Ovotransferrin possesses SOD-like superoxide anion scavenging activity that is promoted by copper and manganese binding

The embryo of oviparous species is confronted by a highly oxidative stress generating as it grows and must rely on effective antioxidant system for protection. Proteins of avian egg albumin have been suggested to play the major redox-modulatory role during embryo development. Recently, we found that ovotransferrin (OTf) undergoes distinct thiol-linked self-cleavage in a redox-dependent process. In this study, we explore that OTf is SOD mimic protein with a potent superoxide anion (O₂⁻) scavenging activity. The O₂⁻ scavenging activity was investigated using the natural xanthine/xanthine oxidase (X/XOD) coupling system. OTf exhibited O₂⁻ scavenging activity in a dose-dependent manner and showed remarkably higher scavenging activity than the known antioxidants, ascorbate or serum albumin. The isolated half-molecules of OTf exhibited higher scavenging activity than the intact molecule, whereas the N-lobe showed much greater activity. OTf dramatically quenched the O₂⁻ flux but had no effect on the urate production in the X/XOD system, indicating its unique specificity to scavenge O₂⁻ but not oxidase inhibition. Strikingly, metal-bound OTf exhibited greater O₂⁻ dismutation capacity than the apo-protein, ranging from moderate (Zn²⁺-OTf and Fe²⁺-OTf) to high (Mn²⁺-OTf and Cu²⁺-OTf) activity with the Cu²⁺-OTf being the most potent scavenger. In a highly sensitive fluorogenic assay, the metal-bound OTf exhibited significant increase in the rate of H₂O₂ production in the X/XOD reaction than the apo-OTf, providing evidence that Zn²⁺-, Mn²⁺- and Cu²⁺-OTf possess SOD mimic activity. This finding is the first to describe that OTf is an O₂⁻ scavenging molecule, providing insight into its novel SOD-like biological function, and heralding a fascinating opportunity for its potential candidacy as antioxidant drug.     

Examination of the Inhibitory Effect of Norathyriol in Formylmethionyl-Leucyl-Phenylalanine-Induced Respiratory Burst in Rat Neutrophils

Norathyriol, aglycone of a xanthone C-glycoside mangiferin isolated from Tripterospermum lanceolatum, concentration dependently inhibited the formylmethionyl-leucyl-phenylalanine (fMLP)-induced superoxide anion (O₂^˙−) generation and O₂ consumption in rat neutrophils. In cell-free oxygen radical generating system, norathyriol inhibited the O₂^˙− generation during dihydroxyfumaric acid (DHF) autoxidation and in hypoxanthine-xanthine oxidase system. fMLP-induced transient elevation of [Ca²⁺]_i and the formation of inositol trisphosphate (IP₃) were significantly inhibited by norathyriol (30 μM) (about 30 and 46% inhibition, respectively). Norathyriol concentration dependently suppressed the neutrophil cytosolic phospholipase C (PLC). In contrast with the marked attenuation of fMLP-induced protein tyrosine phosphorylation (about 70% inhibition at 10 μM norathyriol), norathyriol only slightly modulated the phospholipase D (PLD) activity as determined by the formation of phosphatidic acid (PA) and, in the presence of ethanol, phosphatidylethanol (PEt). Norathyriol did not modulate the intracellular cyclic AMP level. In the presence of NADPH, the phorbol 12-myristate 13-acetate (PMA)-activated particulate NADPH oxidase activity was suppressed by norathyriol in a concentration-dependent manner and the inhibition was noncompetitive with respect to NADPH. Norathyriol inhibited the iodonitrotetrazolium violet (INT) reduction in arachidonic acid (AA)-activated cell-free NADPH oxidase system at the same concentration range as those used in the suppression of PMA-activated particulate NADPH oxidase activity. Taken together, these results suggest that the scavenging ability of norathyriol contributes to the reduction of generated O₂^˙−, however, the inhibition of O₂^˙− generation from neutrophils by norathyriol is attributed to the blockade of PLC pathway, the attenuation of protein tyrosine phosphorylation, and to the suppression of NADPH oxidase through the interruption of electrons transport.     

Reactions of captopril and epicaptopril with transition metal ions and hydroxyl radicals: An EPR spectroscopy study

In the present study, using the technique of EPR spin trapping with DMPO a spin trap, we demonstrated formation of thiyl radicals from thiol-containing angiotensin converting enzyme (ACE) inhibitor captopril (CAP) and from its stereoisomer epicaptopril (EPICAP), a non-ACE inhibitor, in the process of ^.OH radical scavenging. Splitting constants of DMPO/thiyl radical adducts were identical for both thiols and were a_N = 15.3 G, and a_H = 16.2 G. Bimolecular rate constants for the reaction of CAP and EPICAP with ^.OH radicals were close to a diffusion-controlled rate (≈ 2 × 10¹⁰ M⁻¹s⁻¹). Our data also show that both CAP and EPICAP reduce Fe(III) ions and that their respective thiyl radicals are formed in this reaction. In the presence of Fe(III), H₂O₂, and CAP, or EPICAP, ^.OH radicals were produced by a thiol-driven Fenton mechanism. Copper(II) ions were also reduced by these thiols, but no thiyl radicals could be detected in these reactions, and no ^.OH or other Fenton oxidants were observed in the presence of H₂O₂. Our data show direct evidence that thiol groups of CAP and EPICAP are involved in scavenging of ^.OH radicals. The direct ^.OH radical scavenging, together with the reductive “repair” of other sites of ^.OH radical attack, may contribute to the known protective effect of CAP against ischemia/reperfusion-induced arrhythmias. The formation of reactive thiyl radicals in the reactions of the studied compounds with ^.OH radicals and with Fe(III) ions may play a role in some of the known adverse effects of CAP.     

Bipyridylium quaternary salts and related compounds. V. Pulse radiolysis studies of the reaction of paraquat radical with oxygen. Implications for the mode of action of bipyridyl herbicides

1. Rate constants for reduction of paraquat ion (1,1′-dimethyl-4,4′-bipyridy-lium, PQ²⁺) to paraquat radical (PQ⁺·) by e⁻_aq and CO₂⁻· have been measured by pulse radiolysis. Reduction by e⁻_aq is diffusion controlled (k = 8.4·10¹⁰ M⁻¹·s⁻¹) and reduction by CO₂⁻· is also very fast k = 1.5·10¹⁰ M⁻¹·s⁻¹).

2. The reaction of paraquat radical with oxygen has been analysed to give rate constants of 7.7·10⁸ M⁻¹·s⁻¹ and 6.5·10⁸ M⁻¹·s⁻¹ for the reactions of paraquat radical with O₂ and O₂⁻·, respectively. The similarity in these rate constants is in marked contrast to the difference in redox potentials of O₂ and O₂⁻· (− 0.59 V and + 1.12 V, respectively).

3. These rate constants, together with that for the self-reaction of O₂⁻·, have been used to calculate the steady-state concentration of O₂⁻· under conditions thought to apply at the site of reduction of paraquat in the plant cell. On the basis of these calculations the decay of O₂⁻· appears to be governed almost entirely by its self-reaction, and the concentration 5 μm away from the thylakoid is still 90% of that at the thylakoid itself. Thus, O₂⁻· persists long enough to diffuse as far as the chloroplast envelope and tonoplast, which are the first structures to be damaged by paraquat treatment. O₂⁻· is therefore sufficiently long-lived to be a candidate for the phytotoxic product formed by paraquat in plants.     

Cobalt-mediated generation of reactive oxygen species and its possible mechanism

Electron spin resonance spin trapping was utilized to investigate free radical generation from cobalt (Co) mediated reactions using 5,5-dimethyl-l-pyrroline (DMPO) as a spin trap. A mixture of Co with water in the presence of DMPO generated 5,5-dimethylpyrroline-(2)-oxy(1) DMPOX, indicating the production of strong oxidants. Addition of superoxide dismutase (SOD) to the mixture produced hydroxyl radical (OH). Catalase eliminated the generation of this radical and metal chelators, such as desferoxamine, diethylenetriaminepentaacetic acid or 1,10-phenanthroline, decreased it. Addition of Fe(II) resulted in a several fold increase in the OH generation. UV and O₂ consumption measurements showed that the reaction of Co with water consumed molecular oxygen and generated Co(II). Since reaction of Co(II) with H₂O₂ did not generate any significant amount of OH radicals, a Co(I) mediated Fenton-like reaction [Co(I) + H₂O₂ → Co(II) + OH + OH⁻] seems responsible for OH generation. H₂O₂ is produced from O₂⁻ via dismutation. O₂⁻ is produced by one-electron reduction of molecular oxygen catalyzed by Co. Chelation of Co(II) by biological chelators, such as glutathione or β-ananyl-3-methyl- -histidine alters, its oxidation–reduction potential and makes Co(II) capable of generating OH via a Co(II)-mediated Fenton-like reaction [Co(II) + H₂O₂ → Co(III) + OH + OH⁻]. Thus, the reaction of Co with water, especially in the presence of biological chelators, glutathione, glycylglycylhistidine and β-ananyl-3-methyl- -histidine, is capable of generating a whole spectrum of reactive oxygen species, which may be responsible for Co-induced cell injury.     

Kinetics of the reactions of nitrogen dioxide with glutathione,cysteine, and uric acid at physiological pH

Nitrogen dioxide (NO₂^•) is a key biological oxidant. It can be derived from peroxynitrite via the interaction of nitric oxide with superoxide, from nitrite with peroxidases, or from autoxidation of nitric oxide. In this study, submicromolar concentrations of NO₂^• were generated in < 1 μs using pulse radiolysis, and the kinetics of scavenging NO₂^• by glutathione, cysteine, or uric acid were monitored by spectrophotometry. The formation of the urate radical was observed directly, while the production of the oxidizing radical obtained on reaction of NO₂^• with the thiols (the thiyl radical) was monitored via oxidation of 2,2′-azino-bis-(3-ethylthiazoline-6-sulfonic acid). At pH 7.4, rate constants for reaction of NO₂^• with glutathione, cysteine, and urate were estimated as 2 × 10⁷, 5 × 10⁷, and 2 × 10⁷ M⁻¹ s⁻¹, respectively. The variation of these rate constants with pH indicated that thiolate reacted much faster than undissociated thiol. The dissociation of urate also accelerated reaction with NO₂^• at pH > 8. The thiyl radical from GSH reacted with urate with a rate constant of 3 × 10⁷ M⁻¹ s⁻¹. The implications of these values are: (i) the lifetime of NO₂^• in cytosol is < 10 μs; (ii) thiols are the dominant ‘sink’ for NO₂^• in cells/tissue, whereas urate is also a major scavenger in plasma; (iii) the diffusion distance of NO₂^• is 0.2 μm in the cytoplasm and < 0.8 μm in plasma; (iv) urate protects GSH against depletion on oxidative challenge from NO₂^•; and (v) reactions between NO₂^• and thiols/urate severely limit the likelihood of reaction of NO₂^• with NO• to form N₂O₃ in the cytoplasm.     

Mechanisms of H2O2-induced oxidative stress in endothelial cells

Hydrogen peroxide, produced by inflammatory and vascular cells, induces oxidative stress that may contribute to endothelial dysfunction. In smooth muscle cells, H₂O₂ induces production of O₂⁻ by activating NADPH oxidase. However, the mechanisms whereby H₂O₂ induces oxidative stress in endothelial cells are poorly understood. We examined the effects of H₂O₂ on O₂⁻ levels on porcine aortic endothelial cells (PAEC). Treatment with 60 μmol/L H₂O₂ markedly increased intracellular O₂⁻ levels (determined by conversion of dihydroethidium to hydroxyethidium) and produced cytotoxicity (determined by propidium iodide staining) in PAEC. Overexpression of human manganese superoxide dismutase in PAEC reduced O₂⁻ levels and attenuated cytotoxicity resulting from treatment with H₂O₂. L-NAME, an inhibitor of nitric oxide synthase (NOS), and apocynin, an inhibitor of NADPH oxidase, reduced O₂⁻ levels in PAEC treated with H₂O₂, suggesting that both NOS and NADPH oxidase contribute to H₂O₂-induced O₂⁻ in PAEC. Inhibition of NADPH oxidase using apocynin and NOS rescue with L-sepiapterin together reduced O₂⁻ levels in PAEC treated with H₂O₂ to control levels. This suggests interaction-distinct NOS and NADPH oxidase pathways to superoxide. We conclude that H₂O₂ produces oxidative stress in endothelial cells by increasing intracellular O₂⁻ levels through NOS and NADPH oxidase. These findings suggest a complex interaction between H₂O₂ and oxidant-generating enzymes that may contribute to endothelial dysfunction.     

The reaction between the superoxide anion radical and cytochrome c

1. 1. The superoxide anion radical (O₂⁻) reacts with ferricytochrome c to form ferrocytochrome c. No intermediate complexes are observable. No reaction could be detected between O₂⁻ and ferrocytochrome c.

2. 2. At 20 °C the rate constant for the reaction at pH 4.7 to 6.7 is 1.4 · 10⁶ M⁻¹ · s⁻¹ and as the pH increases above 6.7 the rate constant steadily decreases. The dependence on pH is the same for tuna heart and horse heart cytochrome c. No reaction could be demonstrated between O₂⁻ and the form of cytochrome c which exists above pH ≈ 9.2. The dependence of the rate constant on pH can be explained if cytochrome c has pKs of 7.45 and 9.2, and O₂⁻ reacts with the form present below pH 7.45 with k = 1.4 · 10⁶ M⁻¹ · s⁻¹, the form above pH 7.45 with k = 3.0 · 10⁵ M⁻¹ · s⁻¹, and the form present above pH 9.2 with k = 0.

3. 3. The reaction has an activation energy of 20 kJ mol⁻¹ and an enthalpy of activation at 25 °C of 18 kJ mol⁻¹ both above and below pH 7.45. It is suggested that O₂⁻ may reduce cytochrome c through a track composed of aromatic amino acids, and that little protein rearrangement is required for the formation of the activated complex.

4. 4. No reduction of ferricytochrome c by HO₂ radicals could be demonstrated at pH 1.2–6.2 but at pH 5.3, HO₂ radicals oxidize ferrocytochrome c with a rate constant of about 5 · 10⁵–5 · 10⁶ M⁻¹ · s⁻¹

.     

Crystal and microparticle effects on MDCK cell superoxide production: oxalate-specific mitochondrial membrane potential changes

We have previously shown that crystals of calcium oxalate (COM) elicit a superoxide (O₂⁻) response from mitochondria. We have now investigated: (i) if other microparticles can elicit the same response, (ii) if processing of crystals is involved, and (iii) at what level of mitochondrial function oxalate acts. O₂⁻ was measured in digitonin-permeabilized MDCK cells by lucigenin (10 μM) chemiluminescence. [¹⁴C]-COM dissociation was examined with or without EDTA and employing alternative chelators. Whereas mitochondrial O₂⁻ in COM-treated cells was three- to fourfold enhanced compared to controls, other particulates (uric acid, zymosan, and latex beads) either did not increase O₂⁻ or were much less effective (hydroxyapatite +50%, p < 0.01), with all at 28 μg/cm². Free oxalate (750 μM), at the level released from COM with EDTA (1 mM), increased O₂⁻ (+50%, p < 0.01). Omitting EDTA abrogated this signal, which was restored completely by EGTA and partially by ascorbate, but not by desferrioxamine or citrate. Omission of phosphate abrogated O₂⁻, implicating phosphate-dependent mitochondrial dicarboxylate transport. COM caused a time-related increase in the mitochondrial membrane potential (Δψ_m) measured using TMRM fluorescence and confocal microscopy. Application of COM to Fura 2-loaded cells induced rapid, large-amplitude cytosolic Ca²⁺ transients, which were inhibited by thapsigargin, indicating that COM induces release of Ca²⁺ from internal stores. Thus, COM-induced mitochondrial O₂⁻ requires the release of free oxalate and contributes to a synergistic response. Intracellular dissociation of COM and the mitochondrial dicarboxylate transporter are important in O₂⁻ production, which is probably regulated by Δψ_m.     

The antioxidant action of N-acetylcysteine: Its reaction with hydrogen peroxide, hydroxyl radical, superoxide, and hypochlorous acid

N-acetylcysteine has been widely used as an antioxidant in vivo and in vitro. Its reaction with four oxidant species has therefore been examined. N-acetylcysteine is a powerful scavenger of hypochlorous acid (H---OCl); low concentrations are able to protect ₁-antiproteinase against inactivation by HOCl. N-acetylcysteine also reacts with hydroxyl radical with a rate constant of 1.36 × 10¹⁰ M⁻¹s⁻¹, as determined by pulse radiolysis. It also reacts slowly with H₂O₂, but no reaction of N-acetylcysteine with superoxide (O₂⁻) could be detected within the limits of our assay procedures.     

Mitochondrial superoxide production during oxalate-mediated oxidative stress in renal epithelial cells

Crystals of calcium oxalate monohydrate (COM) in the renal tubule form the basis of most kidney stones. Tubular dysfunction resulting from COM-cell interactions occurs by mechanism(s) that are incompletely understood. We examined the production of reactive oxygen intermediates (ROI) by proximal (LLC-PK1) and distal (MDCK) tubular epithelial cells after treatment with COM (25–250 μg/ml) to determine whether ROI, specifically superoxide (O₂^•−), production was activated, and whether it was sufficient to induce oxidative stress. Employing inhibitors of cytosolic and mitochondrial systems, the source of ROI production was investigated. In addition, intracellular glutathione (total and oxidized), energy status (ATP), and NADH were measured. COM treatment for 1–24 h increased O₂^•− production 3–6-fold as measured by both lucigenin chemiluminescence in permeabilized cells and dihydrorhodamine fluorescence in intact cells. Using selective inhibitors we found no evidence of cytosolic production. The use of mitochondrial probes, substrates, and inhibitors indicated that increased O₂^•− production originated from mitochondria. Treatment with COM decreased glutathione (total and redox state), indicating a sustained oxidative insult. An increase in NADH in COM-treated cells suggested this cofactor could be responsible for elevating O₂^•− generation. In conclusion, COM increased mitochondrial O₂^•− production by epithelial cells, with a subsequent depletion of antioxidant status. These changes may contribute to the reported cellular transformations during the development of renal calculi.     

The diffusion-controlled reaction of semioxidized tryptophan with the superoxide radical anion

From pulse radiolysis measurements in oxygenated aqueous solution, the semioxidized tryptophan radical (Trp·— formed by the one-electron oxidation of Trp by Br₂^- radical—has been shown to oxidize the superoxide radical anion with a rate constant of k = 2 × 10⁹ M⁻¹ s⁻¹. Proof of this reaction is found in addition of superoxide dismutase (SOD) to the system, which totally eliminates the contribution of the Trp^· + O₂^- mechanism to Trp^· decay. Little, if any, reaction of molecular oxygen with Trp^· may be observed on the time scale of the pulse radiolysis experiment.     

A comparative study of EPR spin trapping and cytochrome c reduction techniques for the measurement of superoxide anions

Superoxide anions (O₂^.−) generated by the reaction of xanthine with xanthine oxidase were measured by the reduction of cytochrome c and by electron paramagnetic resonance (EPR) spectroscopy using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Studies were performed to determine the relative sensitivities of these two techniques for the measurement of O₂^.−. Mixtures of xanthine, xanthine oxidase, DMPO generated two adducts, a transient DMPO-OOH and a smaller but longer-lived DMPO-OH. Both adducts were inhibited by superoxide dismutase (SOD), demonstrating they originated from O₂^.−, and were also significantly decreased when the experiments were performed using unchelated buffers, suggesting that metal ion impurities in unchelated buffers alter the formation or degradation of DMPO-adducts. O₂^.−, generated by concentrations of xanthine as low as 0.05 μM, were detectable using EPR spin trapping. In contrast, mixtures of xanthine, xanthine oxidase, and cytochrome c measured spectrophotometrically at 550 nm demonstrated that concentrations of xanthine above 1 μM were required to produce measurable levels of reduced cytochrome c. These studies demonstrate that spin trapping using DMPO was at least 20-fold more sensitive than the reduction of cytochrome c for the measurement of superoxide anions. However, at levels of superoxide generation where cytochrome c provides a linear measurement of production, EPR spin trapping may underestimate radical production, probably due to degradation of DMPO radical adducts.     

Reactions of the ferri-ferrocytochrome-c system with superoxide/oxygen and CO2/CO2 studied by fast pulse radiolysis

The reduction of ferricytochrome c by O₂⁻ and CO₂⁻ was studied in the pH range 6.6–9.2 and Arrhenius as well as Eyring parameters were derived from the rate constants and their temperature dependence. Ionic effects on the rate indicate that the redox process proceeds through a multiply-positively charged interaction site on cytochrome c. It is shown that the reaction with O₂⁻ and correspondingly with O₂ of ferrocytochrome c) is by a factor of approx. 10³ slower than warranted by factors such as redox potential. Evidence is adduced to support the view that this slowness is connected with the role of water in the interaction between O₂⁻/O₂ and ferri-ferrocytochrome c in the positively charged interaction site on cytochrome c in which water molecules are specifically involved in maintaining the local structure of cytochrome c and participate in the process of electron equivalent transfer.     

Direct measurement of oscillatory generation of superoxide anions by single phagocytes

Phagocytic cells such as neutrophils generate superoxide anions (O₂⁻) within phagocytic vacuoles for killing and digesting microorganisms. Here we report the simultaneous observation of morphological changes and O₂⁻ generation in single phagocytic cells during phagocytosis. Point stimulation of a cell by contact with an opsonized microelectrode at the cell surface induced significant deformation to engulf the electrode, and also induced the O₂⁻ generation which was measured by the electrode. Periodic fluctuations in the magnitude of the O₂⁻ generation were observed in the time course. These oscillations may be caused by metabolic regulation of the formation of NADPH, which is the substrate for the O₂⁻ generation.     

Trehalose: Protector of antioxidant enzymes or reactive oxygen species scavenger under heat stress?

Trehalose is known to protect membranes and macromolecules. Its accumulation has been implicated in allowing plants to tolerate stress, including heat-shock. However, under heat-shock, it is not clear whether trehalose eliminates reactive oxygen species (ROS) directly or indirectly by protecting antioxidant enzymes. In this study, we initially examined the effects of trehalose on the activities of key antioxidant enzymes, including superoxide dismutases (SODs), ascorbate catalases (CATs), and ascorbate peroxidases (APX) from wheat (Triticum aestivum L.), and then measured the ability of trehalose to scavenge hydrogen peroxide (H₂O₂) and superoxide anions (O₂⁻). Our results indicated that trehalose protected SOD activity slightly. However, it inhibited CAT and APX activities under heat stress, with a little protection of CAT activity (only about 7% promotion) at 22 °C. Moreover, trehalose scavenged H₂O₂ and O₂⁻ greatly in a concentration-dependent manner, reaching the maximal scavenging H₂O₂ rate of 95% and O₂⁻ rate of 78%, respectively, at 50 mM trehalose. These results suggest that trehalose plays a direct role in eliminating H₂O₂ and O₂⁻ in wheat under heat stress.     

Particulate formate oxidase from Nitrobacter agilis

1. The nitrite oxidase particles obtained by sonic oscillation of Nitrobacter agilis cells also possessed appreciable formate oxidase activity, ranging from about 25 to 50% of the nitrite oxidase activity depending upon the N. agilis strain. Both activities distributed themselves in the same pattern and proportions during differential centrifugation, and resided solely in the pellet resulting from high-speed centrifugation.

2. Difference spectra of formate-reduced particles or intact cells demonstrated the presence of cytochromes of the c- and a-types like those of the NO₂⁻-reduced material. Under anaerobic conditions NO₃⁻ or fumarate acted as an alternate electron acceptor in place of O₂ in formate oxidation. Under aerobic conditions increasing NO₃⁻ concentrations resulted in (a) an increased role of NO₃⁻ as a terminal electron acceptor compared to O₂, (b) a greater total enzymatic transfer of electrons from formate than if O₂ were the sole electron acceptor, and (c) a partial inhibition of O₂ uptake suggestive of a competition for electrons by the two acceptors. The formate oxidase system failed to catalyze consistently the transfer of electrons to either added mammalian cytochrome c or Fe(CN)₆³⁻. The marked sensitivity of the system to certain inhibitors implicated cytochrome oxidase as an integral part of the formate oxidase. The system was also inhibited significantly by a variety of chelating agents, indicating a metal component in the formate dehydrogenase or early portion of the electron transfer sequence.

3. The stoichiometry of the formate oxidase system was shown to approach the theoretical value of 2 moles of CO₂ evolved per mole of O₂ or per 2 moles of formate consumed.

4. To a limited extent, phosphorylation occurred concomittantly with the oxidation of formate in the presence of the cell-free particulate system.     

Effects of tocopherols and 2,2'-carboxyethyl hydroxychromans on phorbol-ester-stimulated neutrophils

Tocopherol vitamers [e.g., alpha-, gamma- and delta-tocopherol (-TOC, γ-TOC and δ-TOC, respectively)] and their water-soluble 2,2′-carboxyethyl hydroxychroman metabolites (e.g., -, γ- and δ-CEHC) all possess antioxidant properties. As a consequence, and similarly to other natural antioxidants, vitamin E compounds may be useful in preventing inflammatory and oxidative-stress-mediated diseases. In this study, we investigated the concentration-dependent effect of tocopherols and water-soluble metabolites on a key event in oxidative stress, for example, the oxidative burst in neutrophils. It was found that not only -TOC but also γ-TOC and δ-TOC as well as -, γ- and δ-CEHC at physiological concentrations inhibit superoxide anion (O₂^•−) production in phorbol-ester-stimulated neutrophils. This effect was mediated by the inhibition of the translocation and activation of protein kinase C (PKC) enzyme, which is the key event in the phorbol-ester signaling. Importantly, CEHCs were stronger inhibitors of PKC as compared with the vitamer precursors, and the gamma forms of both tocopherol and CEHC showed the highest inhibitory activities. Tocopherols, but not CEHCs, directly inhibit the fully activated nicotine–adenine–dinucleotide phosphate (NADPH) oxidase. However, none of the test compounds was able to directly scavenge O₂^•− when tested in a cell-free system. In conclusion, vitamin E compounds can control the neutrophil oxidative burst through the negative modulation of PKC-related signaling and NADPH oxidase activity. As an original finding, we observed that CEHC metabolites might contribute to regulate PKC activity in these cells. These results may have important implications in the anti-inflammatory and antioxidant role of vitamin E compounds.     

A nitrite reductase with cytochrome oxidase activity from Micrococcus denitrificans

The formation of nitrite reductase and cytochrome c in Micrococcus denitrificans was repressed by O₂. The purified nitrite reductase utilized reduced forms of cytochrome c, phenazine methosulphate, benzyl viologen and methyl viologen, respectively, as electron donors. The enzyme was inhibited by KCN, NaN₃ and NH₂OH each at 1 mM, whereas CO and bathocuproin, diethyl dithiocarbamate, o-phenanthroline and ,'-dipyridyl at 1 mM concentrations were relatively ineffective. The purified enzyme contains cytochromes, probably of the c and a₂ types, in one complex. A K_m of 46 μM for NO₂⁻ and a pH optimum of 6.7 were recorded for the enzyme. The molecular weight of the enzyme was estimated to be around 130000, and its anodic mobility was 6.8·10⁻⁶ cm²·sec⁻¹·V⁻¹ at pH 4.55.

The most highly purified nitrite reductase still exhibited cytochrome c oxidase activity with a K_m of 27 μM for O₂. This activity was also inhibited by KCN, NaN₃ and NH₂OH and by NO₂⁻.

A constitutive cytochrome oxidase associated with membranes was also isolated from cells grown anaerobically with NO₂⁻. It was inhibited by smaller amounts of KCN, NaN₃ and NH₂OH than the cytochrome oxidase activity of the nitrite reductase enzyme and also differed in having a pH optimum of about 8 and a K_m for O₂ of less than 0.1 μM. Spectroscopically, cytochromes b and c were found to be associated with the constitutive oxidase in the particulate preparation. Its activity was also inhibited by NO₂⁻.

The physiological role of the cytochrome oxidase activity associated with the purified nitrite reductase is likely to be of secondary importance for the following reasons: (a) it accounts for less than 10% of total cytochrome c oxidase activity of cell extracts; (b) the constitutive cytochrome c oxidase has a smaller K_m for O₂ and would therefore be expected to function more efficiently especially at low concentrations of O₂.     

Histochemical Localization of Superoxide Dismutase Activity in Rat Brain

Histochemical localization of superoxide anion (O₂^·−) scavenging activity in rat brain was visualized by the tissue-blotting technique. The activity was thought to mainly depend on Cu/Zn-SOD, because the localization of the activity was identical with the immunohistochemistry of Cu/Zn-SOD and the localization of its mRNA in the brain. Moreover, the activity was dramatically decreased after treatment of Cu (I) chelater. The activity was detected in pyramidal cells of the cortex, granular, and mitral cells of the olfactory bulbs, pyramidal cell layer CA1 to CA3, and dentate gyrus of hippocampus formation and granular cells of the cerebellum. Moreover, the activity was detected in the pontine nuclei of brain stem. Olfactory bulbs, hippocampus, and cerebellum were believed to be bestowed high brain functions, i.e., long-term potentiation and long-term depression. A part of the function was regulated by a retrograde neurotransmitter, nitric oxide (^·NO). Our findings suggest that the SOD is colocalized with NO synthase in olfactory bulbs, hippocampus, and cerebellum, where ^·NO plays the important roles. In contrast, low SOD activity was observed in the axonal neurofiber bundles, although the regions contain a lot of membrane lipids, which was thought to be peroxidized by O₂^·− and related radicals such as ^·OH in the regions. From these findings, it was suggested that the SOD did not only play a role in protecting the neurons from endogenously formed O₂^·−, but also play a role in preservation of beneficial natures of ^·NO in the brain.