monodehydroascorbate reductase in the chloroplasts. The level of dehydroascorbate reductase did not alter. Catalase activity decreased. The photosynthetic pigment composition of cold-acclimated spinach was characterized by increased levels of the xanthophylls lutein + zeaxanthin and violaxanthin. The observed changes are discussed in terms of their possible relevance for plant resistance to photoinhibition at chilling temperatures.Abbreviations DHA dehydroascorbate - GSH reduced glutathione - MDA monodehydroascorbate - SOD superoxide dismutase The authors thank the Deutsche Forschungsgemeinschaft for financial support of this study.     

Isolation of the respiratory burst oxidase: The role of a flavoprotein component

The article reviews the enzymatic and electron transfer properties of a low-potential FAD-dependent flavoprotein that is a component of the NADPH-dependent O ₂ ^– · -generating respiratory burst oxidase of phagocytes. Current methods available for isolation of the respiratory burst oxidase and the flavo-protein component of the complex are also reviewed. These studies and data obtained from affinity-labeling of respiratory burst oxidase components, suggest that the flavoprotein has a molecular weight of 65–67 kD. The prevailing evidence suggests that the flavoprotein functions as a dehydrogenase/electron transferase and can directly catalyse NADPH-dependent O ₂ ^– · formation when isolated. However, in neutrophil plasma membranes, the prevailing evidence suggests that the flavoprotein functions primarily to transfer electrons from NADPH to cytochromeb _–245 and that this latter redox component is the catalytic side of O ₂ ^– · formation. A working model for the arrangement of the flavorprotein and cytochromeb _–245 components of the respiratory burst oxidase in neutrophil membranes is proposed.     

Purification and characterization of an NADH oxidase from extremely thermophilic anaerobic bacterium Thermotoga hypogea

Thermotoga hypogea is an extremely thermophilic anaerobic bacterium capable of growing at 90°C. It was found to be able to grow in the presence of micromolar molecular oxygen (O₂). Activity of NADH oxidase was detected in the cell-free extract of T. hypogea, from which an NADH oxidase was purified to homogeneity. The purified enzyme was a homodimeric flavoprotein with a subunit of 50 kDa, revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It catalyzed the reduction of O₂ to hydrogen peroxide (H₂O₂), specifically using NADH as electron donor. Its catalytic properties showed that the NADH oxidase had an apparent V_max value of 37 mol NADH oxidized min^–1 mg^–1 protein. Apparent K_m values for NADH and O₂ were determined to be 7.5 M and 85 M, respectively. The enzyme exhibited a pH optimum of 7.0 and temperature optimum above 85°C. The NADH-dependent peroxidase activity was also present in the cell-free extract, which could reduce H₂O₂ produced by the NADH oxidase to H₂O. It seems possible that O₂ can be reduced to H₂O by the oxidase and peroxidase, but further investigation is required to conclude firmly if the purified NADH oxidase is part of an enzyme system that protects anaerobic T. hypogea from accidental exposure to O₂.     

Determination of superoxide dismutase-like activity in some divalent metal saccharinates

The superoxide-dismutase-like activity of a series of divalent metal saccharinates of general stoichiometry [M^II(Sac)₂(H₂O)₄]·2H₂O (with M^II=Mn,Fe,Co,Ni,Cu,Zn) has been investigated using the nitroblue tetrazolium O ₂ ⁻ reduction assay. The results show that all these complexes possess the capability to dismutate the superoxide anion generated in the xanthine/xanthine oxidase system. Interestingly, the greatest activity is shown by the corresponding copper complex. The results are discussed and compared with those obtained for native superoxide dismutase, which was tested under the same experimental conditions. Dedicated to Prof. Pedro J. Aymonino on the occasion of his 65^th birthday.     

Chemolithoautotrophy in the marine,magnetotactic bacterial strains MV-1 and MV-2

Magnetite-producing magnetotactic bacteria collected from the oxic–anoxic transition zone of chemically stratified marine environments characterized by O₂/H₂S inverse double gradients, contained internal S-rich inclusions resembling elemental S globules, suggesting they oxidize reduced S compounds that could support autotrophy. Two strains of marine magnetotactic bacteria, MV-1 and MV-2, isolated from such sites grew in O₂-gradient media with H₂S or thiosulfate (S₂O₃^2–) as electron sources and O₂ as electron acceptor or anaerobically with S₂O₃^2– and N₂O as electron acceptor, with bicarbonate (HCO₃^–)/CO₂ as sole C source. Cells grown with H₂S contained S-rich inclusions. Cells oxidized S₂O₃^2– to sulfate (SO₄^2–). Both strains grew microaerobically with formate. Neither grew microaerobically with tetrathionate (S₄O₆^2–), methanol, or Fe²⁺ as FeS, or siderite (FeCO₃). Growth with S₂O₃^2– and radiolabeled ¹⁴C-HCO₃^– showed that cell C was derived from HCO₃^–/CO₂. Cell-free extracts showed ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) activity. Southern blot analyses indicated the presence of a form II RubisCO (cbbM) but no form I (cbbL) in both strains. cbbM and cbbQ, a putative post-translational activator of RubisCO, were identified in MV-1. MV-1 and MV-2 are thus chemolithoautotrophs that use the Calvin–Benson–Bassham pathway. cbbM was also identified in Magnetospirillum magnetotacticum. Thus, magnetotactic bacteria at the oxic–anoxic transition zone of chemically stratified aquatic environments are important in C cycling and primary productivity.     

Asparagine and glutamine metabolism in Rhodopseudomonas acidophila

Rhodopseudomonas acidophila strain 7050 achieved balanced growth when provided with either asparagine or glutamine as nitrogen source. Under these growth conditions R. acidophila synthesized a mixed amidase which exhibited similar activity (223–422 nmol/min·mg protein) against either nitrogen source. Determination of the free intracellular amino acid pools show that deamidation of asparagine and glutamine resulted in elevated levels of both aspartate and glutamate. Cell-free extracts of R. acidophila showed significant aminotransferase activity, particulary glutamine-oxaloacetate aminotransferase (89.7–209.3 nmol/min·mg protein), glycine oxaloacetate aminotransferase (135–227 nmol/min ·mg protein), alanine glyoxylate aminotransferase (66.3–163.2 nmol/min·mg protein) and serineglyoxylate aminotransferase (57.1–68.4 nmol/min ·mg protein). Short term labelling experiments using ¹⁴C-glyoxylate show that glycine plays an important role in amino nitrogen transfer in R. acidophila and that the enzymes for the metabolism of glyoxylate via glycine, serine and hydroxypyruvate were present in cell-free extracts. These data confirm that R. acidophila can satisfy all its' nitrogen requirements by transamination.Abbreviations GDH glutamate dehydrogenase - GS glutamine synthetase - GOGAT glutamate synthase - MSO methionine sulfoximine - GOT glutamate—oxaloacetate aminotransferase - GPT glutamate-pyruvate aminotransferase - AGAT alanineglyoxylate aminotransferase - GOAT glycine-oxaloacetate aminotransferase - GOGAT glycine-2-oxoglutarate aminotransferase - AOAT alanine-oxaloacetate aminotransferase - SGAT serineglyoxylate aminotransferase - INH isonicotinylhydrazide