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PubMed1993

Characterization of the structural gene encoding a copper-containing nitrite reductase and homology of this gene to DNA of other denitrifiers.

A copper-containing nitrite reductase gene (nirU) from Pseudomonas sp. strain G-179 was found in a 1.9-kb EcoRI-BamHI DNA fragment. The coding region contained information for a polypeptide of 379 amino acids. The encoded protein had 78% identity in amino acid sequence to the nitrite reductase purified from Achromobacter cycloclastes. The ligands for type 1 copper- and type 2 copper-binding sites found in A. cycloclastes were also found in Pseudomonas sp. strain G-179, suggesting that these binding sites are conserved. Upstream from the promoter, two putative fnr boxes were found, suggesting that an FNR-like protein may be involved in regulation of the nitrite reductase gene under anaerobic conditions. When the 1.9-kb clone was used to probe Southern blots for similar sequences in DNAs from different denitrifiers, hybridization bands were seen for 15 of 16 denitrifiers known to have nitrite reductase containing copper. Except for Pseudomonas stutzeri JM300, all denitrifiers tested that have nitrite reductases containing heme c,d1 showed no or weak hybridization to this probe. Thus, this structural gene may be useful as a probe to detect denitrifiers with copper-containing nitrite reductases.

Amino Acid Sequence↗

PubMed2002

Involvement of the PrrB/PrrA two-component system in nitrite respiration in Rhodobacter sphaeroides 2.4.3: evidence for transcriptional regulation.

Rhodobacter sphaeroides strain 2.4.3 is capable of diverse metabolic lifestyles, including denitrification. The regulation of many Rhodobacter genes involved in redox processes is controlled, in part, by the PrrBA two-component sensor-regulator system, where PrrB serves as the sensor kinase and PrrA is the response regulator. Four strains of 2.4.3 carrying mutations within the prrB gene were isolated in a screen for mutants unable to grow anaerobically on medium containing nitrite. Studies revealed that the expression of nirK, the structural gene encoding nitrite reductase, in these strains was significantly decreased compared to its expression in 2.4.3. Disruption of prrA also eliminated the ability to grow both photosynthetically and anaerobically in the dark on nitrite-amended medium. Complementation with prrA restored the wild-type phenotype. The PrrA strain exhibited a severe decrease in both nitrite reductase activity and expression of a nirK-lacZ fusion. Nitrite reductase activity in the PrrA strain could be restored to wild-type levels by using nirK expressed from a heterologous promoter, suggesting that the loss of nitrite reductase activity in the PrrA and PrrB mutants was not due to problems with enzyme assembly or the supply of reductant. Inactivation of prrA had no effect on the expression of the gene encoding NnrR, a transcriptional activator required for the expression of nirK. Inactivation of ccoN, part of the cbb(3)-type cytochrome oxidase shown to regulate the kinase activity of PrrB, also caused a significant decrease in both nirK expression and Nir activity. This was unexpected, since PrrA-P accumulates in the ccoN strain. Together, these results demonstrate that PrrBA plays an essential role in the regulation of nirK.

Bacterial Proteins↗

PubMed1991

Vascular nitric oxide-generating activities for organic nitrites and organic nitrates are distinct.

Like organic nitrates, the organic nitrites are assumed to undergo vascular metabolic activation to nitric oxide (NO) to produce vasodilating activity, but this assumption has never been examined. In this study, isobutyl nitrite and amyl nitrite were incubated with subcellular fractions of bovine coronary arterial smooth muscle at 37 degrees C, and NO generation was assessed by redox chemiluminescence assay of headspace NO. Bovine vascular subfractions exhibited substantial and significant catalytic activity for NO generation towards both organic nitrite substrates, and NO-generating activity could be inhibited by heating and irradiation. These observations suggest that organic nitrites are converted enzymatically to NO in vascular smooth muscle. Analysis of marker enzyme activities suggested that the primary NO-generating activity was associated with the cytosol, whereas a distinct, but relatively minor NO-generating activity was also identified in the microsomal fraction. The cytosolic and microsomal NO-generating activities exhibited different temperature dependencies for heat-induced inhibition of activity. The molecular weights of the NO-generating enzymes, determined by radiation inactivation target-size analysis, were 263 kDa (95% confidence interval: 236-298 kDa) for the cytosolic enzyme and 79 kDa (95% confidence interval: 54-148 kDa) for the microsomal enzyme. Previous studies in this laboratory have shown that the representative organic nitrate, nitroglycerin (NTG), is metabolized to NO by an enzyme associated with the plasma membrane of bovine vascular smooth muscle cells. Thus, the enzymes responsible for vascular bioconversion of organic nitrates and organic nitrites to NO are apparently different.(ABSTRACT TRUNCATED AT 250 WORDS)

Amyl Nitrite↗

PubMed1979

Cyanide intoxication in sheep: enhancement of efficacy of sodium nitrite, sodium thiosulfate, and cobaltous chloride.

For treatment of cyanide intoxication of ruminants, the present recommended doses of sodium nitrite (5 mg/kg of body weight) and sodium thiosulfate (25 to 50 mg/kg) are smaller than those recommended for other animals; the decrease is partially attributed to the greater susceptibility of ruminants to the toxic effects of sodium nitrite. Based on the high tissue concentration and activity rate of rhodanese in ruminants, sulfur donors such as sodium thiosulfate could be utilized more efficaciously. Doses of sodium nitrite and sodium thiosulfate (up to 22 and 660 mg/kg, respectively) were evaluated in the present studies. Adjustment of the antidotal combination provided almost three times the protection afforded by the previously recommended doses. Moreover, under the conditions tested, the newly adjusted dose levels of sodium thiosulfate alone were more effective than the previously used antidotal combination of sodium nitrite and sodium thiosulfate and this protective effect was enhanced by cobaltous chloride (10.6 mg/kg) or sodium nitrite. The present recommended therapeutic approach to cyanide intoxication in sheep should be based primarily on administration of a much higher dose of sodium thiosulfate in combination with sodium nitrite or cobaltous chloride (or both).

Animals↗

PubMed1980

Induction of liver tumors in Wistar rats by sodium nitrite given in pellet diet.

Sodium nitrate was given to male noninbred Wistar rats at levels of 800 ppm and 1,600 ppm in a pellet diet for 646 experimental days. The first tum or was found on day 441 in the liver of a rat given a diet containing 800 ppm sodium nitrite. On day 646, liver tumors were found in 1 of 22 rats (4.5%) on an 800-ppm sodium nitrite diet and in 5 of 19 rats (26.3%) on a 1,600-ppm sodium nitrite diet. The incidence of liver tumors in the rats fed 1,600 ppm sodium nitrite was significantly different from that in controls as judged by the t-test (P < 0.05). A hepatocellular carcinoma and a hemangioendothelial sarcoma of the liver were found on day 646 in 2 rats fed 1,600 ppm sodium nitrite. One mammary tumor but no liver tumors were found in the 19 control rats. The concentration of sodium nitrite decreased after preparation of the pellet diet, but it was still at least 70% of the initial amount when the pellets were given to the rats. Volatile N-nitroso compounds, especially dimehylnitrosamine, at ppm levels were detected in the pellet diet with a gas chromatography-thermal energy analyzer.

Adenoma↗

PubMed1982

Absence of DNA damage in liver of rats given high doses of cimetidine and sodium nitrite.

A correlation between cimetidine and gastric cancer has been suggested. Nitrosation of cimetidine in the presence of nitrite and HCl and mutagenic activity as well as DNA damage in mammalian cells displayed by nitrosocimetidine, as these phenomena occur in vitro, were the supporting hypothesis. Previous studies have shown that liver DNA damage was a well correlated index of potential carcinogenic activity of N-nitroso compounds and that such a damage was found after long-term simultaneous oral administration of aminopyrine and nitrite in rats. In this work, liver DNA fragmentation was investigated by three different techniques: DNA alkaline elution, DNA alkaline denaturation followed by hydroxylapatite chromatography and a new viscometric method markedly more sensitive than the above mentioned ones in detecting DNA damage. Evidence of DNA damage was not gained in any of the groups of rats treated with high single or successive oral daily doses of cimetidine (250 mg/kg) along with nitrite (80 mg/kg) in approximately equimolar amounts. Cimetidine and nitrite given alone were also ineffective. The lowering of gastric pH, obtained with fasting and histamine administration before giving cimetidine and nitrite combination in a single dose, did not favor the induction of liver DNA fragmentation neither in the above condition nor even when the amount of cimetidine was lowered to 125 mg/kg in order to obtain an approximately 2-fold molar amount of nitrite.

Animals↗

PubMed1981

Kinetics and mechanism of the oxidation of human deoxyhemoglobin by nitrites.

The reactions of human hemoglobin with sodium nitrite and ethyl nitrite in deoxygenated media have been examined in kinetic detail. Nitrous acid has been identified from kinetic pH dependence studies as the principal oxidant of hemoglobin in reactions with sodium nitrite. Nitrosylhemoglobin is produced concurrently with methemoglobin as a result of reductive release of nitric oxide from nitrous acid. However, oxidation of hemoglobin by nitric oxide competes with association, and this process is proposed to arise from the action of the nitric oxide dimer on hemoglobin. Ethyl nitrite, which serves as a model for nitrous acid, reacts with hemoglobin at rates that are at least 10 times slower than those extrapolated for nitrous acid, and hydrolysis of the alkyl nitrite is not competitive with oxidation of hemoglobin. The composite experimental results are interpreted to describe alkyl nitrite, and, presumably, nitrous acid association with hemoglobin followed by rate-limiting electron transfer resulting in nitric oxide and alkoxide (or hydroxide) production. Proton transfer resulting in alcohol (or water) formation occurs subsequent to the rate-limiting step as do reactions of hemoglobin with nitric oxide.

Hemoglobin A↗

PubMed1987

Genetic etiology of gastric carcinoma: II. Segregation analysis of gastric pH, nitrate, and nitrite.

A study of gastric pH, nitrate, and nitrite in 110 families collected as part of a cohort from the Narino region of Colombia is presented. All three traits are familial and have a significant linearly increasing age trend. Gastric pH has a clear bimodal distribution but does not show Mendelian segregation. The nitrate distribution is slightly skewed, but generational heterogeneity explains the data best. Gastric nitrite is also biomodal with a clear break at concentration 1.08 micrograms/ml, and 74% of the observations at zero concentration; it shows a recessive Mendelian segregation with significant residual spouse correlation. This model also fits the data best when nitrite is dichotomized into detected (measurable) and undetected values. The estimated frequency of the recessive allele is .57, so that an estimated 32% of the population sampled are recessives. Recessives whose spouses have measurable nitrite have an estimated penetrance of 99.3% at age 30 years, whereas those whose spouses have zero or undetected nitrite have a penetrance of only 8.8% at age 30 years. It appears that gastric nitrite, and, from our previous study of these families, chronic atrophic gastritis are important biologic markers for the early identification of persons predisposed to gastric cancer.

Adult↗

PubMed1985

Uptake of inhaled n-butyl nitrite and in vivo transformation in rats.

The uptake of butyl nitrite by rats (500 g, one rat/chamber) was determined over a 5-min exposure period. About 44% of the starting amount (771-3855 ppm) of n-butyl nitrite was consumed in 5 min. Three rats per exposure concentration were individually studied for 0-150 min after exposure. Concentrations of blood methemoglobin and plasma nitrate, nitrite, butyl nitrite, and butyl alcohol were examined. No free nitrite ion or butyl nitrite was detectable in the plasma at any time (6.5-150 min) following the exposure. Concentrations at 6.5 min of butyl alcohol, nitrate ion, and methemoglobin increased with increasing exposure concentrations. Plasma concentrations of butyl alcohol were detectable only briefly at 6.5 and 20 min. Methemoglobin levels decreased linearly at higher concentrations and as an approximate first-order process at lower concentrations (less than 15 g/L of whole blood). By 20 min after the inhalation period, plasma nitrate concentrations had decreased from peak levels to higher than baseline steady-state nitrate concentrations. Nitrosothiols were not detectable in the plasma protein fraction.

Animals↗

PubMed1995

Expression of spinach nitrite reductase in Escherichia coli: site-directed mutagenesis of predicted active site amino acids.

Spinach ferredoxin-nitrite reductase is a chloroplast enzyme that contains a coupled [Fe4S4]-siroheme-active site and catalyzes the six-electron reduction of nitrite to ammonia. An expression system which produced enzymatically active spinach nitrite reductase (NiR) in Escherichia coli was developed in order to study the structure-function relationships of the coupled active site using site-directed mutagenesis. The spinach NiR cDNA, without the sequences encoding the chloroplast transit peptide, was expressed as a beta-galactosidase fusion containing five additional amino acids at the N-terminus. The expressed NiR in aerobic cultures was mostly insoluble and inactive. After optimizing growth conditions, active NiR represented 0.5-1.0% of the total protein. E. coli-expressed NiR was purified approximately 200-fold to homogeneity as indicated by SDS-polyacrylamide gel electrophoresis. The expressed NiR enzyme was recognized by rabbit anti-spinach NiR antibody as visualized by Western blot analysis. The absorption spectrum of the E. coli-expressed NiR was identical to authentic spinach NiR with a Soret and alpha band at 386 and 573 nm, respectively, and a A278/A386 = 1.9. The addition of nitrite to the oxidized enzyme preparation produced the characteristic shifts in the spectrum. The specific activity for the methyl viologen-dependent reduction of nitrite of E. coli-expressed NiR was 100 U/mg and the Km determined for nitrite was 0.3 mM, which are in agreement with reported values for this enzyme. These results indicate that the E. coli-expressed NiR is fully comparable to spinach NiR in purity, catalytic activity, and physical state.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1998

A conserved tryptophan at the ferredoxin-binding site of ferredoxin:nitrite oxidoreductase.

Treatment of spinach leaf ferredoxin-dependent nitrite reductase with N-bromosuccinimide (NBS), under conditions where slightly less than 1 mol of tryptophan is modified per mole of nitrite reductase, inhibits the catalytic activity of the enzyme by ca. 80% without any effect on substrate binding or other enzyme properties. Complex formation between nitrite reductase and ferredoxin completely protects the enzyme against this inhibition. Transient kinetic measurements show that the second-order rate constant for reduction of NBS-modified nitrite reductase by reduced ferredoxin is approximately four-fold larger than that observed for the native, unmodified enzyme. Also, reduction of NBS-modified nitrite reductase by the 5-deazariboflavin radical shows a different kinetic pattern than that observed with the native enzyme, suggesting that tryptophan modification increases access of the radical to the low-potential [4Fe-4S] cluster of the enzyme, decreases the accessibility to the siroheme group of the enzyme, or both. The tryptophan that is modified has been identified as the absolutely conserved W92. A methionine, M73, that is also modified by NBS, has been identified. The ferredoxin-binding site on spinach nitrite reductase thus appears to include W92 and perhaps M73, in addition to the previously identified R375, R556, and K436.

Amino Acid Sequence↗

PubMed1994

A rapid and simple method for the measurement of nitrite and nitrate in plasma by high performance capillary electrophoresis.

Quantification of nitric oxide in vivo is difficult because of its short half-life. Instead the oxidised products, nitrite and nitrate, are commonly used as indices of nitric oxide generation. We describe here a simple method for the measurement of nitrite and nitrate in plasma using high performance capillary electrophoresis. Plasma standard curves gave regression coefficients of 0.98 for nitrite and 0.99 for nitrate with an intra-assay coefficient of variance of 4.6% for 50 microM nitrite (n = 10) and 1.2% for 50 microM nitrate (n = 10). The assay allows measurement of nitrite and nitrate in a single analysis requiring minimal sample preparation, and with the sensitivity to detect both basal and physiological changes in plasma nitrite and nitrate.

Biomarkers↗

PubMed2001

Domain swing upon His to Ala mutation in nitrite reductase of Pseudomonas aeruginosa.

The nitrite reductase (NIR) from Pseudomonas aeruginosa (NIR-Pa) is a soluble enzyme catalysing the reduction of nitrite (NO2(-)) to nitric oxide (NO). The enzyme is a 120 kDa homodimer, in which the monomers carry a c-heme domain and a d(1)-heme domain. The structures of the enzyme in both the oxidised and reduced state were solved previously and indicate His327 and His369 as putative catalytic residues. The kinetic characterisation of site-directed mutants has shown that the substitution of either one of these two His with Ala dramatically reduces the physiologically relevant reactivity towards nitrite, leaving the reactivity towards oxygen unaffected. The three-dimensional structures of P. aeruginosa NIR mutant H327A, and H369A in complex with NO have been solved by multiple wavelength anomalous dispersion (MAD), using the iron anomalous signal, and molecular replacement techniques. In both refined crystal structures the c-heme domain, whilst preserving its classical c-type cytochrome fold, has undergone a 60 degrees rigid-body rotation around an axis parallel with the pseudo 8-fold axis of the beta-propeller, and passing through residue Gln115. Even though the distance between the Fe ions of the c and d(1)-heme remains 21 A, the edge-to-edge distance between the two hemes has increased by 5 A. Furthermore the distal side of the d(1)-heme pocket appears to have undergone structural re-arrangement and Tyr10 has moved out of the active site. In the H369A-NO complex, the position and orientation of NO is significantly different from that of the NO bound to the reduced wild-type structure. Our results provide insight into the flexibility of the enzyme and the distinction between nitrite and oxidase reduction mechanisms. Moreover they demonstrate that the two histidine residues play a crucial role in the physiological activity of nitrite reduction, ligand binding and in the structural organisation of nitrite reductase from P. aeruginosa.

Alanine↗

PubMed1988

Isolation of cDNA clones coding for spinach nitrite reductase: complete sequence and nitrate induction.

The main nitrogen source for most higher plants is soil nitrate. Prior to its incorporation into amino acids, plants reduce nitrate to ammonia in two enzymatic steps. Nitrate is reduced by nitrate reductase to nitrite, which is further reduced to ammonia by nitrite reductase. In this paper, the complete primary sequence of the precursor protein for spinach nitrite reductase has been deduced from cloned cDNAs. The cDNA clones were isolated from a nitrate-induced cDNA library in two ways: through the use of oligonucleotide probes based on partial amino acid sequences of nitrite reductase and through the use of antibodies raised against purified nitrite reductase. The precursor protein for nitrite reductase is 594 amino acids long and has a 32 amino acid extension at the N-terminal end of the mature protein. These 32 amino acids most likely serve as a transit peptide involved in directing this nuclear-encoded protein into the chloroplast. The cDNA hybridizes to a 2.3 kb RNA whose steady-state level is markedly increased upon induction with nitrate.

Amino Acid Sequence↗

PubMed1983

Biotransformation of nitric oxide, nitrite and nitrate.

Biotransformation of NO, nitrite and nitrate was investigated in rats and mice in a 15NO inhalation experiment and intraperitoneal injection experiments of 15N-nitrite and 15N-nitrate, and the following results were obtained: (1) Rats were forced to inhale 15NO (145 ppm, 123 minutes) or were given an intraperitoneal injection of 15N-nitrite (2 mg animal-1 as 15N) or 15N-nitrate (2mg animal-1 as 15N), and determination of 15N recovery in urine was made up to 48 h later. The results were 55, 53 and 78% of the inhaled or injected 15N, respectively. (2) 15N-nitrate in the urine was converted into a 6-nitro derivative of 3,4-xylenol and its identification and quantitative determination were made by the GC-MS method. As to 15N-urea in the urine, identification and quantitative determination were made by the urease method. 15N was present in the urine of rats after 15NO inhalation in the form of NO3- and urea. 75 and 24% respectively. In the urine of rats injected with 15N-nitrite, about 20% of unidentified 15N-compounds not discovered in the inhalation experiment was found. The content of 15N-urea in the urine after injection with 15N-nitrate was lower than that after injection with 15N-nitrite. (3) When 15N-nitrite (0.617 mg animal-1 as 15N) was injected intraperitoneally in mice, 60.7, 7.8 and 0.3% of the injected 15N were found in the urine, feces and exhaled gas (NO, NO2 and NH3 in the gas were caught) up to 48 h after injection respectively, and 1.6% was found in the body 48 h after injection, but the remaining 30% of 15N could not be recovered.

Animals↗

PubMed1986

Analysis of the energy metabolism after incubation of Saccharomyces cerevisiae with sulfite or nitrite.

After addition of 5 mM sulfite or nitrite to glucose-metabolizing cells of Saccharomyces cerevisiae a rapid decrease of the ATP content and an inversely proportional increase in the level of inorganic phosphate was observed. The concentration of ADP shows only small and transient changes. Cells of the yeast mutant pet 936, lacking mitochondrial F1 ATPase, after addition of 5 mM sulfite or nitrite exhibit changes in ATP, ADP and inorganic phosphate very similar to those observed in wild type cells. They key enzyme of glucose degradation, glyceraldehyde-3-phosphate dehydrogenase was previously shown to be the most sulfite- or nitrite-sensitive enzyme of the glycolytic pathway. This enzyme shows the same sensitivity to sulfite or nitrite in cells of the mutant pet 936 as in wild type cells. It is concluded that the effects of sulfite or nitrite on ATP, ADP and inorganic phosphate are the result of inhibition of glyceraldehyde-3-phosphate dehydrogenase and not of inhibition of phosphorylation processes in the mitochondria. Levels of GTP, UTP and CTP show parallel changes to ATP. This is explained by the presence of very active nucleoside monophosphate kinases which cause a rapid exchange between the nucleoside phosphates. The effects of the sudden inhibition of glucose degradation by sulfite or nitrite on levels of ATP, ADP and inorganic phosphate are discussed in terms of the theory of Lynen (1942) on compensating phosphorylation and dephosphorylation in steady state glucose metabolizing yeast.

Adenosine Diphosphate↗

PubMed2001

Direct determination of nitrite traces in high ionic-strength samples by electrostatic ion chromatography using diluted acid solutions as eluent.

An ion-chromatographic (IC) system with high selectivity for separation of nitrite is described. It is analogous to the EIC (electrostatic IC) previously reported and was established using 3-(N,N-dimethylstearylammonio)propanesulfonate (C23H49NO3S, a sulfobetaine type of zwitterionic surfactants) as the stationary phase and dilute aqueous HCl solutions as the mobile phase. Five inorganic anions, sulfate, chloride, bromide, nitrate, and nitrite were chosen as the model analytes and were analyzed using this EIC system. Sulfate was always eluted first, followed by chloride, bromide and nitrate. Nitrite, however, could be eluted either before or after nitrate, depending on the concentration of HCl in the eluent. An elution order nitrate< nitrite was always obtained simply by using >3 mmol L(-1) HCl as the eluent. For nitrite the detection limit was better than 2.1 x 10(-7) mol L(-1) (100 microL sample injection volume, S/N=3, UV at 210 nm). Bromide and nitrate could also be separated under these HPLC conditions. The detection limit for bromide was 7.2 x 10(-8) mol L(-1) and for nitrate 6.5 x 10(-8) mol L(-1). Both nitrite and nitrate in real seawater samples were successfully determined with direct sample injection using this EIC system.

Acids↗

PubMed2004

Increased production and excretion of urea in the kuruma shrimp (Marsupenaeus japonicus) exposed to combined environments of increased ammonia and nitrite.

Marsupenaeus japonicus (6.37 +/- 1.29 g) individually exposed to 9 different combined solutions of ambient ammonia (C) and nitrite (C') ammonia at 0.003 [control], 0.39, and 1.49 mmol/L combined with nitrite at 0.001 [control], 0.38, and 1.49 mmol/L in 30 ppt were examined for nitrogenous excretion accumulations of ammonia, nitrite, urea, and uric acid in tissues after 48 hours. M. japonicus exposed to 0.39 mmol/L ammonia-0.38 mmol/L nitrite displayed higher levels of urea-nitrogen (UNE) and organic-N (ONE) excretion by a factor of 2.2 and 5.7, respectively, compared with shrimp exposed only to 0.39 mmol/L ammonia. Exposure to 0.39 mmol/L ammonia-0.38 mmol/L nitrite resulted in lower levels of hemolymph uric acid (HUA), gill ammonia (GAM), gill urea (GUE), gill uric acid (GUA), hepatopancreas ammonia (HPAM), hepatopancreas urea (HPUE), and hepatopancreas uric acid (APUA), respectively, compared with shrimp exposed only to 0.39 mmol/L ammonia. We concluded that M. japonicus exposed to combined environments of ammonia and nitrite display increased nitrogen metabolism and production of urea-N and other organic-N.

Ammonia↗