Formation of Hydrogen Peroxide by Group N Streptococci and Its Effect on Their Growth and Metabolism

The formation of hydrogen peroxide by group N streptococci was found to occur through the action of a reduced nicotinamide adenine dinucleotide (NADH) oxidase which catalyzed the oxidation of NADH by molecular oxygen. The enzyme was activated by flavine adenine dinucleotide. Whereas some of the hydrogen peroxide formed was removed through the action of an NADH peroxidase, sufficient accumulated in media to inhibit the growth, respiration, and viability of these organisms. The amount of hydrogen peroxide which accumulated varied among strains, and this variation could be related to differences in the properties of the NADH oxidase present.     

Determination of hydrogen peroxide generated by reduced nicotinamide adenine dinucleotide oxidase

Hydrogen peroxide can be conveniently determined using horseradish peroxidase (HRP) and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid). However, interference occurs among assay components in the presence of reduced nicotinamide adenine dinucleotide (NADH) that is also a substrate of NADH oxidase. So, depletion of NADH is required before using the HRP method. Here, we report simple and rapid procedures to accurately determine hydrogen peroxide generated by NADH oxidase. All procedures developed were based on the extreme acid lability of NADH and the stability of hydrogen peroxide, because NADH was decomposed at pH 2.0 or 3.0 for 10 min, while hydrogen peroxide was stable at pH 2.0 or 3.0 for at least 60 min. Acidification and neutralization were carried out by adjusting sample containing NADH up to 30 microM to pH 2.0 for 10 min before neutralizing it back to pH 7.0. Then, hydrogen peroxide in the sample was measured using the HRP method and its determination limit was found to be about 0.3 microM. Alternatively, hydrogen peroxide in samples containing NADH up to 100 microM could be quantitated using a modified HRP method that required an acidification step only, which was found to have a determination limit of about 3 microM hydrogen peroxide in original samples.     

NAD (P) H-dependent reduction of nicotinamide N-oxide by an unique enzyme system consisting of liver microsomal NADPH-cytochrome C reductase and cytosolic aldehyde oxidase

NAD (P) H-dependent reduction of nicotinamide N-oxide was investigated with rabbit liver preparations. Microsomes, microsomal NADPH-cytochrome c reductase or cytosolic aldehyde oxidase alone exhibited no nicotinamide N-oxide reductase activity in the presence of NADPH or NADH. However, when the microsomal preparations were combined with the cytosolic enzyme, a significant N-oxide reductase activity was observed in the presence of the reduced pyridine nucleotide. The activity was enhanced by FAD or methyl viologen. Cytosol alone supplemented with NADPH or NADH exhibited only a slight, but when combined with microsomes, a significant N-oxide reductase activity. Based on these facts, we propose a new electron transfer system consisting of NADPH-cytochrome c reductase and aldehyde oxidase, which exhibits nicotinamide N-oxide reductase activity in the presence of the reduced pyridine nucleotide.     

Pyridine Nucleotide Transhydrogenase from Azotobacter vinelandii

A method is described for the partial purification of pyridine nucleotide transhydrogenase from Azotobacter vinelandii (ATCC 9104) cells. The most highly purified preparation catalyzes the reduction of 300 mumoles of nicotinamide adenine dinucleotide (NAD(+)) per min per mg of protein under the assay conditions employed. The enzyme catalyzes the reduction of NAD(+), deamino-NAD(+), and thio-NAD(+) with reduced nicotinamide adenine dinucleotide phosphate (NADPH) as hydrogen donor, and the reduction of nicotinamide adenine dinucleotide phosphate (NADP(+)) and thio-NAD(+) with reduced NAD (NADH) as hydrogen donor. The reduction of acetylpyridine AD(+), pyridinealdehyde AD(+), acetylpyridine deamino AD(+), and pyridinealdehydedeamino AD(+) with NADPH as hydrogen donor was not catalyzed. The enzyme catalyzes the transfer of hydrogen more readily from NADPH than from NADH with different hydrogen acceptors. The transfer of hydrogen from NADH to NADP(+) and thio-NAD(+) was markedly stimulated by 2'-adenosine monophosphate (2'-AMP) and inhibited by adenosine diphosphate (ADP), adenosine triphosphate (ATP), and phosphate ions. The transfer of hydrogen from NADPH to NAD(+) was only slightly affected by phosphate ions and 2'-AMP, except at very high concentrations of the latter reagent. In addition, the transfer of hydrogen from NADPH to thio-NAD(+) was only slightly influenced by 2'-AMP, ADP, ATP, and other nucleotides. The kinetics of the transhydrogenase reactions which utilized thio-NAD(+) as hydrogen acceptor and NADH or NADPH as hydrogen donor were studied in some detail. The results suggest that there are distinct binding sites for NADH and NAD(+) and perhaps a third regulator site for NADP(+) or 2'-AMP. The heats of activation for the transhydrogenase reactions were determined. The properties of this enzyme are compared with those of other partially purified transhydrogenases with respect to the regulatory functions of 2'-AMP and other nucleotides on the direction of flow of hydrogen between NAD(+) and NADP(+).     

Activation of the superoxide-generating NADPH oxidase of intestinal lymphocytes produces highly reactive free radicals from sulfite.

The objective of this study was to investigate the ability of immune cells of the small intestine to produce highly reactive free radicals from the food additive sulfites. These free radicals were characterized with a spin-trapping technique using the spin traps 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO). In the presence of glucose, purified lymphocytes from intestinal Peyer's patches (PP) and mesenteric lymph nodes (MLN) were stimulated with phorbol 12-myristate 13-acetate (PMA) to produce superoxide and hydroxyl DEPMPO radical adducts. The formation of these adducts was inhibited by superoxide dismutase or diphenyleneiodonium chloride, indicating that these cells produced superoxide radical during reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation. With the treatment of sodium sulfite, PMA-stimulated PP lymphocytes produced a DEPMPO-sulfite radical adduct and an unknown radical adduct. When DEPMPO was replaced with DMPO, DMPO-sulfite and hydroxyl radical adducts were detected. The latter adduct resulted from DMPO oxidation by sulfate radical, which was capable of oxidizing formate or ethanol. Oxygen consumption rates were further increased after the addition of sulfite to PMA-stimulated lymphocytes, suggesting the presence of sulfiteperoxyl radical. Taken together, oxidants generated by stimulated lymphocytes oxidized sulfite to sulfite radical, which subsequently formed sulfiteperoxyl and sulfate radicals. The latter two radicals are highly reactive, contributing to increased oxidative stress, which may lead to sulfite toxicity, altered functions in intestinal lymphocytes, or both.     

Procyanidins from grape pomace are suitable inhibitors of human endothelial NADPH oxidase

Procyanidins have been associated with a reduced risk of cardiovascular diseases such as atherosclerosis. However, the molecular mechanisms underlying this benefit are not fully understood. Increased reactive oxygen species (ROS) production generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is a common problem in different cardiovascular diseases. Our objective was to evaluate the effects of procyanidin-rich fractions from distilled grape pomace on NADPH oxidase activity in human umbilical vein endothelial cells (HUVEC). Three differently polymerized and galloylated procyanidin fractions were analyzed for their NADPH oxidase inhibitory activity in cell lysates and in HUVEC cultures. All of the three fractions, up to 1 μg/ml, equally inhibited isolated NADPH oxidase in HUVEC lysates in a concentration-dependent manner and independently of any superoxide anion scavenging activities. The procyanidin fractions even blocked NADPH oxidase activity in intact HUVEC, inhibiting ROS production at both extra- and intracellular levels. The fractions achieved the same effects that known NADPH oxidase inhibitors, such as diphenylene iodonium and apocynin, but they presented better hydrosolubility. Our results demonstrated that procyanidin from grape pomace inhibit human endothelial NADPH oxidase regardless of their polymerization degree and galloylation percentage. Therefore, procyanidins are suitable NADPH oxidase inhibitors which could serve as models for therapeutic alternatives for cardiovascular diseases.     

Role of plant respiratory burst oxidase homologs in stress responses

Plant respiratory burst oxidase homologs (Rbohs), which are also named nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs), are the homologs of mammalian phagocyte gp91^phox. As a unique among other reactive oxygen species (ROS) production mechanisms in plants, NADPH oxidases can integrate different signal transduction pathways, such as calcium, protein phosphorylation catalysed by protein kinases, nitric oxide, and lipid messengers. Coupling with genetic studies, the ability of plant NADPH oxidases to integrate different signal transduction pathways with ROS production demonstrates their involvement in many important biological processes in cells, such as morphogenesis and development, and stress responses. Here, we focus on several current studies concerning the role of plant NADPH oxidases in stress responses.     

New approaches to NAD(P)H regeneration in the biosynthesis systems

Nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH), as two kinds of well-known cofactor, are widely used in the most of enzymatic redox reactions, playing an important role in industrial catalysis. In general, supply of NAD(P)H is a major challenged factor in redox fermentation systems due to its high cost and low stability, which have stimulated the development of NADH regeneration systems in recent years. Until now, a series of NAD(P)H regeneration systems have been developed. This review focuses primarily on new approaches of NAD(P)H cofactor regeneration in the biosynthesis systems, such as single cell in vivo NADH regeneration system, double cell coupling NADH regeneration system, in vitro enzyme-coupled NADH regeneration system, microbial cell surface display NADH regeneration system. Finally, the prospect and tendency of NADH regeneration are discussed.     

Masking of Bacillus megaterium KM membrane reduced nicotinamide adenine dinucleotide oxidase and solubilization studies

Solubilization of a reduced nicotinamide adenine dinucleotide (NADH)-2,6 dichlorophenol indophenol (DCIP) oxidoreductase associated with the membrane NADH oxidase system of Bacillus megaterium KM was effected by treatment with 0.2% sodium deoxycholate, 8 m urea, or buffer (pH 9.0) in the presence of ethyl-enediaminetetraacetate. These treatments inactivated membrane NADH oxidase. It was found that membrane NADH oxidase and NADH-DCIP oxidoreductase were masked in membranes. Several procedures, including brief sonic oscillation, treatment with 0.05% deoxycholate, prolonged stirring at 4 C with 10% glycerol, and washing in the absence of Mg(2+), unmasked the oxidase and oxidoreductase activities. It was necessary to study the masking and unmasking of these activities to quantitate adequately the effects of solubilization procedures. Further information on the localization of oxidase and oxidoreductase in subcellular fractions and the effects of electron transport inhibitors on NADH oxidation was also obtained.     

A hydrogen peroxide-forming NADH oxidase that functions as an alkyl hydroperoxide reductase in Amphibacillus xylanus

The Amphibacillus xylanus NADH oxidase, which catalyzes the reduction of oxygen to hydrogen peroxide with beta-NADH, can also reduce hydrogen peroxide to water in the presence of free flavin adenine dinucleotide (FAD) or the small disulfide-containing Salmonella enterica AhpC protein. The enzyme has two disulfide bonds, Cys128-Cys131 and Cys337-Cys340, which can act as redox centers in addition to the enzyme-bound FAD (K. Ohnishi, Y. Niimura, M. Hidaka, H. Masaki, H. Suzuki, T. Uozumi, and T. Nishino, J. Biol. Chem. 270:5812-5817, 1995). The NADH-FAD reductase activity was directly dependent on the FAD concentration, with a second-order rate constant of approximately 2.0 x 10(6) M(-1) s(-1). Rapid-reaction studies showed that the reduction of free flavin occurred through enzyme-bound FAD, which was reduced by NADH. The peroxidase activity of NADH oxidase in the presence of FAD resulted from reduction of peroxide by free FADH(2) reduced via enzyme-bound FAD. This peroxidase activity was markedly decreased in the presence of oxygen, since the free FADH(2) is easily oxidized by oxygen, indicating that this enzyme system is unlikely to be functional in aerobic growing cells. The A. xylanus ahpC gene was cloned and overexpressed in Escherichia coli. When the NADH oxidase was coupled with A. xylanus AhpC, the peroxidase activity was not inhibited by oxygen. The V(max) values for hydrogen peroxide and cumene hydroperoxide reduction were both approximately 150 s(-1). The K(m) values for hydrogen peroxide and cumene hydroperoxide were too low to allow accurate determination of their values. Both AhpC and NADH oxidase were induced under aerobic conditions, a clear indication that these proteins are involved in the removal of peroxides under aerobic growing conditions.     

Partial purification and some properties of delta1-pyrroline-5-carboxylate reductase from Escherichia coli.

delta1-Pyrroline-5-carboxylate (PCA) reductase [L-proline:NAD(P)+5-oxidoreductase, EC 1.5.1.2] has been purified over 200-fold from Escherichia coli K-12. It has a molecular weight of approximately 320,000. PCA reductase mediates the pyridine nucleotide-linked reduction of PCA to proline but not the reverse reaction (even at high substrate concentrations). The partially purified preparation is free of competing pyridine nucleotide oxidase, PCA dehydrogenase, and proline oxidase activities. The Michaelis constant (Km) values for the substrate, PCA, with reduced nicotinamide adenine dinucleotide phosphate (NADPH) or NADH as cofactor are 0.15 and 0.14 mM, respectively. The Km values determined for NADPH and NADH are 0.03 and 0.23 mM, respectively. Although either NADPH or NADH can function as cofactor, the activity observed with NADPH is severalfold greater. PCA reductase is not repressed by growth in the presence of proline, but it is inhibited by the reaction end products, proline and NADP.     

A new spin-labelled analogue of nicotinamide adenine dinucleotide

The preparation of a spin-labelled analogue of nicotinamide adenine dinucleotide, 3-(4′,4′,5′,5′-tetramethyl-3′-oxide-1′-oxyl-2′-imidazolinyl) pyridine adenine dinucleotide, is descrebed. This compound was obtained by treatment of 3-carboxaldehyde pyridine adenine dinucleotide with 2,3-dimethyl-2,3-dihydroxylaminobutane followed by oxidation with lead dioxide. The interpretation of the particular electron spin resonance spectra of this nitronylnitroxide (five lines) in terms of the rotational correlation time of the radical is shown to be possible. The high stability of this compound makes its use in NAD⁺-dependent biological systems feasible.     

A novel strategy involved in [corrected] anti-oxidative defense: the conversion of NADH into NADPH by a metabolic network

The reduced nicotinamide adenine dinucleotide phosphate (NADPH) is pivotal to the cellular anti-oxidative defence strategies in most organisms. Although its production mediated by different enzyme systems has been relatively well-studied, metabolic networks dedicated to the biogenesis of NADPH have not been fully characterized. In this report, a metabolic pathway that promotes the conversion of reduced nicotinamide adenine dinucleotide (NADH), a pro-oxidant into NADPH has been uncovered in Pseudomonas fluorescens exposed to oxidative stress. Enzymes such as pyruvate carboxylase (PC), malic enzyme (ME), malate dehydrogenase (MDH), malate synthase (MS), and isocitrate lyase (ICL) that are involved in disparate metabolic modules, converged to create a metabolic network aimed at the transformation of NADH into NADPH. The downregulation of phosphoenol carboxykinase (PEPCK) and the upregulation of pyruvate kinase (PK) ensured that this metabolic cycle fixed NADH into NADPH to combat the oxidative stress triggered by the menadione insult. This is the first demonstration of a metabolic network invoked to generate NADPH from NADH, a process that may be very effective in combating oxidative stress as the increase of an anti-oxidant is coupled to the decrease of a pro-oxidant.     

Ischemia-activated microglia induces neuronal injury via activation of gp91phox NADPH oxidase

Although glial cells play a major role in the pathogenesis of many neurological diseases by exacerbating neuronal and non-neuronal cell death, the mechanisms involved are unclear. We examined the effects of microglia-(MCM) or astrocyte-(ACM) conditioned media obtained by chemical ischemia on the neuronal injury in SH-SY5Y cells. Chemical ischemia was induced by the treatment with NaN₃ and 2-deoxy-d-glucose for 2 h. MCM-treated SH-SY5Y cells showed reduced the viability, increased caspase-3 activity, decreased Bcl-2/Bax ratio, and increased cytochrome c release, increased inflammatory cytokines, and increased reactive oxygen species (ROS) generation. MCM also increased gp91phox nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which was inhibited by NADPH oxidase inhibitor, apocynin, and gp91phox siRNA. However, ACM did not show any significant changes. The results suggest that microglia activated by ischemic insult may increase reactive oxygen species generation via activation of gp91phox NADPH oxidase, resulting in neuronal injury.     

Exogenous hydrogen sulfide delays the senescence of human umbilical vein endothelial cells by lessening oxidative stress

The present study was aimed to investigate the effect of pretreatment with hydrogen sulfide (H2S) on human umbilical vein endothelial cells (HUVECs) senescence and the underlying mechanism. Cultured HUVECs at twelfth and fourth passages were taken as old and young groups, respectively. Sodium hydrosulfide (NaHS, donor of H2S) group was treated with NaHS from fourth to twelfth passage. The cell senescence was determined by senescence-associated β-galactosidase (SA β-gal) staining. DAPI fluorescent dye was used to detect cellular apoptosis. Western blot was used to analyze the expression levels of xanthine oxidase (XOD), manganese-superoxide dismutase (Mn-SOD) and the subunits p67(phox) of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in the HUVECs. Colorimetric method was used to detect SOD activity and cellular hydrogen peroxide (H2O2) level. The results showed that, compared with young group, the old group exhibited higher SA β-gal positive rate and cellular apoptosis, while NaHS pretreatment decreased SA β-gal positive rate and cellular apoptosis. Compared with the young group, the old group showed increased expression levels of XOD and p67(phox), as well as lower Mn-SOD expression level. With the pretreatment of NaHS, the up-regulations of XOD and p67(phox) levels and down-regulation of Mn-SOD level were inhibited. Compared with the young group, the old group showed lower SOD activity and higher H2O2 level, whereas NaHS pretreatment reversed the changes of SOD activity and H2O2 level. These results suggest that H2S delays senescence of HUVECs through lessening oxidative stress.     

Generation of superoxide anion and hydrogen peroxide during redox cycling of 5-(4-nitrophenyl)-penta-2,4-dienal by mammalian microsomes and enzymes

5-(4-Nitrophenyl)penta-2,4-dienal (NPPD) stimulated NADPH-supported oxygen consumption by rat liver microsomes in a concentration-dependent manner. The NPPD stimulation of O2 uptake was not inhibited by metyrapone and was decreased in the presence of NADP+ and p-hydroxymercuribenzoate. These observations suggest that the NPPD initial reduction step is mediated by NADPH-cytochrome P-450 reductase and not by cytochrome P-450. Spin-trapping studies using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) revealed the formation of superoxide anion upon incubation of NPPD, NADPH, DMPO and rat liver microsomes. Hydrogen peroxide generation was also detected in these incubations, thus confirming redox cycling of NPPD under aerobic conditions. NPPD stimulated oxygen consumption, superoxide anion formation and hydrogen peroxide generation by rat kidney, testes and brain microsomes. Other enzymes capable of nitroreduction (NADH dehydrogenase, xanthine oxidase, glutathione reductase, and NADP+ ferredoxin oxidoreductase) were also found to stimulate redox cycling of NPPD. The ability of NPPD to induce superoxide anion and hydrogen peroxide formation might play a role in its reported mutagenicity.     

Interactions between NADPH oxidase and voltage-gated proton channels: why electron transport depends on proton transport

Leukocytes kill microbes by producing reactive oxygen species, using a multi-component enzyme complex, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Electrons pass from intracellular NADPH through a redox chain within the enzyme, to reduce extracellular O2 to O2-. Electron flux is electrogenic, and rapidly depolarizes the membrane potential. Excessive depolarization can turn off electron transport by self-inhibition, but this is prevented by proton flux that balances the electron flux. Although the membrane potential depolarizes by approximately 100 mV during the respiratory burst (NADPH oxidase activity), NADPH oxidase activity is independent of voltage in this range, which permits optimal function and prevents self-inhibition.     

A Novel Strategy Involved Anti-Oxidative Defense: The Conversion of NADH into NADPH by a Metabolic Network

The reduced nicotinamide adenine dinucleotide phosphate (NADPH) is pivotal to the cellular anti-oxidative defence strategies in most organisms. Although its production mediated by different enzyme systems has been relatively well-studied, metabolic networks dedicated to the biogenesis of NADPH have not been fully characterized. In this report, a metabolic pathway that promotes the conversion of reduced nicotinamide adenine dinucleotide (NADH), a pro-oxidant into NADPH has been uncovered in Pseudomonas fluorescens exposed to oxidative stress. Enzymes such as pyruvate carboxylase (PC), malic enzyme (ME), malate dehydrogenase (MDH), malate synthase (MS), and isocitrate lyase (ICL) that are involved in disparate metabolic modules, converged to create a metabolic network aimed at the transformation of NADH into NADPH. The downregulation of phosphoenol carboxykinase (PEPCK) and the upregulation of pyruvate kinase (PK) ensured that this metabolic cycle fixed NADH into NADPH to combat the oxidative stress triggered by the menadione insult. This is the first demonstration of a metabolic network invoked to generate NADPH from NADH, a process that may be very effective in combating oxidative stress as the increase of an anti-oxidant is coupled to the decrease of a pro-oxidant.