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In Vitro Stability of Nitrate Reductase from Wheat Leaves: II. Isolation of Factors from Crude Extract Which Affect Stability of Highly Purified Nitrate Reductase.

When a crude extract from 8-day-old wheat (Triticum aestivum L. cv. Olympic) leaves was fractionated by a combination of ammonium sulfate precipitation and Sephadex G-100 chromatography the presence of three factors which have a marked effect on the stability of highly purified nitrate reductase was revealed. Two of these factors (I and III) have a positive effect and the other factor (II) has a negative effect on stability. Factors I and III can each overcome the instability-promoting effect of II; however, this was apparently not due to a direct effect on factor II.Both factors I and III have been subjected to further purification. Factor I can be separated into at least four fractions, each with stability-promoting activity. Factor III appears to be a single factor.The in vitro activity and stability of nitrate reductase in crude extracts were found to vary diurnally. Stability and activity were highest 4 hours after the start of the light period and both were minimal 1 to 3 hours after the end of the light period. When crude extract was fractionated as described above and an assessment made of the relative amounts of I, II, and III, there appeared to be a distinct diurnal variation in their levels. Factors I and III were highest when in vitro nitrate reductase activity and stability were highest. Factor II was apparently out of phase in that maximum activity coincided with the time of minimum in vitro nitrate reductase activity and stability.

Journal Article↗

Expression of leaf nitrate reductase genes from tomato and tobacco in relation to light-dark regimes and nitrate supply.

The influence of light-dark cycles and nitrate supply on nitrate reductase (NR) mRNA levels was studied in two plant species, tobacco (Nicotiana tabacum) and tomato (Lycopersicon esculentum) using specific NR DNA probes. In the same series of experiments, changes in the levels of NR protein (NRP) by enzyme-linked immunosorbent assay and changes in the level of NADH-nitrate reductase activity (NRA) were also followed. During a light-dark cycle, it was found that in both tomato and tobacco, NR mRNA accumulation increased rapidly during the dark period and reached a maximum at the beginning of the day, while NRP reached a peak 2 and 4 hours after mRNA peaked, for tomato and tobacco, respectively. At the end of the day, the amount of mRNA was decreased by a factor of at least 100 compared to sunrise in both species. These results demonstrate that light is involved, although probably not directly, in the regulation of the NR gene expression at the mRNA level. The peak of NRA in tobacco coincided with the peak in NR mRNA accumulation (i.e. sunrise), whereas in tomato the peak of NRA was approximately 5 to 6 hours after sunrise. There is no obvious correlation between NRP and NRA levels during the day. In nitrogen starvation experiments, a rapid decrease of NRP and NRA was detected, while NR mRNA levels were not significantly altered. Upon nitrate replenishment, nitrogen-starved plants accumulated NR mRNA rapidly. These results suggest that the availability of nitrogen affects the expression of NR activity at the transcriptional as well as at the post-transcriptional levels.

Journal Article↗

The 'vasovagal effect' of nitrates: an under-recognised complication of nitrate use.

Nitrates are useful drugs in cardiovascular medicine. They have a presumed vasovagal effect, which is a rare but often under-recognised complication that is partly caused by central inhibition of the sympathetic nervous system. However, no consistent variable associated with the presumed vasovagal effects following nitrate administration can be identified, and there are only limited reports of the phenomenon in the literature. In fact, the true prevalence of such a complication is unknown, because no data from observational studies exist, although it would seem likely to be a rare event. A greater awareness and recognition of this unusual effect of nitrate use is necessary in view of the increasing use of nitrates. Fortunately, recovery is generally the rule with supportive management.

Adult↗

Effect of nitrate on the synthesis and decay of nitrate reductase of Neurospora.

1. A method was developed to examine the turnover of nitrate reductase by the use of tungstate. 2. Evidence is presented which suggests that the disappearance of nitrate reductase activity from Neurospora mycelia exposed to non-inducing conditions is due to the disappearance of the enzyme protein(s) from the mycelia, and not merely due to the disappearance of its (their) catalytic power. 3. The presence of NO(3) (-) in the culture medium slows down the rate of degradation of nitrate reductase in Neurospora in vivo.

Animals↗

Posttranslational regulation of nitrate reductase strongly affects the levels of free amino acids and nitrate, whereas transcriptional regulation has only minor influence.

Diurnal variations in nitrate reductase (NR) activity and nitrogen metabolites were examined in wild-type Nicotiana plumbaginifolia and transformants with various degrees of NR deregulation. In the C1 line, NR was only deregulated at the transcriptional level by placing the NR gene under the control of the cauliflower mosaic virus 35S RNA promoter. In the Del8 and S521D lines, NR was additionally deregulated at the posttranslational level either by a deletion mutation in the N-terminal domain or by a mutation of the regulatory phosphorylation site (serine-521). Posttranslational regulation was essential for pronounced diurnal variations in NR activity. Low nitrate content was related to deregulation of NR, whereas the level of total free amino acids was much higher in plants with fully deregulated NR. Abolishing transcriptional and posttranslational regulation (S521D plants) resulted in an increase of glutamine and asparagine by a factor of 9 and 14, respectively, compared with wild type, whereas abolishing transcriptional regulation (C1 plants) only resulted in increases of glutamine and asparagine by factors <2. Among the minor amino acids, isoleucine and threonine, in particular, showed enhanced levels in S521D. Nitrate uptake rates were the same in S521D and wild type as determined with (15)N feeding. Deregulation of NR appears to set the level of certain amino acids, whereas diurnal variations were still determined by light/dark. Generally, deregulation of NR at the transcriptional level did not have much influence on metabolite levels, but additional deregulation at the posttranslational level resulted in profound changes of nitrogen metabolite levels.

Amino Acids↗

Detection of genes for membrane-bound nitrate reductase in nitrate-respiring bacteria and in community DNA.

A nested PCR primed by four degenerate oligonucleotides was developed for the specific amplification of sequences from the narG gene encoding the membrane-bound nitrate reductase. This approach was used to amplify fragments of the narG gene from five Pseudomonas species previously shown to be able to express the membrane-bound nitrate reductase and from community DNA extracted from a freshwater sediment. Amino acid sequences encoded by the narG fragments were compared to one another, and to the corresponding regions of related enzymes. This comparison indicates that the amplification protocols are specific for their intended targets. Sequences amplified from community DNA were tightly clustered, which may indicate a degree of homogeneity in the sediment community. The PCR primers and amplification protocols described will be useful in future studies of nitrate respiring populations.

Amino Acid Sequence↗

Nitrate reduction in so-called nitrate reductase (NR) negative strains of the genus Mycobacterium.

The goal of our present investigations was to complete our knowledge concerning the actual ability to reduce nitrate within the genus Mycobacterium and to eliminate previously reported results like "weakly reaction" or "variable reactions". The influence of conditions like reaction temperature and reaction time, substrate concentration, H+-donors, ammonia and the presence of the nitrate reductase were studied. The results can be summated as follows: 1. The nitrate reductase is more widespread within the genus Mycobacterium than it has been reported by other previous investigators. - 2. Previously doubtful results can be understood. - 3. By means of proper experimental conditions M. chelonei ssp. borstelense can be easily separated from M. chelonei ssp. chelonei, although, according to Bergey's Manual of Determinative Bacteriology, they are recommended as synonyms.

Ammonia↗

Adsorption and desorption of polydimethylsiloxane, PCBs, cadmium nitrate, copper sulfate, nickel sulfate and zinc nitrate by river surface sediments.

An investigation into the adsorption and desorption of polydimethylsiloxane, PCBs, cadmium nitrate, copper sulfate, nickel nitrate and zinc nitrate by river sediments was carried out using either a flow-through system or a semi-static system. The material balance in the sediment compartment could be explained by the equation, dCs/dT= K1Cw-K2Cs. The adsorption rate constants (K1), desorption rate constants (K2) and concentration factors (K1/K2) were calculated. For hydrophobic chemicals, the K1's were independent of water solubility, but the K2's were relatively related to water solubility. For both hydrophobic chemicals and heavy metals, the concentration factors per fraction organic carbon were similar to the soil sorption coefficients (Koc), expressed on a organic carbon basis.

Adsorption↗

Nitration and hydroxylation of aromatic amino acid and guanine by the air pollutant peroxyacetyl nitrate.

Peroxyacetyl nitrate (PAN) is a common gaseous photochemical compound in polluted air and cigarette smog. The toxicity of PAN has been found to depend on three pathways: (1) its oxidizing property that mimics peroxide or peroxynitrite; (2) its nitrating and hydroxylating properties similar to peroxynitrite; and (3) its acetylating property like acetic anhydride. The present investigations were intended to focus on the reactions of PAN with aromatic amino acids and guanine. When PAN interacted with tyrosine and guanine the major products were 3-nitrotyrosine, 3, 5-dinitrotyrosine, 8-hydroxyguanine and 8-nitroguanine. These compounds have been used as indicators for the presence of peroxynitrite in previous studies. When PAN interacted with phenylalanine, the products were 3-nitrotyrosine, 4-nitrophenylalanine, p-tyrosine, o-tyrosine and m-tyrosine. 5-Hydroxytryptophan is produced from the reaction of PAN with tryptophan. Furthermore, the formation of nitrated tyrosines was also found in the PAN-treated HL-60 cells. A high yield of dityrosine was formed when PAN and peroxynitrite were reacted with tyrosine, probably through free radical oxidation. We also found that peroxynitrite and PAN are similar in their oxidizing activity. From these findings, we suggest that peroxynitrite may be considered as the reactive intermediate of PAN.

Air Pollutants↗

Inhibition of peroxynitrite dependent tyrosine nitration by hydroxycinnamates: nitration or electron donation?

Peroxynitrite is a cytotoxic species generated by the reaction between superoxide and nitric oxide. In this study the ability of hydroxycinnamate antioxidants to decrease peroxynitrite-mediated nitration of tyrosine was investigated. The results obtained show that all compounds were able to inhibit nitration of tyrosine. The potency of inhibitory activity was in the order; caffeic acid > or = chlorogenic acid > or = ferulic acid > p-coumaric acid > ocoumaric acid > m-coumaric acid. Trolox, which was included in the study for comparative purposes, had an activity between that of ferulic acid and p-coumaric acid. The data obtained suggest that hydroxycinnamates can act by one of two possible mechanisms: preferential nitration for monophenolates and electron donation by catecholates.

Antioxidants↗

Nitrate transport: a key step in nitrate assimilation.

The nitrate assimilation pathway has been the matter of intensive research during the past decade. Many genes involved in low and high affinity nitrate uptake have been identified in fungi, algae and, more recently, in plants. The plant genes so far isolated are transcriptionally regulated; their inducibility by nitrate seems to be a common feature, shared by their homologs in fungi and algae. A number of questions remain to be elucidated regarding the physiological roles of these transporters and the regulation of their expression.

Amino Acid Sequence↗

Differential effects of nitrated ricin and nitrated and dithionite-reduced ricin on protein-synthesis inhibition and transmembrane tramsport in eukaryotic cells.

Ricin, was nitrated with tetranitromethane and reduced with sodium dithionite. Of the 8.0 nitro groups incorporated, 3.2 were on the A chain and 5.1 were on the B chain. Nitrated ricin1 was somewhat less active than nitrated and reduced ricin1 in inhibiting protein synthesis in vitro, but both were highly inhibitory. However, the modified toxins were less than 1% as active as ricin in inhibiting protein synthesis in cultured cells. Indirect immunofluorescence assays demonstrated tha both modified toxins were specifically bound to the cell surface and could be displaced by galactose.

Animals↗

Effect of nitration on the activity of bovine erythrocyte Cu,Zn-superoxide dismutase (BESOD) and a kinetic analysis of its dimerization-dissociation reaction as examined by subunit exchange between the native and nitrated BESODs.

Bovine erythrocyte Cu,Zn-superoxide dismutase (BESOD) is a dimeric enzyme composed of identical subunits associated through unusually strong non-covalent interactions. The state of the unique tyrosyl residue (Tyr 108) of BESOD was examined, and the kinetics of subunit exchange was studied using Tyr 108 as a probe. UV-absorption difference spectra demonstrate that Tyr 108 is exposed to the solvent, and that the accessibilities to ethanol, ethylene glycol, and polyethylene glycol 600 are 53.5, 52.0, and 44.6%, respectively. Tyr 108 was fully nitrated by tetranitromethane. The pK(a) values of the phenolic hydroxyl group of native and nitrated Tyr 108 were determined to be 11.3 and 7.9, whereas those of model compounds, L-tyrosine and 3-nitro-L-tyrosine, are 9.8 and 6.8, respectively. When the native and nitrated BESODs of an equal concentration were mixed, the hybrid dimer was formed. From the amount of hybrid dimer formed, the rate constant (k(-1)) of the subunit dissociation at pH 7.8, 25 degrees C was assessed to be (4.17 +/- 0.13) x 10(-6) s(-1). The activation energy of the subunit dissociation at pH 7.8 was determined to be 117 +/- 4 kJ.mol(-1). The k(-1) value remains constant at BESOD concentrations ranging from 0.62 to 6.8 micro M and at pH ranging from 6.0 to 10.0, but increased remarkably with a decrease in the dielectric constant of the reaction mixture. It is suggested that hydrophobic interaction may play a significant role in the subunit interaction.

Animals↗

Increased levels of nitrate and surfactant protein a nitration in the pulmonary edema fluid of patients with acute lung injury.

Levels of nitrite (NO2-) and nitrate (NO3-) were measured in pulmonary edema fluid and plasma from 34 patients with early acute lung injury (ALI) and 20 patients with hydrostatic pulmonary edema. Pulmonary edema fluid from patients with ALI had significantly higher levels of NO2- + NO3- compared with pulmonary edema fluid from patients with hydrostatic pulmonary edema (108 +/- 13 microM versus 66 +/- 9 microM; means +/- SEM; p < 0.05). In addition, patients with shock had higher plasma NO2- + NO3- levels than those without shock (79 +/- 11 microM versus 53 +/- 12 microM, p < 0.05). Acidemia and increased anion gap, markers of systemic hypoperfusion, were also associated with twofold higher plasma NO2- + NO3- levels (p < 0.01). Increased levels of NO2- + NO3- in edema fluid samples were associated with slower rates of alveolar fluid clearance. Nitrated pulmonary surfactant protein A (SP-A) was also detected in the edema fluid of patients with ALI after immunoprecipitation with a specific antibody against this protein. Previously, we have shown that nitration of SP-A impairs its host- defense properties. In aggregate, the results of this study indicate that reactive oxygen-nitrogen species may play a role in the pathogenesis of human ALI.

Body Fluids↗

Nitrate tolerance, oxidative stress, and mitochondrial function: another worrisome chapter on the effects of organic nitrates.

A shortcoming in the clinical use of organic nitrates is the development of tolerance. Recent data have suggested that the denitrification of organic nitrates is mediated by mitochondrial aldehyde dehydrogenase and that dysfunction of this enzyme is an important cause of tolerance. In this issue of the JCI, evidence in support of this hypothesis is presented in an in vivo model of nitrate tolerance (see the related article beginning on page 482).

Free Radicals↗

Highly sensitive reaction of nitrate with brucine and 3-methyl-2-benzothiazolinone hydrazone hydrochloride for the determination of nitrate in environmental samples.

A modified and highly sensitive spectrophotometric method for the determination of nitrate in trace quantities in environmental samples is described. The method is based on the reaction of nitrate ion with brucine and 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) in sulfuric acid medium to yield a violet-colored product which is stable for over two days. The optimum photometric range for the determination of nitrate is 0.04-0.16 microg cm(-3) and the Sandell's sensitivity being 0.000279 microg cm(-2). The proposed method is applied to various water samples and the results indicate that the reaction is highly sensitive than the original brucine method.

Benzothiazoles↗

[Changes in nitrate levels and nitrates in carrot roots during storage in an earthen clamp].

The aim of this study was the determine the nitrate and nitrite contents in edible parts of 5 varieties of carrot, before clamping and after uncovering, in fresh carrot and dry matter. The colorimetric methods of Griess with modified by Romminger and Straus were used. Dry matter was determined with a drier. Statistical significance of differences in the levels of the studied compounds was calculated by a two-ways analysis of variance. In the studied carrot before clamping average 406.6 mg KNO3/kg and 4.1 mg NaNO2/kg of fresh product was found. These amounts considerably admissible values. After 6-month storage a statistically significant decrease in nitrate levels of Karlena, Perfekcja and Amager was found, in dry matter, whereas a statistically significant increase in Koral variety, while no statistically significant difference in the nitrate level did not changes, was found in Bercoro variety. As concerns nitrites a statistically significant decrease was stated in all carrot varieties.

Colorimetry↗

The reaction between organic nitrates and sulfhydryl compounds. A possible model system for the activation of organic nitrates.

The rate of loss of the sulfhydryl group, determined with the Ellman reagent, was used to derive second order rate constants for the reaction of a series of organic nitrates with a series of sulfhydryl compounds. For the organic nitrates, increases in the rate of reaction with cysteine, in general, ran parallel both with increases in pharmacological potency (flow in the Langendorff heart) and with increases in total clearance. Cysteine was the most active sulfhydryl compound examined, which is compatible with a possible role as an important nitrate receptor. Under some conditions the rate of loss of the sulfhydryl group was much greater than the rate of formation of nitrite ion. This indicates the presence of a reaction intermediate, probably a thionitrate. It is suggested that, in vivo, a thionitrate could function as an important intermediate in the activation of guanylate cyclase.

Animals↗