Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.7.1.2 (nitrate reductase)
3,861 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) can be efficiently mineralized with anaerobic domestic sludge, but the initial enzymatic processes involved in its transformation are unknown. To test the hypothesis that the initial reaction involves reduction of nitro group(s), we designed experiments to test the ability of a nitrate reductase (EC 1.6.6.2) to catalyze the initial reaction leading to ring cleavage and subsequent decomposition. A nitrate reductase from Aspergillus niger catalyzed the biotransformation of RDX most effectively at pH 7.0 and 30 degrees C under anaerobic conditions using NADPH as electron donor. LC/MS (ES-) chromatograms showed the formation of hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) and methylenedinitramine as key initial products of RDX, but neither the dinitroso neither (DNX) nor trinitroso (TNX) derivatives were observed. None of the above detected products persisted, and their disappearance was accompanied by the accumulation of nitrous oxide (N20), formaldehyde (HCHO), and ammonium ion (NH4+). Stoichiometric studies showed that three NADPH molecules were consumed, and one molecule of methylenedinitramine was produced per RDX molecule. The carbon and nitrogen mass balances were 96.14% and 82.10%, respectively. The stoichiometries and mass balance measurements supported a mechanism involving initial transformation of RDX to MNX via a two-electron reduction mechanism. Subsequent reduction of MNX followed by rapid ring cleavage gave methylenedinitramine which in turn decomposed in water to produce quantitatively N2O and HCHO. The results clearly indicate that an initial reduction of a nitro group by nitrate reductase is sufficient for the decomposition of RDX.
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PMID:Biotransformation of hexahydro-1,3,5-trinitro-1,3,5-tiazine catalyzed by a NAD(P)H: nitrate oxidoreductase from Aspergillus niger. 1214 90

mAb NL7 was raised against purified flavocytochrome b(558), important in host defense and inflammation. NL7 recognized the gp91(phox) flavocytochrome b(558) subunit by immunoblot and bound to permeabilized neutrophils and neutrophil membranes. Epitope mapping by phage display analysis indicated that NL7 binds the (498)EKDVITGLK(506) region of gp91(phox). In a cell-free assay, NL7 inhibited in vitro activation of the NADPH oxidase in a concentration-dependent manner, and had marginal effects on the oxidase substrate Michaelis constant (K(m)). mAb NL7 did not inhibit translocation of p47(phox), p67(phox), or Rac to the plasma membrane, and bound its epitope on gp91(phox) independently of cytosolic factor translocation. However, after assembly of the NADPH oxidase complex, mAb NL7 bound the epitope but did not inhibit the generation of superoxide. Three-dimensional modeling of the C-terminal domain of gp91(phox) on a corn nitrate reductase template suggests close proximity of the NL7 epitope to the proposed NADPH binding site, but significant separation from the proposed p47(phox) binding sites. We conclude that the (498)EKDVITGLK(506) segment resides on the cytosolic surface of gp91(phox) and represents a region important for oxidase function, but not substrate or cytosolic component binding.
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PMID:Functional epitope on human neutrophil flavocytochrome b558. 1279 37

The nitrate assimilation pathway represents a useful model system in which to study the contribution of a mycorrhizal fungus to the nitrogen nutrition of its host plant. In the present work we cloned and characterized the nitrate reductase gene (tbnr1) from Tuber borchii. The coding region of tbnr1 is 2,787 nt in length, and it encodes a protein of 929 amino acids. Biochemical and Northern-blot analyses revealed that nitrate assimilation in T. borchii is an inducible system that responds mainly to nitrate. Furthermore, we cloned a nitrate reductase cDNA (tpnr1) from Tilia platyphyllos to set up a quantitative real-time PCR assay that would allow us to determine the fungal contribution to nitrate assimilation in ectomycorrhizal tissue. Using this approach we demonstrated that the level of tbnr1 expression in ectomycorhizae is eight times higher than in free-living mycelia, whereas tpnr1 transcription was found to be down-regulated after the establishment of the symbiosis. Enzymatic assays showed that NADPH-dependent nitrite formation markedly increases in ectomycorrhizae. These findings imply that the fungal partner plays a fundamental role in nitrate assimilation by ectomycorrhizae. Amino acid determination by HPLC revealed higher levels of glutamate, glutamine and asparagine in symbiotic tissues compared with mycelial controls, thus suggesting that these amino acids may represent the compounds that serve to transfer nitrogen to the host plant.
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PMID:Characterization of the Tuber borchii nitrate reductase gene and its role in ectomycorrhizae. 1289 21

Nitrate reductase was purified from and characterized in a bloom-forming unicellular calcifying alga, Emiliania huxleyi (Haptophyceae). The molecular masses of the native form and the subunit were 514 and 85 kDa, respectively, showing that the enzyme is a hexamer composed of 6 homologous subunits. The Km values for NADH and NO3- were 40 microM and 104 microM, respectively. Activity of the reduction of nitrate was very high with reduced methylviologen and NADH, but no activity was observed with NADPH or reduced flavin mononucleotide; oxidation of NADH was very high with cytochrome c but did not occur with ferricyanide. These results indicate that Emiliania nitrate reductase is NADH-specific (EC 1.6.6.1), and that among algae and plants its subunit structure and kinetic properties are unique.
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PMID:Characterization of NADH: nitrate reductase from the coccolithophorid Emiliania huxleyi (Lohman) Hay & Mohler (Haptophyceae). 1292 15

With respect to cofactor requirements, NADH, and FMNH(2) were equally effective as electron donors for nitrate reductase obtained from leaves of maize, marrow, and spinach, when the cofactors were supplied in optimal concentrations. The concentration of FMNH(2) required to obtain half-maximal activity was from 40- to 100-fold higher than for NADH. For maximal activity with the corn enzyme, 0.8 millimolar FMNH(2) was required. In contrast, NADPH was functional only when supplied with NADP:reductase and exogenous FMN (enzymatic generation of FMNH(2)).All attempts to separate the NADH(2)- and FMNH(2)-dependent nitrate reductase activities were unsuccessful and regardless of cofactor used equal activities were obtained, if cofactor concentration was optimal. Unity of NADH to FMNH(2) activities were obtained during: A) purification procedures (4 step, 30-fold); B) induction of nitrate reductase in corn seedlings with nitrate; and C) inactivation of nitrate reductase in intact or excised corn seedlings. The NADH- and FMNH(2)-dependent activities were not additive.A half-life for nitrate reductase of approximately 4 hours was estimated from the inactivation studies with excised corn seedlings. Similar half-life values were obtained when seedlings were incubated at 35 degrees in a medium containing nitrate and cycloheximide (to inhibit protein synthesis), or when both nitrate and cycloheximide were omitted.In those instances where NADH activity but not FMNH(2) activity was lost due to treatment (temperature, removal of sulfhydryl agents, addition of p-chloromercuribenzoate), the loss could be explained by inactivation of the sulfhydryl group (s) required for NADH activity. This was verified by reactivation with exogenous cysteine.Based on these current findings, and previous work, it is concluded that nitrate reductase is a single moiety with the ability to utilize either NADH or FMNH(2) as cofactor. However the high concentration of FMNH(2) required for optimal activity suggests that in vivo NADH is the electron donor and that nitrate reductase in higher plants should be designated NADH:nitrate reductase (E.C. 1.6.6.1).
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PMID:Some characteristics of nitrate reductase from higher plants. 1665 64

Under conditions of controlled pH, nitrate and ammonium are equally effective in supporting the growth of young soybean (Glycine max var. Bansei) and sunflower (Helianthus annuus L. var., Mammoth Russian) plans. Soybean contains an active nitrate reductase in roots and leaves, but the low specific activity of this enzyme in sunflower leaves indicates a dependency upon the roots for nitrate reduction. Suppression of nitrate reductase activity in sunflower leaves may be due to high concentrations of ammonia received from the roots. Nitrate reductase activity in leaves of nitrate-supplied soybean and sunflower follows closely the distribution of nitrate reductase. For the roots of both species, glutamic acid dehydrogenase activity was greater with ammonium than with nitrate. The glutamic acid dehydrogenase of ammonium roots is wholly NADH-dependent, whereas that of nitrate roots is active with NADH and NADPH. In leaves, an NADPH-dependent glutamic acid dehydrogenase appears to be responsible for the assimilation of translocated ammonia and ammonia formed by nitrate reduction.In soybean roots ammonia is actively incorporated into amides, much of which remains in the roots. Sunflower roots are less active in amide formation but transfer much of it, together with ammonia, into the shoots. Glutamine synthetase activity in leaves is 20- to 40-fold lower than in roots.Glucose-6-phosphate dehydrogenase activity appears to be correlated with the activity of the nitrate reducing system in roots, but not in leaves.
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PMID:Influence of ammonium and nitrate nutrition on enzymatic activity in soybean and sunflower. 1665 12

Total pyridine nucleotide concentration of root tissue for young soybean (Glycine max var. Bansei) and sunflower (Helianthus annuus L. var. Mammoth Russian) plants is the same with either ammonium or nitrate, but nitrate results in an increased proportion of total oxidized plus reduced NADP (NADP[H]) seemingly at the expense of NAD. The activity of NADH- and NADPH-dependent forms of glutamic acid dehydrogenase is correlated with the ratio of total oxidized plus reduced NAD to NADP(H). The low NAD: NADH ratio maintained in nitrate roots despite active NADH utilization via nitrate reductase and glutamic acid dehydrogenase may be the result of nitrate-stimulated glycolysis. Nitrate roots also maintain a high level of NADPH, presumably by the stimulatory effect of nitrate utilization on glucose-6-phosphate dehydrogenase activity. In the presence of nitrate rather than ammonium, the highly active nitrate-reducing leaves of soybean show a greater proportion of total pyridine nucleotide in the form of NADP(H) than do the inactive leaves of sunflower.For all tissues examined, ammonium nutrition yields a higher concentration of total adenine nucleotide than is found with nitrate. The data indicate the production of a higher level of metabolites that enter into purine synthesis with ammonium than with nitrate. Glutamine synthetase activity can be correlated with the concept that enzymes utilizing ATP for biosynthetic purposes increase in activity in accordance with the energy level of the cell.
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PMID:Influence of ammonium and nitrate nutrition on the pyridine and adenine nucleotides of soybean and sunflower. 1665 13

Nitrate simultaneously induced NADH- and NADPH-nitrate reductase activities in rice seedlings. Chloramphenicol, other organic nitro-compounds such as o-nitroaniline and 2,4-dinitrophenol and nitrite also induced nitrate reductase in rice seedlings. The nitrate- or nitrite-induced nitrate reductase could accept electrons more efficiently from NADH than NADPH. However, when this enzyme was induced by organic nitro-compounds, it could accept electrons more efficiently from NADPH than NADH.
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PMID:Nitrate reductase of rice seedlings and its induction by organic nitro-compounds. 1665 98

In a study on 3-day maize (Zea mays) seedlings, grown on nitrate, requirements were established for the maximum extraction and optimum stabilization of nitrate reductase in vitro. With the primary root, 5 mm cysteine were required in the extraction medium, but for the scutellum, which has a high level of endogenous thiol, the use of additional thiol resulted in a reduced yield of a more labile enzyme. Activity of the root and scutella nitrate reductase was obtained with either NADH or NADPH, but that of the root enzyme with NADPH was only demonstrated in the absence of phosphate.Before leaf expansion, the nitrate reductase in the maize seedling was mainly in the scutellum. The enzyme present in the primary root was predominantly in the apical region (0-2 mm). In contrast, glutamate dehydrogenase was concentrated in the mature basal region of the root (30-60 mm). A high level of nitrate (approximately 100 mm) was required to saturate the induction of nitrate reductase in the root tip, mature root, and scutellum. The concentration of nitrate required to give half the maximum level of enzyme induced was the same for each region (29 mm).After leaf expansion, more than 90% of the nitrate reductase was in the shoot, mainly in the leaf blade, and a marked decrease occurred in the level of the enzyme in the scutellum. A large proportion of the glutamate dehydrogenase was still found in the root.
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PMID:The distribution and characteristics of nitrate reductase and glutamate dehydrogenase in the maize seedling. 1665 30

Preliminary work revealed that nitrate reductase in crude extracts prepared from leaves of certain corn genotypes as well as soybeans could utilize NADPH as well as NADH as the electron donor. Isoelectric focusing and diethylaminoethyl cellulose chromatography confirmed previous findings that NADH and NADPH activities could not be separated, which suggests the involvement of a single enzyme. Nitrate reduction with both cofactors varies with plant species, plant age, and assay conditions. The ability of the nitrate reductase from a given genotype to utilize NADPH was associated with the amount of NADPH-phosphatase in the extract. While diethylaminoethyl cellulose chromatography of plant extracts separated nitrate reductase from the bulk (90%) of the phosphatase and caused a decrease in the NADPH activity, the residual level of phosphatase was sufficient to account for the apparent NADPH nitrate reductase activity. Addition of KH(2)PO(4) and KF, inhibitors of NADPH-phosphatase activity in in vitro assays, caused a drastic reduction or abolishment of NADPH-mediated nitrate reductase activity but were without effect on NADH nitrate reductase activity. It is concluded that NADPH-nitrate reduction, in soybean and certain corn genotypes, is an artifact resulting from the conversion of NADPH to NADH by a phosphatase and that the enzyme in leaf tissue is NADH-dependent (E.C.1.6.6.1).
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PMID:Specificity for nicotinamide adenine dinucleotide by nitrate reductase from leaves. 1665 48


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