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PubMed1986

Cloning and nitrate induction of nitrate reductase mRNA.

Nitrate is the major source of nitrogen taken from the soil by higher plants but requires reduction to ammonia prior to incorporation into amino acids. The first enzyme in the reducing pathway is a nitrate-inducible enzyme, nitrate reductase (EC 1.6.6.1). A specific polyclonal antiserum raised against purified barley nitrate reductase has been used to immunoprecipitate in vivo labeled protein and in vitro translation products, demonstrating that nitrate induction increases nitrate reductase protein and translatable mRNA. A partial cDNA clone for barley nitrate reductase has been isolated and identified by hybrid-selected translation. RNA blot-hybridization analysis shows that nitrate induction also causes a marked increase in the steady-state level of nitrate reductase mRNA.

Journal Article↗

PubMed1975

Nitrate Uptake and Assimilation by Wheat Seedlings during Initial Exposure to Nitrate.

Nitrate uptake, reduction, and translocation were examined in intact, 14-day-old, nitrogen-depleted wheat (Triticum vulgare var. Knox) seedlings during a 9-hour exposure to 0.2 mm Ca (NO(3))(2). The nitrate uptake rate was low during the initial 3-hour period, increased during the 3- to 6-hour period, and then declined. By the 3rd hour, 14% of the absorbed nitrate had been reduced, and this increased to 36% by the 9th hour. Shoots accumulated reduced (15)N more rapidly than roots and the ratio of reduced (15)N to (15)N-nitrate was higher in the shoots. A significant proportion of the total reduction occurred in the root system under these experimental conditions. Accumulation of (15)N in ethanol-insoluble forms was evident in both roots and shoots by the 3rd hour and, after 4.5 hours, increased more rapidly in shoots than in roots.An experiment in which a 3-hour exposure to 0.2 mm Ca ((15)NO(3))(2) was followed by a 12-hour exposure to 0.2 mm Ca ((14)NO(3))(2) revealed a half-time of depletion of root nitrate of about 2.5 hours. A large proportion of this depletion, however, was due to loss of (15)N-nitrate to the ambient (14)N-nitrate solution. The remaining pool of (15)N-nitrate was only slowly available for reduction. Total (15)N translocation to the shoot was relatively efficient during the first 3 hours after transfer to Ca ((14)NO(3))(2) but it essentially ceased after that time in spite of significant pools of (15)N-nitrate and alpha-amino-(15)N remaining in the root tissue.

Journal Article↗

PubMed1984

Effects of nitrogen dioxide and nitrate nutrition on growth and nitrate assimilation in bean leaves.

The influence of nutrient nitrate level (0-20 millimolar) on the effects of NO(2) (0-0.5 parts per million) on growth, K, photosynthetic pigment, N contents, and the activities of enzymes of N assimilation was studied in bean (Phaseolus vulgaris L. cv Kinghorn Wax) leaves. Exposing 7-day old bean seedlings for 5 days continuously to 0.02 to 0.5 parts per million NO(2) increased plant height, fresh weight, chlorophyll, carotenoid, organic N and nitrate contents, and nitrate reductase and glutamate synthase activities in the leaves of seedlings supplied with no external N. At 20 millimolar nitrate, most of the parameters examined were inhibited except for organic N and nitrate contents and glutamate synthase activity which increased in most cases. Generally, with an increase in NO(2) concentration, the stimulatory effect declined and/or the inhibitory effect increased. A 3-hour exposure of 12-day-old bean seedlings to 0.1 to 2.0 parts per million NO(2) increased nitrate content and nitrate reductase activity at each nutrient nitrate level except for a slight inhibition of enzyme activity during exposure to 2.0 parts per million NO(2) at 20 millimolar nitrate. The experiments demonstrated that the effect of NO(2) is strongly influenced by nutrient N level and that NO(2) is assimilated into organic nitrogenous compounds to serve as a source of N, only to a limited extent.

Journal Article↗

PubMed1990

Genetic evidence that NarL function is not required for nitrate regulation of nitrate assimilation in Klebsiella pneumoniae M5al.

We cloned the narL gene, required for nitrate induction of respiratory nitrate reductase synthesis, from Klebsiella pneumoniae. The E. coli narL gene product shares sequence similarity with the response regulator proteins of two-component regulatory systems. We found that narL(+)-containing plasmids restored nitrate regulation of anaerobic respiratory gene expression in appropriate Escherichia coli hosts. The K. pneumoniae narL region encoded a protein whose migration in Laemmli gels was indistinguishable from that of the narL product of E. coli. We constructed a narL::Km mutant of K. pneumoniae. This mutation abolished nitrate induction of respiratory nitrate reductase synthesis but had no effect on nitrate induction of assimilatory nitrate and nitrite reductase synthesis. We conclude that K. pneumoniae has distinct nitrate-responsive regulators for controlling respiratory and assimilatory gene expression.

Cloning, Molecular↗

PubMed1993

The herbicide sensitivity gene CHL1 of Arabidopsis encodes a nitrate-inducible nitrate transporter.

This paper reports the identification and functional expression of a gene that is involved in nitrate uptake in plants, a process essential for the assimilation of nitrate and the biological removal of nitrate from the soil solution. The CHL1 gene of Arabidopsis, which when mutated confers resistance to the herbicide chlorate and a decrease in nitrate uptake, was isolated and found to encode a protein with 12 putative membrane-spanning segments. Injection of CHL1 mRNA into Xenopus oocytes produces a nitrate- and pH-dependent membrane depolarization, inward current, and nitrate uptake. These data show that the CHL1 gene encodes an electrogenic nitrate transporter. CHL1 mRNA is found predominantly in roots and displays nitrate- and pH-dependent regulation.

Amino Acid Sequence↗

PubMed1988

The reproducibility of the conversion of nitrate to nitrite in human saliva after a nitrate load.

We investigated the reproducibility of the inter- and intra-individual variations in the conversion of nitrate to nitrite in saliva. Saliva samples were collected from 20 volunteers just before and at regular intervals after a nitrate load on four non-consecutive days within a period of 2 months. On three occasions beetroot juice was the nitrate source and on one occasion a nitrate solution was given. Despite large day-to-day variations it was possible to discriminate between subjects with a consistently high or low nitrate conversion after a nitrate load. Neither saliva sampling before a nitrate load nor single saliva samples are sufficient to obtain clear information about individual capacities for nitrate-nitrite conversion.

Ascorbic Acid↗

PubMed2001

Nitration and oxidation of a hydrophobic tyrosine probe by peroxynitrite in membranes: comparison with nitration and oxidation of tyrosine by peroxynitrite in aqueous solution.

It has been reported that peroxynitrite will initiate both oxidation and nitration of tyrosine, forming dityrosine and nitrotyrosine, respectively. We compared peroxynitrite-dependent oxidation and nitration of a hydrophobic tyrosine analogue in membranes and tyrosine in aqueous solution. Reactions were carried out in the presence of either bolus addition or slow infusion of peroxynitrite, and also using the simultaneous generation of superoxide and nitric oxide. Results indicate that the level of nitration of the hydrophobic tyrosyl probe located in a lipid bilayer was significantly greater than its level of oxidation to the corresponding dimer. During slow infusion of peroxynitrite, the level of nitration of the membrane-incorporated tyrosyl probe was greater than that of tyrosine in aqueous solution. Evidence for hydroxyl radical formation from decomposition of peroxynitrite in a dimethylformamide/water mixture was obtained by electron spin resonance spin trapping. Mechanisms for nitration of the tyrosyl probe in the membrane are discussed. We conclude that nitration but not oxidation of a tyrosyl probe by peroxynitrite is a predominant reaction in the membrane. Thus, the local environment of target tyrosine residues is an important factor governing its propensity to undergo nitration in the presence of peroxynitrite. This work provides a new perspective on selective nitration of membrane-incorporated tyrosine analogues.

Bicarbonates↗

PubMed1983

Mammalian nitrate biosynthesis: incorporation of 15NH3 into nitrate is enhanced by endotoxin treatment.

Incorporation of an oral dose of [15N]ammonium acetate into urinary [15N]nitrate has been demonstrated in the rat. Investigation of the regulation of nitrate synthesis has shown that Escherichia coli lipopolysaccharide potently stimulates urinary nitrate excretion (9-fold increase). It was further shown that the enhanced rate of nitrate excretion by lipopolysaccharide was due not to a reduction in nitrate metabolic loss but rather to an increased rate of synthesis. This conclusion was based on finding a proportionally increased incorporation of [15N]ammonium into nitrate nitrogen with lipopolysaccharide treatment. Nitrate biosynthesis was also increased by intraperitoneal injection of carrageenan and subcutaneous injection of turpentine. It is proposed that the pathway of nitrate biosynthesis may be the result of oxidation of reduced nitrogen compounds by oxygen radicals generated by an activated reticuloendothelial system.

Acetates↗

PubMed1985

Mammalian nitrate biosynthesis: mouse macrophages produce nitrite and nitrate in response to Escherichia coli lipopolysaccharide.

Escherichia coli lipopolysaccharide (LPS)-induced nitrate biosynthesis was studied in LPS-sensitive C3H/He and LPS-resistant C3H/HeJ mice. Intraperitoneal injection of 15 micrograms of LPS led to a temporary 5- to 6-fold increase in blood nitrate concentration in the C3H/He strain. Levels of nitrate excreted in the urine were also increased. In contrast, no increase was observed in the C3H/HeJ strain with LPS injections up to 175 micrograms. Furthermore, thioglycolate-elicited peritoneal macrophages from C3H/He, but not from C3H/HeJ mice, produced nitrite (60%) and nitrate (40%) when cultured with LPS (10 micrograms/ml). T-lymphocyte addition/depletion experiments showed the presence of T cells enhanced this response. However, LPS did not cause nitrite or nitrate production in cultures of spleen lymphocytes from either strain. LPS-induced nitrate synthesis was also observed with nude mice and CBA/N mice, indicating that neither functional T lymphocytes nor LPS-responsive B lymphocytes were required for the response in vivo. This was consistent with the in vitro results showing macrophages alone were competent. Mycobacterium bovis infection of C3H/He and C3H/HeJ mice resulted in a large increase in nitrate production over the course of the infection for both strains, suggesting T-lymphocyte-mediated activation of macrophages as a potent stimulus for nitrate biosynthesis. The synthesis of nitrite is significant in that it can directly participate in the endogenous formation of nitrosamines and may also be involved in some aspect of the chemistry of cytotoxicity.

Animals↗

PubMed1996

Nitrate and nitrate are transported by different specific transport systems and by a bispecific transporter in Chlamydomonas reinhardtii.

Nitrate transport mutants from Chlamydomonas reinhardtii and strains derived from them upon transformation with plasmids containing the C. reinhardtii nar2/Nrt2;1 or nar2/Nrt2;2 genes have been used to study nitrate and nitrite transport systems. Mutants lacking nitrate assimilation clustered genes showed a high affinity nitrite transporter activity (system 3), which was subject to ammonium inhibition and appeared to be independent of a functional nar2 gene. Transformants carrying nar2/Nrt2;2 recovered a high affinity nitrate transporter activity (system 2) and showed nitrite transport activities with properties similar to those in nonrecovered high affinity nitrate transporter activity (system 1) together with a considerably enhanced nitrite transport activity. Nitrite transport mediated by system 1 was very sensitive to inhibition by nitrate at microM concentrations. Results strongly suggest that three nitrate assimilation related high affinity transport systems operate in C. reinhardtii: one specific for nitrite, a second one encoded by nar2/Nrt2;2 specific for nitrate, and another one encoded by nar2/Nrt2;1, which is bispecific for these two anions.

Animals↗

PubMed1998

Influence of carbon source on nitrate removal by nitrate-tolerant Klebsiella oxytoca CECT 4460 in batch and chemostat cultures.

The nitrate-tolerant organism Klebsiella oxytoca CECT 4460 tolerates nitrate at concentrations up to 1 M and is used to treat wastewater with high nitrate loads in industrial wastewater treatment plants. We studied the influence of the C source (glycerol or sucrose or both) on the growth rate and the efficiency of nitrate removal under laboratory conditions. With sucrose as the sole C source the maximum specific growth rate was 0.3 h-1, whereas with glycerol it was 0.45 h-1. In batch cultures K. oxytoca cells grown on sucrose or glycerol were able to immediately use sucrose as a sole C source, suggesting that sucrose uptake and metabolism were constitutive. In contrast, glycerol uptake occurred preferentially in glycerol-grown cells. Independent of the preculture conditions, when sucrose and glycerol were added simultaneously to batch cultures, the sucrose was used first, and once the supply of sucrose was exhausted, the glycerol was consumed. Utilization of nitrate as an N source occurred without nitrite or ammonium accumulation when glycerol was used, but nitrite accumulated when sucrose was used. In chemostat cultures K. oxytoca CECT 4460 efficiently removed nitrate without accumulation of nitrate or ammonium when sucrose, glycerol, or mixtures of these two C sources were used. The growth yields and the efficiencies of C and N utilization were determined at different growth rates in chemostat cultures. Regardless of the C source, yield carbon (YC) ranged between 1.3 and 1.0 g (dry weight) per g of sucrose C or glycerol C consumed. Regardless of the specific growth rate and the C source, yield nitrogen (YN) ranged from 17.2 to 12.5 g (dry weight) per g of nitrate N consumed. In contrast to batch cultures, in continuous cultures glycerol and sucrose were utilized simultaneously, although the specific rate of sucrose consumption was higher than the specific rate of glycerol consumption. In continuous cultures double-nutrient-limited growth appeared with respect to the C/N ratio of the feed medium and the dilution rate, so that for a C/N ratio between 10 and 30 and a growth rate of 0.1 h-1 the process led to simultaneous and efficient removal of the C and N sources used. At a growth rate of 0.2 h-1 the zone of double limitation was between 8 and 11. This suggests that the regimen of double limitation is influenced by the C/N ratio and the growth rate. The results of these experiments were validated by pulse assays.

Culture Media↗

PubMed1973

In vitro formation of nitrate reductase using extracts of the nitrate reductase mutant of Neurospora crassa, nit-1, and Rhodospirillum rubrum.

In vitro formation of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-nitrate reductase (NADPH: nitrate oxido-reductase, EC 1.6.6.2) has been attained by using extracts of the nitrate reductase mutant of Neurospora crassa, nit-1, and extracts of either photosynthetically or heterotrophically grown Rhodospirillum rubrum, which contribute the constitutive component. The in vitro formation of NADPH-nitrate reductase is characterized by the conversion of the flavin adenine dinucleotide (FAD) stimulated NADPH-cytochrome c reductase, contributed by the N. crassa nit-1 extract from a slower sedimenting form (4.5S) to a faster sedimenting form (7.8S). The 7.8S NADPH-cytochrome c reductase peak coincides in sucrose density gradient profiles with the NADPH-nitrate reductase, FADH(2)-nitrate reductase and reduced methyl viologen (MVH)-nitrate reductase activities which are also formed in vitro. The constitutive component from R. rubrum is soluble (both in heterotrophically and photosynthetically grown cells), is stimulated by the addition of 10(-4) M Na(2)MoO(4) and 10(-2) M NaNO(3) to cell-free preparations, and has variable activity over the pH range from 3.0 to 9.5. The activity of the constitutive component in some extracts showed a threefold stimulation when the pH was lowered from 6.5 to 4.0. The constitutive activity appears to be associated with a large molecular weight component which sediments as a single peak in sucrose density gradients. However, the constitutive component from R. rubrum is dialyzable and is insensitive to trypsin and protease. These results demonstrate that R. rubrum contains the constitutive component and suggests that it is a low molecular weight, trypsin- and protease-insensitive factor which participates in the in vitro formation of NADPH nitrate reductase.

Cell-Free System↗

PubMed1968

Regulation of nitrate assimilation and nitrate respiration in Aerobacter aerogenes.

The influence of growth conditions on assimilatory and respiratory nitrate reduction in Aerobacter aerogenes was studied. The level of nitrate reductase activity in cells, growing in minimal medium with nitrate as the sole nitrogen source, was much lower under aerobic than anaerobic conditions. Further, the enzyme of the aerobic cultures was very sensitive to sonic disintegration, as distinct from the enzyme of anaerobic cultures. When a culture of A. aerogenes was shifted from anaerobic growth in minimal medium with nitrate and NH(4) (+) to aerobiosis in the same medium, but without NH(4) (+), the production of nitrite stopped instantaneously and the total activity of nitrate reductase decreased sharply. Moreover, there was a lag in growth of about 3 hr after such a shift. After resumption of growth, the total enzymatic activity increased again slowly and simultaneously became gradually sensitive to sonic disintegration. These findings show that oxygen inactivates the anaerobic nitrate reductase and represses its further formation; only after a de novo synthesis of nitrate reductase with an assimilatory function will growth be resumed. The enzyme in aerobic cultures was not significantly inactivated by air, only by pure oxygen. The formation of the assimilatory enzyme complex was repressed, however, by NH(4) (+), under both aerobic and anaerobic conditions. The results indicate that the formation of the assimilatory enzyme complex and that of the respiratory enzyme complex are regulated differently. We suggest that both complexes have a different composition, but that the nitrate reductase in both cases is the same protein.

Ammonium Chloride↗

PubMed1982

Volatile N-nitrosamines, nitrate and nitrite in urine and saliva of healthy volunteers after administration of large amounts of nitrate.

Twelve healthy volunteers ingested orally 7-10.5 g of NH4NO3 in a single dose and 12 others received 9.5 g of NaNO3 intravenously in about 60 min. Samples of blood, saliva and urine were collected just before administration of nitrate and at various times afterwards during a 24 h period. Saliva and urine were analysed for volatile NA, nitrate and nitrite. Blood was analysed for nitrate. Neither in urine nor in saliva were NA other than NDMA detected. Of the 188 urine samples, only 13% contained more than 0.1 microgram NDMA/kg, the highest level being 0.5 microgram/kg. In 92% of the 179 saliva samples, less than 0.5 microgram NDMA/1 was found. The saliva of 1 person contained 0.7-8.9 microgram NDMA/1. Nitrate levels in blood rose sharply during the first hour after intravenous nitrate administration and 2 hours after oral intake, reaching peak values of ca. 6 mmol/1. Peak values for NO-3 + NO-2 in saliva, which were reached 2-6 hours after intake, varied strongly between individuals, ranging from 4 to 43 mmol/1. Nitrite was detected in 26% of the urine samples. There was no correlation between NDMA contents and nitrite in urine. An average of 75% of administered nitrate was excreted in urine in 24 h. However, nitrate contents in blood, urine and saliva after 24 h were still higher than before the nitrate intake.

Adult↗

PubMed1999

Effect of nitrate and incubation conditions on the production of catalase and nitrate reductase by staphylococci.

The objective of this work was to study the production of catalase and nitrate reductase by staphylococci in order to understand their role in lipid oxidation during sausage manufacturing. Catalase and nitrate reductase were measured in resting cells and supernatants of staphylococci grown in different conditions. All staphylococci (except S. warneri) synthetized nitrate reductase. In static condition, the synthesis was maximal during exponential growth phase, whereas in shaking condition, the synthesis was maximal at the beginning of stationary phase. The production of nitrate reductase was increased in presence of nitrate, this effect was particularly important for the two S. carnosus strains which exhibited the highest activity. For all staphylococci, the production of catalase was maximal at the end of the exponential growth phase. The lowest amount of catalase was produced by S. warneri and the highest by S. carnosus. Only S. xylosus 873 and S. saprophyticus 852 released high amounts of catalase in the supernatant growth. Staphylococci produced higher amounts of catalase in shaking conditions. Addition of nitrate in the growth media favoured the synthesis of catalase, with a pronounced effect for S. carnosus. Nitrate also favoured the release of catalase.

Animals↗

PubMed2004

Nitrate reductase regulation in tomato roots by exogenous nitrate: a possible role in tolerance to long-term root anoxia.

The mechanism of nitrate reductase (NR) regulation under long-term anoxia in roots of whole plants and the putative role of nitrate in anoxia tolerance have been addressed. NR activity in tomato roots increased significantly after 24 h of anaerobiosis and increased further by 48 h, with a concomitant release of nitrite into the culture medium. Anoxia promoted NR activation through dissociation of the 14-3-3 protein inhibitor and NR dephosphorylation. After 24 h of anoxia, the total amount of NR increased slightly up to 48 h. However, NR-mRNA levels remained constant between 0 h and 24 h of root anoxia and decreased after 48 h. This is probably due to the inhibition of NR degradation and the accumulation of its native form. NR was slightly dephosphorylated in the absence of oxygen and nitrate. Under anoxia, NR dephosphorylation was modulated by nitrate-controlled NR activity. In addition, the presence of nitrate prevents anoxic symptoms on leaves and delays wilting by 48 h during root anoxia. In the absence of nitrate, plants withered within 24 h, as they did with tungstate treatment, an inhibitor of NR activity. Thus, anoxia tolerance of tomato roots could be enhanced by nitrate reduction.

Adaptation, Physiological↗

PubMed1994

5' proximal regions of Arabidopsis nitrate reductase genes direct nitrate-induced transcription in transgenic tobacco.

Nitrate reductase (NR) is the first enzyme in nitrate assimilation, a critical process for plant survival. The regulation of NR gene expression is complex, involving both internal and external factors. Of these, nitrate induction of NR gene expression has been studied most extensively and is well conserved among bacteria, fungi, and higher plants. We are interested in understanding the mechanism of nitrate induction of higher plant NR genes. Here we describe promoter analyses of the 5' flanking regions of the Arabidopsis NR genes, NR1 and NR2, with respect to nitrate induction of gene expression. To facilitate these analyses, a nitrate induction procedure using T1 transgenic tobacco plants was established. Approximately 1.5-kb 5' flanking regions of the two Arabidopsis NR genes (NR1 and NR2) were fused to a reporter gene and its expression in transgenic plants was analyzed. Deletion analyses of these regions show that 238- and 188-bp 5' flanking regions of the NR1 and NR2, respectively, contain sequences responsive to nitrate induction.

Arabidopsis↗

PubMed2004

Protective effect of salivary nitrate and microbial nitrate reductase activity against caries.

To test the hypothesis that a combination of high salivary nitrate and high nitrate-reducing capacity are protective against dental caries, 209 children attending the Dental Institute, Barts and The London NHS Trust were examined. Salivary nitrate and nitrite levels, counts of Streptococcus mutans and Lactobacillus spp., and caries experience were recorded. Compared with control subjects, a significant reduction in caries experience was found in patients with high salivary nitrate and high nitrate-reducing ability. Production of nitrite from salivary nitrate by commensal nitrate-reducing bacteria may limit the growth of cariogenic bacteria as a result of the production of antimicrobial oxides of nitrogen, including nitric oxide.

Adolescent↗