NAD(P)H oxidation elicits anion superoxide formation in radish plasmalemma vesicles

Radish plasmalemma-enriched fractions show an NAD(P)H-ferricyanide or NAD(P)H-cytochrome c oxidoreductase activity which is not influenced by pH in the 4.5-7.5 range. In addition, at pH 4.5-5.0, NAD(P)H elicits an oxygen consumption (NAD(P)H oxidation) inhibited by catalase or superoxide dismutase (SOD), added either before or after NAD(P)H addition. Ferrous ions stimulate NAD(P)H oxidation, which is again inhibited by SOD and catalase. Hydrogen peroxide does not stimulate NADH oxidation, while it does stimulate Fe2+-induced NADH oxidation. NADH oxidation is unaffected by salicylhydroxamic acid and Mn2+, is stimulated by ferulic acid, and inhibited by KCN, EDTA and ascorbic acid. Moreover, NADH induces the conversion of epinephrine to adrenochrome, indicating that anion superoxide is formed during its oxidation. These results provide evidence that radish plasma membranes contain an NAD(P)H-ferricyanide or cytochrome c oxidoreductase and an NAD(P)H oxidase, active only at pH 4.5-5.0, able to induce the formation of anion superoxide, that is then converted to hydrogen peroxide. Ferrous ions, sparking a Fenton reaction, would stimulate NAD(P)H oxidation.     

Salicylhydroxamic acid-stimulated NADH oxidation by purified plasmalemma vesicles from wheat roots

Purified, right side-out plasmalemma vesicles were isolated from 7-day-old roots of dark-grown wheat ( Triticum aestivum L. cv. Drabant) by aqueous polymer two-phase partitioning. The oxygen consumption by these vesicles at pH 6.5 in the presence of 1 m M NADH [12–29 nmol (mg protein)⁻¹min⁻¹] was 66% inhibited by 1 m M KCN and ca 40% by 1 m M EDTA. It was unaffected by rotenone, antimycin A, carbonyl cyanide trifluoromethoxyphenylhydrazone (FCCP), mersalyl, chlorotetracycline + Ca²⁺, and EGTA. Salicylhydroxamic acid (SHAM) and its analogue, m -chlorobenzhydroxamic acid, stimulated the rate of oxygen consumption 10–20 fold in the presence of 1 m M NAD(P)H with an apparent K_m (SHAM) of ca 40 μ M (with NADH). The dependence of O₂ consumption on NADH concentration in the presence of SHAM (2 m M ) was sigmoidal, possibly due to endogenous catalase activity, and half-maximal rate was obtained at 1.5 m M . In the absence of SHAM the rate increased with increasing acidity and no pH optimum was detectable between pH 4.5 and 8.5. In the presence of SHAM an optimum was observed at pH 6.5 and 0.8 mol of H₂O₂ was produced for every 1 mol O₂ consumed. Endogenous catalase converted this H₂O₂ to O₂ and after complete conversion the stoichiometry was 2 mol NADH consumed for every mol O₃. SHAM was not consumed in the reaction. The possible involvement of a cytochrome P-450/420 system is discussed.     

Vanadate stimulates oxidation of NADH to generate hydrogen peroxide

Vanadate in the polymeric form of decavanadate, but not other forms, stimulated oxidation of NADH to NAD⁺ NADPH was also oxidized with comparable rates. This oxidation of NADH was accompanied by uptake of oxygen and generated hydrogen peroxide with the following stoichiometry: NADH + H⁺ + O₂ → NAD⁺ + H₂O₂. The reaction followed second-order kinetics. The rate was dependent on the concentration of both NADH and vanadate and increased with decreasing pH. The reaction had an obligatory requirement for phosphate ions. Esr studies in the presence of the spin trap dimethyl pyrroline N oxide indicated the involvement of Superoxide anion as an intermediate. The reaction was sensitive to Superoxide dismutase and other scavengers of superoxide anions.     

Fusicoccin stimulates the H+-ATPase of plasmalemma in isolated membrane vesicles from radish

The effect of fusicoccin on the plasmalemma H+-ATPase has been investigated in a membrane fraction from 24 h old radish seedlings, in which Mg:ATP-dependent H+-transport is mediated only by the plasmalemma H+-ATPase. Fusicoccin stimulated the plasmalemma H+-ATPase - i.e. Mg:ATP-dependent intravesicular acidification, hyperpolaryzation of delta psi and ATPase activity -, when these activities were measured at the physiologically relevant pHs of 7.3 to 7.6. No effect of FC on the plasmalemma H+-ATPase was evident at pH 6.6.     

NADH oxidation in phospholipid-enriched cytoplasmic membrane vesicles from Escherichia coli

NADH oxidation in Escherichia coli cytoplasmic membrane vesicles enriched in anionic phospholipids by de novo synthesis of lipid in the vesicles from acyl-CoA esters and sn-glycerol 3-phosphate has been studied. NADH-oxidase but not NADH-dehydrogenase activity was found to decrease during synthesis and accumulation of phospholipid in the vesicles. Density gradient fractionation showed that NADH-oxidase activity was reduced to approximately 30% in vesicles with a 3-6 fold increase in anionic phospholipid, whereas vesicles with a greater than 10-fold increase in phospholipid had virtually no NADH oxidase activity.     

Hydrogen peroxide production by roots and its stimulation by exogenous NADH

H₂O₂ production by roots of young seedlings was monitored using a non-destructive in vivo assay at pH 5.0. A particularly high rate of H₂O₂ production was measured in the roots of soybean (Glycine max L. cv. Labrador) seedlings which were used for further investigation of the physiological and enzymological properties of apoplastic H₂O₂ production. In the soybean root H₂O₂ production can be stimulated 10-fold by exogenous NADH or NADPH. This response displays typical features of a peroxidase-catalyzed oxidase reaction using NAD(P)H as electron donor for the reduction of O₂ to H₂O₂. Comparative measurements showed that the NADH-induced H₂O₂ production of the roots resembles the H₂O₂-forming activity of horseradish peroxidase with respect to NADH and O₂ concentration requirements and sensitivity to inhibition by KCN, NaN₃, superoxide dismutase and catalase. NADH-induced H₂O₂ production can be observed with similar intensity in all regions of the root, in agreement with the distribution of apoplastic peroxidase activity. In contrast, the activity responsible for the basal H₂O₂ production in the absence of exogenous NADH was mainly confined to a short subapical zone of the root and differs from the NADH-induced reaction by insensitivity to inhibition by superoxide dismutase and a strikingly lower requirement for O₂. It is concluded that the basal H₂O₂ production of the root is mediated by an enzyme different from peroxidase, possibly a plasma membrane O₂^?-producing oxidase.     

Hydrogen peroxide formation during iron deposition in horse spleen ferritin using O2 as an oxidant

The reaction of Fe2+ with O2 in the presence of horse spleen ferritin (HoSF) results in deposition of FeOH3 into the hollow interior of HoSF. This reaction was examined at low Fe2+/HoSF ratios (5-100) under saturating air at pH 6.5-8.0 to determine if H2O2 is a product of the iron deposition reaction. Three methods specific for H2O2 detection were used to assess H2O2 formation: (1) a fluorometric method with emission at 590 nm, (2) an optical absorbance method based on the reaction H2O2 + 3I- + 2H+ = I3- + 2H2O monitored at 340 nm for I3- formation, and (3) a differential pulsed electrochemical method that measures O2 and H2O2 concentrations simultaneously. Detection limits of 0.25, 2.5, and 5.0 microM H2O2 were determined for the three methods, respectively. Under constant air-saturation conditions (20% O2) and for a 5-100 Fe2+/HoSF ratio, Fe2+ was oxidized and the resulting Fe3+ was deposited within HoSF but no H2O2 was detected as predicted by the reaction 2Fe2+ + O2 + 6H2O = 2Fe(OH)3 + H2O2 + 4H+. Two other sets of conditions were also examined: one with excess but nonsaturating O2 and another with limiting O2. No H2O2 was detected in either case. The absence of H2O2 formation under these same conditions was confirmed by microcoulometric measurements. Taken together, the results show that under low iron loading conditions (5-100 Fe2+/HoSF ratio), H2O2 is not produced during iron deposition into HoSF using O2 as an oxidant. This conclusion is inconsistent with previous, carefully conducted stoichiometric and kinetic measurements [Xu, B., and Chasteen, N. D. (1991) J. Biol. Chem. 266, 19965], predicting that H2O2 is a quantitative product of the iron deposition reaction with O2 as an oxidant, even though it was not directly detected. Possible explanations for these conflicting results are considered.     

Oxygen consumption by purified plasmalemma vesicles from wheat roots: Stimulation by NADH and salicylhydroxamic acid (SHAM)

Plasmalemma vesicles from wheat (Triticum aestivum L.) roots consumed O₂ and the addition of 1 mM NADH increased the rate ~ 3-fold (to 15-30 nmol O₂·mg⁻¹·min⁻¹). The NADH-dependent O₂ uptake was abolished by catalase. In the presence of salicylhydroxamic acid (SHAM), an inhibitor of the alternative oxidase pathway in plant mitochondria, NADH-dependent O₂ consumption was stimulated 10–20-fold (to 200–400 nmol·mg^1̄·min⁻¹). Catalase also abolished this stimulation, which was KCN-sensitive but antimycin A-insensitive, and the production of H₂O₂ during SHAM-stimulated NADH-dependent O₂ uptake was demonstrated. Irrespective of the mechanism, SHAM-stimulated respiration by root plasmalemma makes it difficult to interpret results on root respiration obtained using KCN and SHAM.     

Hydrogen peroxide formation in cultured rose cells in response to UV-C radiation

Suspension-cultured rose ( Rosa damascena Mill. cv. Gloire de Guilan) cells irradiated with UV-C (254 nm. 558 J m⁻²) showed a transient production of H₂O₂ as measured by chemiluminescence of luminol in the presence of peroxidase (EC 1.1 1.1.7). The peak concentration of H₂O₂, which occurred at about 60–90 min after irradiation, was 8–9 μ M . The time course for the appearance of H₂O₂ matched that for UV–induced K⁺ efflux. Treatments that inhibited the UV-induced efflux of K⁺, including heat and overnight incubation with cycloheximide and diethylmaleate, also inhibited the appearance of H₂O₂. The converse was not always true, since catalase (EC 1.11.1.6. and salicylhydroxamic acid, which inhibited luminescence, did not stop K⁺ efflux. We conclude that H₂O₂ synthesis depends on K⁺ efflux. Because H₂.O₂ in the extracellular space is required for lignin synthesis in many plant tissues, we suggest that the UV–stimulated production of H₂O₂ is an integral part of a defensive lignin synthesis.     

Hydrogen peroxide linked to lysine oxidase activity facilitates biofilm differentiation and dispersal in several gram-negative bacteria

The marine bacterium Pseudoalteromonas tunicata produces an antibacterial and autolytic protein, AlpP, which causes death of a subpopulation of cells during biofilm formation and mediates differentiation, dispersal, and phenotypic variation among dispersal cells. The AlpP homologue (LodA) in the marine bacterium Marinomonas mediterranea was recently identified as a lysine oxidase which mediates cell death through the production of hydrogen peroxide. Here we show that AlpP in P. tunicata also acts as a lysine oxidase and that the hydrogen peroxide generated is responsible for cell death within microcolonies during biofilm development in both M. mediterranea and P. tunicata. LodA-mediated biofilm cell death is shown to be linked to the generation of phenotypic variation in growth and biofilm formation among M. mediterranea biofilm dispersal cells. Moreover, AlpP homologues also occur in several other gram-negative bacteria from diverse environments. Our results show that subpopulations of cells in microcolonies also die during biofilm formation in two of these organisms, Chromobacterium violaceum and Caulobacter crescentus. In all organisms, hydrogen peroxide was implicated in biofilm cell death, because it could be detected at the same time as the killing occurred, and the addition of catalase significantly reduced biofilm killing. In C. violaceum the AlpP-homologue was clearly linked to biofilm cell death events since an isogenic mutant (CVMUR1) does not undergo biofilm cell death. We propose that biofilm killing through hydrogen peroxide can be linked to AlpP homologue activity and plays an important role in dispersal and colonization across a range of gram-negative bacteria.     

Hydrogen peroxide affects ion channels in lily pollen grain protoplasts

Ion homeostasis plays a central role in polarisation and polar growth. In several cell types ion channels are controlled by reactive oxygen species (ROS). One of the most important cells in the plant life cycle is the male gametophyte, which grows under the tight control of both ion fluxes and ROS balance. The precise relationship between these two factors in pollen tubes has not been completely elucidated, and in pollen grains it has never been studied to date. In the present study we used a simple model – protoplasts obtained from lily pollen grains at the early germination stage – to reveal the effect of H₂O₂ on cation fluxes crucial for pollen germination. Here we present direct evidence for two ROS‐sensitive currents on the pollen grain plasma membrane: the hyperpolarisation‐activated calcium current, which is strongly enhanced by H₂O₂, and the outward potassium current, which is modestly enhanced by H₂O₂. We used low concentrations of H₂O₂ that do not cause an intracellular oxidative burst and do not damage cells, as demonstrated with fluorescent staining.     

Hydrogen peroxide causes greater oxidation in cellular RNA than in DNA

Human A549 lung epithelial cells were challenged with 18O-labeled hydrogen peroxide ([18O]-H2O2), the total RNA and DNA extracted in parallel, and analyzed for 18O-labeled 8-oxo-7,8-dihydroguanosine ([18O]-8-oxoGuo) and 8-oxo-7,8-dihydro-2'-deoxyguanosine ([18O]-8-oxodGuo) respectively, using high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-MS/MS). [18O]-H2O2 exposure resulted in dose-response formation of both [18O]-8-oxoGuo and [18O]-8-oxodGuo and 18O-labeling of guanine in RNA was 14-25 times more common than in DNA. Kinetics of formation and subsequent removal of oxidized nucleic acids adducts were also monitored up to 24 h. The A549 showed slow turnover rates of adducts in RNA and DNA giving half-lives of approximately 12.5 h for [18O]-8-oxoGuo in RNA and 20.7 h for [18O]-8-oxodGuo in DNA, respectively.     

Hydrogen peroxide for disulfide bridge formation in methionine-containing peptides.

Two methionine-containing peptides, endothelin 1 and the 1-16 fragment of the receptor of the plasminogen activator 1 for human urokinase, were synthesized and cyclized by hydrogen peroxide. Endothelin 1 was obtained by using regioselective and random schemes of disulfide bond formation. The conditions of cyclization that provided the target products in high purity were found. The general potential of disulfide bond formation by means of hydrogen peroxide was demonstrated for methionine-containing peptides. The method resulted in target products containing insignificant quantities of the corresponding Met-sulfoxide derivatives.     

Hydrogen peroxide in the aqueous humor and cataract formation in human diabetes

Hydrogen peroxide in the aqueous humor was measured in cataractous eyes from normal subjects and in cataractous eyes from diabetic subjects. The level of H2O2 in the aqueous humor was significantly higher in diabetes than in the idiopathic forms. It is likely that in the eye, impaired enzymic defenses lead to the accumulation of reactive species of O2, such as H2O2, which induces lipid peroxidation. This mechanism may be involved, as a direct consequence of retinal damage, in the pathogenesis of cataract in diabetes.     

Hydrogen peroxide involvement in formation and development of adventitious roots in cucumber

Hydrogen peroxide (H₂O₂), an active oxygen species, is widely generated in many biological systems. The present study demonstrates that H₂O₂ was generated in seedling explants after the primary roots were removed, and it mediates the auxin response prior to adventitious root formation in cucumber (Cucumis sativus L. Ganfeng 8). When compared with the controls, treatment of cucumber seedling explants after primary roots removal with either 20–40 mM H₂O₂ or 10 μM IAA significantly increased the number of adventitious roots, and treatment with 10–50 mM H₂O₂ significantly increased the fresh weight of adventitious roots. The effects of H₂O₂ on promoting the formation and growth of adventitious roots were eliminated by 2 mM ascorbic acid, 100 U CAT or 1 μM DPI, and the effects of IAA were eliminated by 4 mM ascorbic acid, 100 U CAT or 5 μM DPI. Treatment with either 4 mM ascorbic acid or 1–5 μM DPI inhibited the formation and growth of adventitious roots, and these inhibitory effects were partly reversed by exogenous H₂O₂.Furthermore, a higher concentration of endogenous H₂O₂ was detected in seedling explants 3 h after the primary roots were removed. However, in 10 μM DPI-treated seedling explants, the concentration of endogenous H₂O₂ was markedly reduced by DPI. Results obtained suggest that H₂O₂ may function as a signaling molecule, involved in the formation and development of adventitious roots in cucumber.     

Hydrogen peroxide and superoxide radical formation in anaerobic broth media exposed to atmospheric oxygen.

Fourteen different broth media were autoclaved under anaerobic conditions and then exposed to atmospheric oxygen. The hydrogen peroxide and superoxide radical formation as well as the bactericidal effect of the media were studied. The rate of killing of Peptostreptococcus anaerobius VPI 4330-1 was high in media that rapidly autoxidized and accumulated hydrogen peroxide. In actinomyces broth (BBL), 50% of the cells were killed within 2 min, and in Brewer thioglycolate medium (Difco), 50% were killed within 11 min, whereas more than 50% of the cells survived for more than 2 h in Clausen medium (Oxoid), fluid thioglycolate medium (BBL), and thioglycolate medium without dextrose or indicator (Difco). Only media that contained phosphate and glucose had a tendency to accumulate hydrogen peroxide. A solution of phosphate and glucose autoxidized when it had been heated to 120 degrees C for at least 5 min and when the pH of the solution was higher than 6.5. Transitional metal ions catalyzed the autoxidation, but they were not necessary for the reaction to occur. Of the other substances heated in phosphate buffer, only alpha-hydroxycarbonyl compounds autoxidized with accumulation of hydrogen peroxide. Superoxide dismutase decreased the autoxidation rate of most of the broth media. This indicated that superoxide radicals were generated in these media.