Showing papers on "Nitrite published in 1981"

Assays for differentiation of glutathione S-transferases.

Abstract: Publisher Summary This chapter provides the spectrophotometric, titrimetric, nitrite, and cyanide assay for the differentiation of glutathione S-transferases. Spectrophotometric assays depend upon a direct change in the absorbance of the substrate when it is conjugated with glutathione (GSH). Because each of the reactions is catalyzed at a finite rate in the absence of enzyme, care is needed to reduce nonenzymatic catalysis by minimizing substrate concentrations and by decreasing pH wherever necessary. Titrimetric assay is based on the principle that the conjugation of alkyl halides with GSH can be measured titrimetrically. Although acid production accompanies many of the transferase catalyzed reactions in which thioethers are formed, titrimetry is only used when more convenient assays are not available. Nitrite assay is based on the principle that nitrite is released when GSH reacts with nitroalkanes or with organic nitrate esters. The nitrite can be assayed as the limiting factor in a diazotization reaction with sulfanilamide that produces a readily quantitatable pink dye. Cyanide assay is based on the fact that when glutathione transferases catalyze the attack of the glutathione thiolate ion on the electrophilic sulfur atom of several organic thiocyanates, it results in the formation of an asymmetric glutathionyl disulfide and cyanide. Cyanide can be readily quantitated by a calorimetric method.

Kinetic Explanation for Accumulation of Nitrite, Nitric Oxide, and Nitrous Oxide During Bacterial Denitrification

Abstract: The kinetics of denitrification and the causes of nitrite and nitrous oxide accumulation were examined in resting cell suspensions of three denitrifiers. An Alcaligenes species and a Pseudomonas fluorescens isolate characteristically accumulated nitrite when reducing nitrate; a Flavobacterium isolate did not. Nitrate did not inhibit nitrite reduction in cultures grown with tungstate to prevent formation of an active nitrate reductase; rather, accumulation of nitrite seemed to depend on the relative rates of nitrate and nitrite reduction. Each isolate rapidly reduced nitrous oxide even when nitrate or nitrite had been included in the incubation mixture. Nitrate also did not inhibit nitrous oxide reduction in Alcaligenes odorans, an organism incapable of nitrate reduction. Thus, added nitrate or nitrite does not always cause nitrous oxide accumulation, as has often been reported for denitrifying soils. All strains produced small amounts of nitric oxide during denitrification in a pattern suggesting that nitric oxide was also under kinetic control similar to that of nitrite and nitrous oxide. Apparent Km values for nitrate and nitrite reduction were 15 μM or less for each isolate. The Km value for nitrous oxide reduction by Flavobacterium sp. was 0.5 μM. Numerical solutions to a mathematical model of denitrification based on Michaelis-Menten kinetics showed that differences in reduction rates of the nitrogenous compounds were sufficient to account for the observed patterns of nitrite, nitric oxide, and nitrous oxide accumulation. Addition of oxygen inhibited gas production from 13NO3− by Alcaligenes sp. and P. fluorescens, but it did not reduce gas production by Flavobacterium sp. However, all three isolates produced higher ratios of nitrous oxide to dinitrogen as the oxygen tension increased. Inclusion of oxygen in the model as a nonspecific inhibitor of each step in denitrification resulted in decreased gas production but increased ratios of nitrous oxide to dinitrogen, as observed experimentally. The simplicity of this kinetic model of denitrification and its ability to unify disparate observations should make the model a useful guide in research on the physiology of denitrifier response to environmental effectors.

Relationship between cyclic guanosine 3':5'-monophosphate formation and relaxation of coronary arterial smooth muscle by glyceryl trinitrate, nitroprusside, nitrite and nitric oxide: effects of methylene blue and methemoglobin.

Abstract: The purpose of the present study was to determine time course relationship between cyclic GMP accumulation and relaxation in bovine coronary artery and evaluate the effects of recently identified inhibitors, methylene blue and methemoglobin, on these relationships. Arterial strips were suspended in specially mounted tissue baths which permitted continuous recording of isometric tension until rapid freeze-clamping for subsequent determination of cyclic GMP levels by radioimmunoassay. Relaxation and cyclic GMP levels were measured in submaximally contracted strips at zero time (untreated) or 5-sec to 5-min intervals after exposure to 0.5 microliter of nitric oxide, 1 microM glyceryl trinitrate, 1 microM sodium nitroprusside of 1 mM sodium nitrite in the absence or presence of 10 mM methylene blue or 1 microM methemoglobin. Cyclic GMP accumulation preceded onset of relaxation elicited by nitric oxide and glyceryl trinitrate and temporally correlated with relaxation induced by sodium nitroprusside and sodium nitrite. Methylene blue simultaneously inhibited cyclic GMP accumulation and relaxation induced by all four relaxants. In contrast to methylene blue, methemoglobin abolished cyclic GMP accumulation and relaxation elicited by nitric oxide without altering responses to glyceryl trinitrate, sodium nitroprusside and sodium nitrite. These findings are consistent with and strongly support an involvement of cyclic GMP formation in vascular smooth muscle relaxation elicited by nitrogen oxide-containing vasodilators.

Nitrous Oxide Production by Nondenitrifying Soil Nitrate Reducers

Abstract: Of 214 soil bacterial isolates able to reduce NO3-, 209 produced nitrous oxide (N2O), even though only 46 were respiratory denitrifiers (competent to carry out complete reduction of NO3- to N gases). Nitrite or NH4+ was the major product of NO3- reduction by the nondenitrifying organisms, but typically about 5 to 10% and up to 34% of the NO3- reduced by them was released as N2O during a 2-week incubation. Bacillus and Enterobacter were the most commonly observed genera of nondenitrifying N2O producers. Fermentative NO2- reduction and N2O production by a Bacillus sp. and a Citrobacter sp. were characterized in pure culture studies. Dinitrogen (N2) was not produced in detectable quantities by these organisms. When added to autoclaved soil, they accumulated more N2O than two denitrifying pseudomonads, since the latter consume and produce N2O. In tryptic soy broth (TSB), which allows active fermentative growth, NH4+ was apparently the major product of NO3- reduction. In nutrient broth (NB), NO2- accumulated. Added NH4+ did not inhibit N2O production or apparent reduction to NH4+, indicating that these processes are not assimilatory. The effect of added glucose on N2O production varied with the organism and media composition. Nitrous oxide production from NO2- by these organisms was shown to be at least partially a biochemical reaction. The N2O evolved slowly in batch cultures and mostly after apparent growth ceased. This is apparently a novel mechanism of N2O generation which differs significantly from respiratory denitrification.

The Family Nitrobacteraceae

Abstract: The biological oxidations of ammonia to nitrite and nitrite to nitrate, collectively referred to as nitrification, are carried out in nature by two physiological groups of Gram-negative, chemolithotrophic bacteria. The organisms in both groups fix carbon dioxide via the Calvin cycle (Campbell, Hellebust, and Watson, 1966) for their major source of cell carbon and derive their energy and reducing power either from the oxidation of ammonia (ammonia-oxidizing bacteria) or nitrite (nitrite-oxidizing bacteria). With the exception of a few strains of Nitrobacter winogradskyi, which can be grown chemoheterotro-phically, the nitrifying bacteria are obligate chemo-lithotrophs.

Dissimilatory reduction of nitrate and nitrite in the bovine rumen: nitrous oxide production and effect of acetylene.

Abstract: 15N tracer methods and gas chromatography coupled to an electron capture detector were used to investigate dissimilatory reduction of nitrate and nitrite by the rumen microbiota of a fistulated cow Ammonium was the only 15N-labeled end product of quantitative significance Only traces of nitrous oxide were detected as a product of nitrate reduction; but in experiments with nitrite, up to 03% of the added nitrogen accumulated as nitrous oxide, but it was not further reduced Furthermore, when 13NO3- was incubated with rumen microbiota virtually no [13N]N2 was produced Acetylene partially inhibited the reduction of nitrite to ammonium as well as the formation of nitrous oxide It is suggested that in the rumen ecosystem nitrous oxide is a byproduct of dissimilatory nitrite reduction to ammonium rather than a product of denitrification and that the latter process is absent from the rumen habitat

A blue protein as an inactivating factor for nitrite reductase from Alcaligenes faecalis strain S-6.

Abstract: A blue protein with a molecule weight of 12,000 containing 1 atom of type I Cu2+ was purified and crystallized from a denitrifying bacterium, Alcaligenes faecalis strain S-6, as an inactivating factor for copper-containing nitrite reductase of the same organism. Inactivation of the enzyme occurred when the enzyme was incubated aerobically with a catalytic amount of the blue protein in the presence of reducing agents such as cysteine and ascorbate. The blue protein acts as a direct electron donor for the enzyme to catalyze the reduction of nitrite, but in the absence of nitrite, the enzyme-reduced blue protein system reacts with oxygen to produce H2O2. A suicide inactivation mechanism of the enzyme due to this H2O2 production is proposed.

Purification and properties of a copper-containing nitrite reductase from a denitrifying bacterium, Alcaligenes faecalis strain S-6.

Abstract: A copper-containing nitrite reductase was purified and crystallized from a potent denitrifying bacterium, Alcaligenes faecalis strain S-6. The enzyme was composed of 4 subunits with a molecular weight of about 30,000, each containing 1 atom of Cu2+. Nitric oxide was identified as a main reduction product from nitrite in the enzyme-catalyzed reaction. The enzyme activity was inhibited strongly by KCN but only slightly by sulfhydryl reagents such as p-chloromercuribenzoate and N-ethylmaleimide.

Nisin: a possible alternative or adjunct to nitrite in the preservation of meats.

Abstract: Nisin at 75 ppm (75 microgram/g) was superior to 150 ppm of nitrite in inhibiting outgrowth of Clostridium sporogenes PA3679 spores in meat slurries, which had been heated to simulate the process used for cooked ham The inhibitory activity of nisin decreased as the spore load or pH of the slurries increased Unlike nitrite, inhibition by nisin was unaffected by high levels of iron either as a constituent of meats or when added as an iron salt In slurries treated with 75 ppm of nisin, refrigerated storage for 56 days resulted in depletion of nisin to a level low enough to allow outgrowth within 3 to 10 days if the slurries were subsequently abused at 35 degrees C In contrast, a combination of 40 ppm of nitrite and either 75 or 100 ppm of nisin almost completely inhibited outgrowth in these slurries The nisin-nitrite combination appeared to have a synergistic effect, and the low concentration of nitrite was sufficient to preserve the color in meats similar to that of products cured with 150 ppm of nitrite

Denitrification and dissimilatory nitrate reduction to ammonium in digested sludge.

Abstract: Acetylene inhibition and 13N methods were used to assay digested sludge for its potential to denitrify and to reduce nitrate to ammonium. At nitrate concentrations below 10 μM, the reduction of N2O to N2 was not inhibited by acetylene concentrations as high as 80 kPa, though at higher nitrate concentrations acetylene was an effective inhibitor. NO, N2O, and N2 were produces immediately after addition of nitrate or nitrite, indicating that denitrifying enzymes were present. NO was maintained at a concentration of 2–5 nM, while nitrate or nitrite were being reduced, but this gas was depleted once the ionic N oxide substrates were exhausted. Acetylene had little effect on appearance and disappearance of NO. It was also noted that NO was readily consumed by chemical reactions in the anaerobic sludge. Added N2O was reduced without a lag, but pasteurized samples did not consume N2O although they produced it. Fresh digested sludge reduced 60–70% of the added 13NO3− to 13NH4+ with the rest of the NO3−-N presumabl...

Nucleic acid related compounds. 34. Non-aqueous diazotization with tert-butyl nitrite. Introduction of fluorine, chlorine, and bromine at C-2 of purine nucleosides

Abstract: Treatment of 2,6-diamino-9-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)purine (1c) with tert-butyl nitrite (TBN) in 60% anhydrous hydrogen fluoride/pyridine at −20 °C gave 2-fluoroadenosine triacetate (2...

Acute Toxicity of Nitrite to Rainbow Trout (Salmo gairdneri): Effects of pH, Nitrite Species, and Anion Species

Abstract: The toxicity of nitrite to rainbow trout is pH-dependent within the range considered acceptable to most freshwater aquatic life (pH 6.5–9.0). Both of the nitrite species, NO2− and HNO2, are toxic. It is recommended that nitrite criteria to protect freshwater aquatic life be based on total nitrite, and that such criteria reflect the pH dependence of nitrite toxicity. Variation in the toxicity of nitrite in the presence of chloride, sulfate, phosphate, and nitrate anions has also been demonstrated. It is concluded that the toxicity of nitrite to fishes, in addition to being pH-dependent, is also dependent in varying degrees upon many of the anions that are commonly found in natural aquatic environments.Key words: nitrite, pH, chloride, phosphate, sulfate, toxicity, rainbow trout, Salmo gairdneri

Production of superoxide radicals by soluble hydrogenase from Alcaligenes eutrophus H16.

Abstract: The soluble hydrogenase (hydrogen-NAD+ oxidoreductase, EC 1.12.1.2) of Alcaligenes eutrophus H16 was shown to be stabilized by oxidation with oxygen and ferricyanide as long as electron donors and reducing compounds were absent. The simultaneous presence of H2, NADH and O2 in the enzyme solution, however, caused an irreversible inactivation of hydrogenase that was dependent on the O2 concentration. The half-life periods of 4 degrees C under partial pressures of 0.1, 5, 20 and 50% O2 were 11, 5, 2.5 and 1.5 h respectively. Evidence has been obtained that hydrogenase produces superoxide free radical anions (O2-.), which were detected by their ability to oxidize hydroxylamine to nitrite. The correlation between O2 concentration, nitrite formation and inactivation rates and the stabilization of hydrogenase by addition of superoxide dismutase indicated that superoxide radicals are responsible for enzyme inactivation. During short-term activity measurements (NAD+ reduction, H2 evolution from NADH), hydrogenase activity was inhibited by O2 only very slightly. In the presence of 0.7 mM-O2 an inhibition of about 20% was observed.

Plasma Corticosteroid Dynamics in Channel Catfish (Ictalurus punctatus) Exposed to Ammonia and Nitrite

Abstract: Plasma corticosteroid concentrations in channel catfish (Ictalurus punctatus) increased in response to increasing concentrations of both ammonia and nitrite. Corticosteroid levels in fish exposed to nitrite increased continuously during a 24-h exposure whereas corticosteroid concentrations of fish exposed to ammonia peaked after 8 h and then decreased for the remainder of the 24-h experiment. Low environmental pH and elevated environmental chloride were effective in preventing elevated corticosteroid levels in fish exposed to ammonia and nitrite, respectively. Based on plasma corticosteroid concentrations, no additive or synergistic toxic interaction was evident in fish simultaneously exposed to environmental ammonia and nitrite. These data indicate that (1) corticosteroids are released into circulation probably in response to a physiological dysfunction brought about by the toxins and not simply their presence in the environment, and (2) the absence of a toxic interaction between ammonia and nitrite as m...

Nitric oxide from nitrite photolysis in the central equatorial Pacific

Abstract: Sunlight photolyzes nitrite in seawater: NO2− + HOH + hv = NO + OH + OH−. We studied nitrite loss and nitric oxide production attributed to this reaction in surface waters of the equatorial Pacific near 170°W. Net photochemical loss rates of 2–15% per day were derived from two different types of laboratory incubation experiments. The net nitrite loss rate in the surface water of this region is calculated to average 4 × 10−13 mol l−l s−l during the day, or ∼6 × 10−2 mol m−2 y−1. Nitric oxide was detected in situ with a floating gas-seawater equilibrator. NO was always detectable in nitrite-containing seawater during the day but was undetectable at night or in nitrite-free water. Near sunrises and sunsets the estimated NO vapor pressure, pNO(sea) covaried with the ambient UV insolation in air according to log pNO(sea) = a log UVair + b. Best-fit values to the in situ data indicate a ≈ 1 with r2 ≥ 0.9; simple kinetic models rationalize a values of O, ½, or 1. During the day, pNO(sea) averaged ∼3.1 × 10−8 atm, corresponding to ∼4.6 × 10−11 M [NO]aq. The ambient atmospheric pNO was ∼104 -fold lower, implying a substantial seawater supersaturation and a sea → air flux. From the stagnant-boundary layer model and our measurements, we estimate ∼2 × 10−16 mol 1−1 s−1 (∼1.3 × 108 molecule cm−2 s−1) of NO efflux in daylight, an insignificant NO loss from the sea. The photochemical NO source and the estimated dark reaction sink are, within the accuracy of the data, in balance. These results provide evidence for the presence of NO, a free radical, in surface seawater. They substantiate that photochemical reactions produce measurable concentrations of reactive intermediates in surface seawater and that these enter into rapid secondary reactions. These processes may reach sufficient intensity to provide significant effects, such as sea → air fluxes.

Nitrogen-13-labeled nitrite and nitrate: distribution and metabolism after intratracheal administration.

Abstract: Radioactive nitrogen-13 from nitrite (NO2-) or nitrate (NO3-) administered intratracheally or intravenously without added carrier to mice or rabbits was distributed evenly throughout most organs and tissues regardless of the entry route or the anion administered. Nitrogen-13 from both anions was distributed uniformly between plasma and blood cells. We found rapid in vivo oxidation of NO2- to NO3- at concentrations of 2 to 3 nanomoles per liter in blood. Over 50 percent oxidation within 10 minutes accounted for the similar nitrogen-13 distributions from both parent ions. The oxidation rates were animal species-dependent. No reduction of 13NO3- to 13NO2- was observed. A mechanistic hypothesis invoking oxidation of 13NO2- by a catalase-hydrogen peroxide complex accounts for the results. These results imply a concentration dependence for the in vivo fate of NO2- or nitrogen dioxide.

The Involvement of Nitric Oxide in the Inhibition of the Phosphoroclastic System in Clostridium sporogenes by Sodium Nitrite

Abstract: The phosphoroclastic system was demonstrated in cell-free extracts of Clostridium sporogenes by the production of carbon dioxide, acetyl phosphate, ATP and reduced NAD in the presence of pyruvate. The kinetics of acetyl phosphate production and NAD reduction were investigated. The addition of sodium nitrite to a suspension of C. sporogenes in glucose medium resulted in a rapid decrease in intracellular ATP concentration which was accompanied by an accumulation of pyruvate in the medium. This accumulation of pyruvate was caused by inhibition of the phosphoroclastic system by nitrite. Nitrite inhibits this system by reaction of nitric oxide, formed from nitrite, with the non-haem iron of pyruvate: ferredoxin oxidoreductase.

Prosthetic groups of the NADH-dependent nitrite reductase from Escherichia coli K12

Abstract: A substantially improved purification of Escherichia coli NADH-dependent nitrite reductase was obtained by purifying it in presence of 1 mM-NO2- and 10 microM-FAD. The enzyme was obtained in 20% yield with a maximum specific activity of 1.04 kat . kg-1: more than 95% of this sample subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis migrated as a single band of protein. This highly active enzyme contained one non-covalently bound FAD molecule, and, probably, 5 Fe atoms and 4 acid-labile S atoms per subunit. No FMN, covalently bound flavin or Mo was detected. The spectrum of the enzyme shows absorption maxima at 386, 455, 530 and about 575 nm with a shoulder at 480--490 nm. The Soret-band/alpha-band absorbance ratio is about 4:1. These spectral features are characteristic of sirohaem, apart from the maximum at 455nm, which is attributed to flavin. The enzyme also catalyses the NADH-dependent reduction of horse heart cytochrome c, 2,6-dichlorophenol-indophenol and K3Fe(CN)6. The presence of sirohaem in E. coli nitrite reductase explains the apparent identity of the cysG and nirB gene of E. coli and inability of hemA mutants to reduce nitrite.

The thermal decomposition of sodium nitrate and the effects of several oxides on the decomposition.

Abstract: The thermal decomposition of sodium nitrate and the effects of several oxides, such as silica, titania, zirconia, alumina, and magnesia, on the decomposition were studied up to 900 °C by means of the thermal analysis, gas analysis, and chemical analysis of the reaction products. The reaction of sodium nitrate and silica was especially investigated in some detail over a wide composition range. The thermal decomposition of sodium nitrate started at about 450 °C. The gases formed were O2, NO, and N2, the formation of N2 being detected above 680 °C The thermal decomposition of sodium nitrate was supposed to consist of the following three successive or concurrent reaction processes, according to the degree of the reaction: (I) the decomposition of sodium nitrate to nitrite and oxygen, (II) the first-order liquid-phase reaction, with some kind of quantitative relationship between nitrate and nitrite, and (III) the formation reaction of sodium oxide, expressed by the Avrami-Erofe’ef equation. The (II) and (III) ...