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1.
Ferric leghemoglobin reductase (FLbR) from soybean ( Glycine max [L.] Merr) nodules catalyzed oxidation of NADH, reduction of ferric leghemoglobin (Lb +3), and reduction of dichloroindophenol (diaphorase activity). None of these reactions was detectable when O 2 was removed from the reaction system, but all were restored upon readdition of O 2. In the absence of exogenous electron carriers and in the presence of O 2 and excess NADH, FLbR catalyzed NADH oxidation with the generation of H 2O 2 functioning as an NADH oxidase. The possible involvement of peroxide-like intermediates in the FLbR-catalyzed reactions was analyzed by measuring the effects of peroxidase and catalase on FLbR activities; both enzymes at low concentrations (about 2 μg/mL) stimulated the FLbR-catalyzed NADH oxidation and Lb +3 reduction. The formation of H 2O 2 during the FLbR-catalyzed NADH oxidation was confirmed using a sensitive assay based on the fluorescence emitted by dichlorofluorescin upon reaction with H 2O 2. The stoichiometry ratios between the FLbR-catalyzed NADH oxidation and Lb +3 reduction were not constant but changed with time and with concentrations of NADH and O 2 in the reaction solution, indicating that the reactions were not directly coupled and electrons from NADH oxidation were transferred to Lb +3 by reaction intermediates. A study of the affinity of FLbR for O 2 showed that the enzyme required at least micromolar levels of dissolved O 2 for optimal activities. A mechanism for the FLbR-catalyzed reactions is proposed by analogy with related oxidoreductase systems. 相似文献
2.
Summary NADH oxidase activity has been detected at the ultrastructural level using cerium ions to trap H 2O 2 generated by the enzyme (via intermediate reactive oxygen species). In an attempt to localize NADH oxidase activity at the light microscope level using the cerium-diaminobenzidine (DAB)-nickel-H 2O 2, the cerium-DAB-cobalt-H 2O 2 or the cerium-alkaline lead procedures, the distribution patterns of the revealed enzyme were found to be identical to those for non-specific alkaline phosphatase and especially 5-nucleotidase activity. With the cerium-DAB-cobalt-H 2O 2 visualization procedure, the distribution pattern of the inial reaction product was similar to that obtained with the other two techniques but much less final reaction product was formed. Incubations for NADH oxidase activity performed in the presence of exogenous catalase or in the absence of catalase or peroxidase inhibitors did not affect the staining intensity, whereas inhibitors of 5-nucleotidase (EDTA) and non-specific alkaline phosphatase (levamisole) always did. Therefore, phosphatases contribute to the formation of the final reaction product. Since NADH initially cannot be hydrolysed by either of these two phosphatases, then presumably nucleotide prophosphatase (E.C.3.6.1.9) cleaves NADH into 5-AMP and nicotinamide mononucleotide in a first step. Both nucleotides can be hydrolysed further by the two monophosphatases. These then generate cerium phosphate which is detected by the DAB-nickel-H 2O 2, DAB-cobalt-H 2O 2 or lead visualization methods. 相似文献
3.
Addition of NADH inhibited the peroxidative loss of scopoletin in presence of horseradish and H 2O 2 and decreased the ratio of scopoletin (consumed):H 2O 2 (added). Concomitantly NADH was oxidized and oxygen was consumed with a stoichiometry of NADH:O 2 of 2:1. On step-wise addition of a small concentration of H 2O 2 a high rate of NADH oxidation was obtained for a progressively decreasing time period followed by termination of the reaction with NADH:H 2O 2 ratio decreasing from about 40 to 10. The rate of NADH oxidation increased linearly with increase in scopoletin concentration. Other phenolic compounds including p-coumarate also supported this reaction to a variable degree. A 418-nm absorbing compound accumulated during oxidation of NADH. The effectiveness of a small concentration of H 2O 2 in supporting NADH oxidation increased in presence of SOD and decreased in presence of cytochrome c, but the reaction terminated even in their presence. The results indicate that the peroxidase is not continuously generating H 2O 2 during scopoletin-mediated NADH oxidation and that both peroxidase and oxidase reactions occur simultaneously competing for an active form of the enzyme. 相似文献
4.
The enzyme horseradish peroxidase (EC 1.11.1.7) catalyses oxidation of NADH. NADH oxidation is prevented by addition of the enzyme superoxide dismutase (EC 1.15.1.1) to the reaction mixture before adding peroxidase but addition of dismutase after peroxidase has little inhibitory effect. Catalase (EC 1.11.1.6) inhibits peroxidase-catalysed NADH oxidation when added at any time during the reaction. Apparently the peroxidase uses hydrogen peroxide (H 2O 2) generated by non-enzymic breakdown of NADH to catalyse oxidation of NADH to a free-radical, NAD., which reduces oxygen to the superoxide free-radical ion, O 2
.-. Some of the O 2
.- reacts with peroxidase to give peroxidase compound III, which is catalytically inactive in NADH oxidation. The remaining O 2
.- undergoes dismutation to O 2 and H 2O 2. O 2
.- does not react with NADH at significant rates. Mn 2+ or lactate dehydrogenase stimulate NADH oxidation by peroxidase because they mediate a reaction between O 2
.- and NADH. 2,4-Dichlorophenol, p-cresol and 4-hydroxycinnamic acid stimulate NADH oxidation by peroxidase, probably by breaking down compound III and so increasing the amount of active peroxidase in the reaction mixture. Oxidation in the presence of these phenols is greatly increased by adding H 2O 2. The rate of NADH oxidation by peroxidase is greatest in the presence of both Mn 2+ and those phenols which interact with compound III. Both O 2
.- and H 2O 2 are involved in this oxidation, which plays an important role in lignin synthesis. 相似文献
5.
The two peroxidase isoenzyme groups (G I and G III) localized in the cell walls of tobacco ( Nicotiana tabacum L.) tissues were compared with respect to their capacity for NADH-dependent H 2O 2 formation. Peroxidases of the G III group are slightly more active than those of the G I group when both are assayed under optimal conditions. This difference is probably not of major regulatory importance. NADH-dependent formation of H 2O 2 required the presence of Mn 2+ and a phenol as cofactors. The addition of H 2O 2 to the reaction mixture accelerated subsequent NADH-dependent H 2O 2 formation. In the presence of both cofactors or Mn 2+ alone, catalase oxidized NADH. However, if the cofactors were absent or if only dichlorophenol was present, catalase inhibited NADH oxidation. No H 2O 2 accumulation occurred in the presence of catalase. Superoxide dismutase inhibited NADH oxidation quite significantly indicating the involvement of the superoxide radical in the peroxidase reaction. These results are interpreted to mean that the reactions whereby tobacco cell wall peroxidases catalyze NADH-dependent H 2O 2 formation are similar to those proposed for horseradish peroxidase (Halliwell 1978 Planta 140: 81-88). 相似文献
6.
The pathways through which NADPH, NADH and H 2 provide electrons to nitrogenase were examined in anaerobically isolated heterocysts. Electron donation in freeze-thawed heterocysts and in heterocyst fractions was studied by measuring O 2 uptake, acetylene reduction and reduction of horse heart cytochrome c. In freeze-thawed heterocysts and membrane fractions, NADH and H 2 supported cyanide-sensitive, respiratory O 2 uptake and light-enhanced, cyanide-insensitive uptake of O 2 resulting from electron donation to O 2 at the reducing side of Photosystem I. Membrane fractions also catalyzed NADH-dependent reduction of cytochrome c. In freeze-thawed heterocysts and soluble fractions from heterocysts, NADPH donated electrons in dark reactions to O 2 or cytochrome c through a pathway involving ferredoxin:NADP reductase; these reactions were only slightly influenced by cyanide or illumination. In freeze-thawed heterocysts provided with an ATP-generating system, NADH or H 2 supported slow acetylene reduction in the dark through uncoupler-sensitive reverse electron flow. Upon illumination, enhanced rates of acetylene reduction requiring the participation of Photosystem I were observed with NADH and H 2 as electron donors. Rapid NADPH-dependent acetylene reduction occurred in the dark and this activity was not influenced by illumination or uncoupler. A scheme summarizing electron-transfer pathways between soluble and membrane components is presented. 相似文献
7.
The rate of ascorbate and nicotinamide adenine dinucleotide plus hydrogen (NADH) cooxidation (i.e., their nonenzymic oxidation
by peroxidase/H 2O 2-generated phenoxyl radicals of three hydroxycinnamates: caffeate, ferulate and p-coumarate) was studied in vitro. The reactions initiated by different sources of peroxidase (EC 1.11.1.7) [isolates from
soybean ( Glycine max L.) seed coat, maize ( Zea mays L.) root-cell wall, and commercial horseradish peroxidase] were monitored. Native electrophoresis of samples and specific
staining for peroxidase activity revealed various isoforms in each of the three enzyme sources. The peroxidase sources differed
both in the rate of H 2O 2-dependent hydroxycinnamate oxidation and in the order of affinity for the phenolic substrates. The three hydroxycinnamates
did not differ in their ability to cooxidize ascorbate, whereas NADH cooxidation was affected by substitution of the phenolic
ring. Thus, p-coumarate was more efficient than caffeate in NADH cooxidation, with ferulate not being effective at all. Metal ions (Zn 2+ and Al 3+) inhibited the reaction of peroxidase with p-coumarate and affected the cooxidation rate of ascorbate and the peroxidase reaction in the same manner with all substrates
used. However, inhibition of p-coumarate oxidation by metal ions did not affect NADH cooxidation rate. We propose that both the ascorbate and NADH cooxidation
systems can function as mechanisms to scavenge H 2O 2 and regenerate phenolics in different cellular compartments, thus contributing to protection from oxidative damage.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
8.
Two strains of Lactobacillus plantarum accumulated H 2O 2 when grown aerobically in a complex glucose based medium. The H 2O 2 accumulation did not occur immediately on exposure of the culture to O 2 but was delayed for a time which, in the case of one strain, was dependent on the amount of inoculum used to seed the culture. The accumulation was always preceded by an increase in the rate of O 2 utilization by the cultures. The latter coincided approximately with an increase in specific activity of NADH oxidase, pyruvate oxidase and NADH peroxidase. H 2O 2 was not a product of NADH oxidase in vitro but was formed in substantial quantities from O 2 during oxidation of pyruvate. The three enzymes were induced by O 2 and H 2O 2; the induction of NADH oxidase responded to lower levels of O 2 (but not of H 2O 2) than the pyruvate oxidase or the NADH peroxidase.Abbreviations MRSG
Mann, Rogosa and Sharpe medium (1960) with glucose as fermentation source
- TPP
thiamin pyrophosphate 相似文献
9.
The oxidation of NADH by mouse liver plasma membranes was shown to be accompanied by the formation of H 2O 2. The rate of H 2O 2 formation was less than one-tenth the rate of oxygen uptake and much slower than the rate of reduction of artificial electron acceptors. The optimum pH for this reaction was 7.0 and the K
m value for NADH was found to be 3×10 –6 M. The H 2O 2-generating system of plasma membranes was inhibited by quinacrine and azide, thus distinguishing it from similar activities in endoplasmic reticulum and mitochondria. Both NADH and NADPH served as substrates for plasma membrane H 2O 2 generation. Superoxide dismutase and adriamycin inhibited the reaction. Vanadate, known to stimulate the oxidation of NADH by plasma membranes, did not increase the formation of H 2O 2. In view of the growing evidence that H 2O 2 can be involved in metabolic control, the formation of H 2O 2 by a plasma membrane NAD(P)H oxidase system may be pertinent to control sites at the plasma membrane. 相似文献
10.
In potato ( Solatium tuberosum L. cv. Bintje and Doré) callus a very active hydrox-amate-stimulated NADH-dependent O 2-uptake develops during the growth of the callus, which is caused by a peroxidase. More than 95% of the peroxidase activity is found in the 40000 g supernatant. The total activity may be as high as 1000 times the respiratory acitivity of the callus tissue. At least two fractions, obtained by Sephadex gel filtration, can be distinguished showing this peroxidase activity, one of about 15 kDa and one > 50 kDa. The main properties of both fractions are: a) Hydroxamate at 0.2–0.5 m M gives half-maximal stimulation. Maximal stimulation is observed with 1–3 m M benzhydroxamate (BHAM) and 1–15 m M salicylhydroxamate (SHAM). Higher concentrations, especially of BHAM, give less or no stimulation. b) Hydroxamates are not consumed during the reaction. c) Both NADH and NADPH can serve as the electron donor for the reaction. The affinity for NAD(P)H is very low (K m near 10 m M). In the absence of hydroxamates NAD(P)H is only slowly oxidized, with an even lower affinity. d) The peroxidase can carry out two reactions: an O 2-consuming and a H 2O 2-consuming reaction. In both reactions one NAD(P)H is consumed. In the first reaction H 2O 2 is formed which can be consumed in the second reaction, resulting in an overall stoichiometry of 2 NADH consumed for each O 2 molecule and in the production of H 2O. e) The reaction is completely blocked by cyanide, superoxide dismutase (EC 1.15.1.1) and (excess) catalase (EC 1.11.1.6), but not by antimycin A or azide. This peroxidase-mediated O 2-uptake might interfere with respiratory measurements. In experiments with isolated mitochondria this interference can be prevented by the addition of catalase to the reaction mixture. The use of high concentrations of hydroxamate is not allowed because of inhibitory effects on the cytochrome pathway. In intact callus tissue hydroxamates only stimulate O 2-uptake in the presence of exogenous NADH. In vivo the peroxidase does not appear to function in O 2-uptake, probably because of its localization (at least partly in the cell wall) and/or its low affinity for NADH. The use of hydroxamates in the determination of cytochrome and alternative pathway activity is discussed. 相似文献
11.
Glucose oxidation by immobilized glucose oxidase (GlO) and catalase (Cat) has been investigated in batch and continuous reactions for operational studies. The macrokinetics of the process depend on coupled reaction steps and diffusion rates. The problem may be approximated by a simple pseudohomogeneous model taking into account both substrates of glucose oxidase and the intermediate reaction product H 2O 2. The effectiveness of both enzymes is enhanced in the coupled reaction path, the overall effectiveness nevertheless is very low. H 2O 2 causes the inactivation of both GlO and Cat. The rates of deactivation depend on the oxidation rates of glucose that give different quasistationary levels of H 2O 2 concentration. As a first approximation, the deactivation rates may be described by first-order reactions with respect to H 2O 2. 相似文献
12.
In the presence of NADH, and the reductase and rubredoxin components of the ω-hydroxylation system of , epinephrine is oxidized to adrenochrome at pH 7.8, and the reaction is strongly inhibited by the addition of superoxide dismutase (SDM). Boiled SDM has no effect on the reaction rate. The oxidation reaction is oxygen-dependent, and approximately 1 mole of H 2O 2 is produced per mole of O 2 consumed. The stoichiometry between NADH oxidation and adrenochrome formation is approximately 2:1. Epoxidation and epinephrine oxidation are mutually competitive reactions, despite the fact that the epoxidation reaction is not stimulated by a superoxide generating system nor inhibited by SDM. 相似文献
13.
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 + + O 2 → NAD + + H 2O 2. 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. 相似文献
14.
Vanadyl (V (IV)) salts autoxidize in neutral aqueous solution yielding O 2− plus vanadate (V (V)) and these, in turn, cause the oxidation of NADH, by a free radical chain reaction. This oxidation of NADH was inhibited by superoxide dismutase, but not by a scavenger of HO.. When H 2O 2 was present V (IV)) caused rapid oxidation of NADH by a process which was unaffected by superoxide dismutase but was inhibited by a scavenger of HO.. This appeared to be dependent upon reduction of H 2O 2 to OH − plus HO., by V (IV)), followed by oxidation of NADH by HO.. Since there are reductants, within cells, capable of reducing V (V)) to V (IV), these reactions are likely to contribute to the toxicity of vanadate. 相似文献
15.
Hydrogen peroxide (H 2O 2) was detected cytochemically, via transmission electron microscopy (TEM), in pumpkin tissues exposed to high-dose gamma ray.
Its reaction with cerium chloride produced electron-dense precipitates of cerium perhydroxides. Their patterns of deposition
in the tissues of both control plants and those irradiated with gamma ray (PIG) were typically found in the plasma membranes
and cell walls. However, gamma irradiation remarkably increased the intensities of cerium perhydroxide deposits (CPDs) in
the plasma membranes and cell walls for all tissue types, but especially the leaves. The only exception was for vessels in
the cotyledons. After gamma irradiation, the (H 2O 2) content in all tissues was higher than in the control samples, except for the cotyledons of PIG, where the (H 2O 2) content was lower than for all others. The increased appearance of CPDs may have been due to the enhancement of (H 2O 2) accumulation by gamma radiation. This accumulation also varied according to the cell or tissue type examined. 相似文献
16.
This work was undertaken to verify whether surface NADH oxidases or peroxidases are involved in the apoplastic reduction of Fe(III). The reduction of Fe(III)-ADP, linked to NADH-dependent activity of horseradish peroxidase (HRP), protoplasts and cells of Acer pseudoplatanus, was measured as Fe(II)-bathophenanthrolinedisulfonate (BPDS) chelate formation. In the presence of BPDS in the incubation medium (method 1), NADH-dependent HRP activity was associated with a rapid Fe(III)-ADP reduction that was almost completely inhibited by superoxide dismutase (SOD), while catalase only slowed down the rate of reduction. A. pseudoplatanus protoplasts and cells reduced extracellular Fe(III)-ADP in the absence of exogenously supplied NADH. The addition of NADH stimulated the reduction. SOD and catalase only inhibited the NADH-dependent Fe(III)-ADP reduction. Mn(II), known for its ability to scavenge O ?2, inhibited both the independent and NADH-dependent Fe(III)-ADP reduction. The reductase activity of protoplasts and cells was also monitored in the absence of BPDS (method 2). The latter was added only at the end of the reaction to evaluate Fe(II) formed. Also, in this case, both preparations reduced Fe(III)-ADP. However, the addition of NADH did not stimulate Fe(III)-ADP reduction but, instead, lowered it. This may be related to a re-oxidation of Fe(II) by H 2O 2 that could also be produced during NADH-dependent peroxidase activity. Catalase and SOD made the Fe(III)-ADP reduction more efficient because, by removing H 2O 2 (catalase) or preventing H 2O 2 formation (SOD), they hindered the re-oxidation of Fe(II) not chelated by BPDS. As with the result obtained by method 1, Mn(II) inhibited Fe(III)-ADP reduction carried out in the presence or absence of NADH. The different effects of SOD and Mn(II), both scavengers of O ?2, may depend on the ability of Mn(II) to permeate the cells more easily than SOD. These results show that A. pseudoplatanus protoplasts and cells reduce extracellular Fe(III)-ADP. Exogenously supplied NADH induces an additional reduction of Fe(III) by the activity of NADH peroxidases of the plasmalemma or cell wall. However, the latter can also trigger the formation of H 2O 2 that, reacting with Fe(II) (not chelated by BPDS), generates hydroxyl radicals and converts Fe(II) to Fe(III) (Fenton's reaction). 相似文献
17.
It has recently been reported that plasmalemma electron transport may be involved in the generation of H + gradients and the uptake of ions into root tissue. We report here on the influence of extracellular NADH and ferricyanide on K + ( 86Rb +) influx, K + ( 86Rb +) efflux, net apparent H + efflux, and O 2 consumption in 2-centimeter corn ( Zea mays [A632 × Oh43]) root segments and intact corn roots. In freshly excised root segments, NADH had no effect on O 2 consumption and K + uptake. However, after the root segments were given a 4-hour wash in aerated salt solution, NADH elicited a moderate stimulation in O 2 consumption but caused a dramatic inhibition of K + influx. Moreover, net apparent H + efflux was significantly inhibited following NADH exposure in 4-hour washed root segments. Exogenous ferricyanide inhibited K+ influx in a similar fashion to that caused by NADH, but caused a moderate stimulation of net H+ efflux. Additionally, both reagents substantially altered K+ efflux at both the plasmalemma and tonoplast. These complex results do not lend themselves to straightforward interpretation and are in contradiction with previously published results. They suggest that the interaction between cell surface redox reactions and membrane transport are more complex than previously considered. Indeed, more than one electron transport system may operate in the plasmalemma to influence, or regulate, a number of transport functions and other cellular processes. The results presented here suggest that plasmalemma redox reactions may be involved in the regulation of ion uptake and the `wound response' exhibited by corn roots. 相似文献
18.
Summary New light microscopic visualization methods were developed for the histochemical detection of non-specific alkaline and acid phosphatase, Mg-, Ca-and Na, K-dependent adenosine triphosphatase, myosin adenosine triphosphatase, glucose-6-phosphatase, 5-nucleotidase and thiamine pyrophosphatase with cerium ions as trapping agents in cryostat and plastic sections. The techniques are based on the conversion of cerium phosphate into cerium perhydroxide by H 2O 2 which decomposes at 55°–60° C into cerium hydroxide and oxygen radicals. These radicals are able to oxidize diaminobenzidine (DAB) to DAB brown. Addition of nickel ions to the DAB-H 2O 2 mixture generates bluish-black stained nickel-DAB complexes. Compared with the classical metal precipitation, azo, azoindoxyl and tetrazolium procedures the H 2O 2-DAB and especially the H 2O 2-DAB-nickel methods provided identical or superior results in catalytic phosphatase histochemistry and immunohistochemistry when using non-specific alkaline phosphatase as the enzyme label. 相似文献
19.
Summary Visualization methods for the light microscopic detection of the activity of oxidases after being localized with cerium ions as reported by Angermüller and Fahimi (1988a, b) are not suitable for the demonstration of H 2O 2-genrating oxidases at sites with low activity. Therefore, the cerium-diaminobenzidine (DAB) visualization procedure of these authors was modified. Nickel or cobalt ions were added to the DAB solution together with small amounts of H 2O 2. Visualization was performed in a one-step-method. This modified visualization technique enables light micro-scopic detection of amino acid oxidase activity in kidney and liver cells where it was found with the original method but the amounts of final reaction product were considerably higher. Moreover, the DAB-nickel-H 2O 2 and DAB-cobalt-H 2O 2 procedures were more sensitive than the cerium-lead method of Angermüller and Fahimi (1988a, b). The method appeared to be specific, because final reaction product was not found after control incubation. Especially the DAB-nickel-H 2O 2 procedure can also be used for immunohistochemistry when glucose oxidase serves as the enzyme label.Supported by the Deutsche Forschungsgemeinschaft (Sfb 174) 相似文献
20.
H 2O 2 production by roots of young seedlings was monitored using a non-destructive in vivo assay at pH 5.0. A particularly high rate of H 2O 2 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 H 2O 2 production. In the soybean root H 2O 2 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 O 2 to H 2O 2. Comparative measurements showed that the NADH-induced H 2O 2 production of the roots resembles the H 2O 2-forming activity of horseradish peroxidase with respect to NADH and O 2 concentration requirements and sensitivity to inhibition by KCN, NaN 3, superoxide dismutase and catalase. NADH-induced H 2O 2 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 H 2O 2 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 O 2. It is concluded that the basal H 2O 2 production of the root is mediated by an enzyme different from peroxidase, possibly a plasma membrane O 2?-producing oxidase. 相似文献
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