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)

The increasing importance of nitric oxide synthase has been underscored by the elucidation of its role in a growing number of normal and pathophysiological processes. Therefore, techniques for detection of nitrite/nitrate, oxidation products of the enzymatic conversion of arginine to citrulline and nitric oxide, should serve as useful tools in defining the contribution of NO synthase to these processes. We have developed a rapid and sensitive fluorometric assay for quantification of nitrite/nitrate based upon the reaction of nitrite with 2,3-diaminonaphthalene to form the fluorescent product, 1-(H)-naphthotriazole. The assay can be used to detect 10 nM nitrite, making it 50-100 times more sensitive than the well-known Griess assay. Moreover, the assay is adaptable to a 96-well plate format, facilitating the handling of a large number of samples including conditioned media from cell culture or the nitrite generated by the purified enzyme. Nitrite/nitrate levels in blood can also be monitored using this assay when it is combined with a filtration step (to remove hemoglobin) followed by conversion of the nitrate to nitrite by nitrate reductase. Thus, this fluorometric method combines speed and sensitivity with the handling of a large number of samples for the quantification of nitrite generated from in vivo and in vitro sources.
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PMID:A fluorometric assay for the measurement of nitrite in biological samples. 750 9

In the strictly aerobic, gram-negative bacterium Vitreoscilla strain C1, oxygen-limited growth conditions create a more than 50-fold increase in the expression of a homodimeric heme protein which was recognized as the first bacterial hemoglobin (Hb). The recently determined crystal structure of Vitreoscilla Hb has indicated that the heme pocket of microbial globins differs from that of eukaryotic Hbs. In an attempt to understand the diverse functions of Hb-like proteins in prokaryotes, we have cloned and characterized the gene (vgb) encoding an Hb-like protein from another strain of Vitreoscilla, V. stercoraria DW. Several silent changes were observed within the coding region of the V. stercoraria vgb gene. Apart from that, V. stercoraria Hb exhibited interesting differences between the A and E helices. Compared to its Hb counterpart from Vitreoscilla strain C1, the purified preparation of V. stercoraria Hb displays a slower autooxidation rate. The differences between Vitreoscilla Hb and V. stercoraria Hb were mapped onto the three-dimensional structure of Vitreoscilla Hb, which indicated that the four changes, namely, Ile7Val, Ile9Thr, Ile10Ser, and Leu62Val, present within the V. stercoraria Hb fall in the region where the A and E helices contact each other. Therefore, alteration in the relative orientation of the A and E helices and the corresponding conformational change in the heme binding pocket of V. stercoraria Hb can be correlated to its slower autooxidation rate. In sharp contrast to the oxygen-regulated biosynthesis of Hb in Vitreoscilla strain C1, production of Hb in V. stercoraria has been found to be low and independent of oxygen control, which is supported by the absence of a fumarate and nitrate reductase regulator box within the V. stercoraria vgb promoter region. Thus, the regulation mechanisms of the Hb-encoding gene appear to be quite different in the two closely related species of Vitreoscilla. The relatively slower autooxidation rate of V. stercoraria Hb, lack of oxygen sensitivity, and constitutive production of Hb suggest that it may have some other function(s) in the cellular physiology of V. stercoraria DW, together with facilitated oxygen transport, predicted for earlier reported Vitreoscilla Hb.
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PMID:Hemoglobin biosynthesis in Vitreoscilla stercoraria DW: cloning, expression, and characterization of a new homolog of a bacterial globin gene. 960 38

Microarray and RNA gel blot analyses were performed to identify Arabidopsis genes that responded to nitrate at both low (250 microM) and high (5 to 10 mM) nitrate concentrations. Genes involved directly or indirectly with nitrite reduction were the most highly induced by nitrate. Most of the known nitrate-regulated genes (including those encoding nitrate reductase, the nitrate transporter NRT1, and glutamate synthase) appeared in the 40 most strongly nitrate-induced genes/clones on at least one of the microarrays of the 5524 genes/clones investigated. Novel nitrate-induced genes were also found, including those encoding (1) possible regulatory proteins, including an MYB transcription factor, a calcium antiporter, and putative protein kinases; (2) metabolic enzymes, including transaldolase and transketolase of the nonoxidative pentose pathway, malate dehydrogenase, asparagine synthetase, and histidine decarboxylase; and (3) proteins with unknown functions, including nonsymbiotic hemoglobin, a senescence-associated protein, and two methyltransferases. The primary pattern of induction observed for many of these genes was a transient increase in mRNA at low nitrate concentrations and a sustained increase when treated with high nitrate concentrations. Other patterns of induction observed included transient inductions after both low and high nitrate treatments and sustained or increasing amounts of mRNA after either treatment. Two genes, AMT1;1 encoding an ammonium transporter and ANR1 encoding a MADS-box factor, were repressed by nitrate. These findings indicate that nitrate induces not just one but many diverse responses at the mRNA level in Arabidopsis.
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PMID:Genomic analysis of a nutrient response in Arabidopsis reveals diverse expression patterns and novel metabolic and potential regulatory genes induced by nitrate. 1094 65

The transcription factor NNR from Paracoccus denitrificans was expressed in a strain of Escherichia coli carrying a plasmid-borne fusion of the melR promoter to lacZ, with a consensus FNR-binding site 41.5 bp upstream of the transcription start site. This promoter was activated by NNR under anaerobic growth conditions in media containing nitrate, nitrite, or the NO(+) donor sodium nitroprusside. Activation by nitrate was abolished by a mutation in the molybdenum cofactor biosynthesis pathway, indicating a requirement for nitrate reductase activity. Activation by nitrate was modulated by the inclusion of reduced hemoglobin in culture media, because of the ability of hemoglobin to sequester nitric oxide and nitrite. The ability of nitrate and nitrite to activate NNR is likely due to the formation of NO (or related species) during nitrate and nitrite respiration. Amino acids potentially involved in NNR activity were replaced by site-directed mutagenesis, and the activities of NNR derivatives were tested in the E. coli reporter system. Substitutions at Cys-103 and Tyr-35 significantly reduced NNR activity but did not abolish the response to reactive nitrogen species. Substitutions at Phe-82 and Tyr-93 severely impaired NNR activity, but the altered proteins retained the ability to repress an FNR-repressible promoter, so these mutations have a "positive control" phenotype. It is suggested that Phe-82 and Tyr-93 identify an activating region of NNR that is involved in an interaction with RNA polymerase. Replacement of Ser-96 with alanine abolished NNR activity, and the protein was undetectable in cell extracts. In contrast, NNR in which Ser-96 was replaced with threonine retained full activity.
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PMID:Heterologous NNR-mediated nitric oxide signaling in Escherichia coli. 1105 88

Plant nitrate reductase (NR) produces nitric oxide (NO) when nitrite is provided as the substrate in the presence of NADH [H. Yamasaki and Y. Sakihama (2000) FEBS Lett. 468, 89-92]. Using a NR-dependent NO producing system, we investigated the effects of NO on the energy transduction system in plant mitochondria isolated from mung bean (Vigna radiata). Plant mitochondria are known to possess two respiratory electron transport pathways-the cytochrome and alternative pathways. When the alternative pathway was inhibited by n-propyl gallate, the addition of NR strongly suppressed respiratory O(2) consumption driven by the cytochrome pathway. In contrast, the alternative pathway measured in the presence of antimycin A was not affected by NO. The extent of the steady-state membrane potential (Deltapsi) generated by respiratory electron transport rapidly declined in response to NO production. The addition of bovine hemoglobin, a quencher of NO, resulted in the recovery of Deltapsi to the uninhibited level. Consistent with its inhibition of Deltapsi, NO produced by NR strongly suppressed ATP synthesis in the mitochondria. These results provide substantial evidence to confirm that the plant alternative pathway is resistant to NO and support the idea that the alternative pathway may lower respiration-dependent production of active oxygens under conditions where NO is overproduced.
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PMID:Inhibitory effects of nitric oxide on oxidative phosphorylation in plant mitochondria. 1138 99

Biological activity of nitric oxide (NO) production was investigated in the unicellular green alga Chlamydomonas reinhardtii. An NO specific electrode detected a rapid increase in signal when nitrite (NO(2)(-)) was added into a suspension of C. reinhardtii intact cells in the dark. The addition of KCN or the NO quencher bovine hemoglobin completely abolished the signal, verifying that the nitrite-dependent increase in signal is due to enzymatic NO production. L-arginine, the substrate for NO synthase, did not induce detectable NO production and the NOS inhibitor N(omega)-nitro-L-arginine showed no inhibitory effect on the nitrite-dependent production of NO. Illuminating cells showed a significant suppressive effect on NO production. When the photosynthetic electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea was present in the suspension, C. reinhardtii cells produced NO after the addition of nitrite even under illumination. Kinetic and microscopic observations, using the intracellular fluorescent NO probe 4,5-diaminofluorescein-2 diacetate, both demonstrated that NO was produced within the cells in response to the addition of nitrite. The Chlamydomonas mutant cc-2929, which lacks nitrate reductase (NR) activity, did not display any of the responses observed in the wild-type cells. The results presented here provide direct in vivo evidence to confirm that NR is involved in the nitrite-dependent NO production in the green alga.
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PMID:Nitric oxide production mediated by nitrate reductase in the green alga Chlamydomonas reinhardtii: an alternative NO production pathway in photosynthetic organisms. 1191 83

Nitric oxide (NO) is a reactive gas involved in many biological processes of animals, plants and microbes. Previous work has demonstrated that NO is formed during hypoxia in alfalfa ( Medicago sativa L.) root cultures and that the levels of NO detected are inversely related to the levels of expression of class-1 hemoglobin expressed in the tissue. The objectives of this study were: to examine whether NO is produced in transgenic maize ( Zea mays L.) cell-suspension cultures exposed to anoxic growth conditions; to determine whether a similar relationship existed between a class-1 hemoglobin and the amount of NO detected under these conditions; and, to estimate the route of formation and breakdown of NO in the tissue. Maize cell-suspension cultures, transformed to express the sense or antisense strands of barley hemoglobin were used to overexpress or underexpress class-1 hemoglobin. A maize cell-suspension culture transformed with an empty vector was used as a control. Up to 500 nmol NO (g FW)(-1) was detected in maize cells exposed to low oxygen tensions for 24 h. The steady-state levels of NO in the different cell lines under anoxic conditions had an inverse relationship to the level of hemoglobin in the cells. There was no detectable NO produced under aerobic growth conditions. Spectroscopic data demonstrated that recombinant maize hemoglobin reacted with NO to form methemoglobin and NO(3)(-). Nitrate was shown to be a precursor of NO in anoxic maize cell-suspension cultures by using (15)NO(3)(-) and electron paramagnetic resonance spectroscopy, suggesting that NO is formed via nitrate reductase during hypoxia. The results demonstrate that NO is produced in plant tissues grown under low oxygen tensions and suggest that class-1 hemoglobins have a significant function in regulating NO levels.
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PMID:Class-1 hemoglobins, nitrate and NO levels in anoxic maize cell-suspension cultures. 1496 9

A nitrate reductase (EC 1.6.6.1)-inactivating factor has been isolated from 8-day-old wheat leaves. The purification schedule involved ammonium sulfate precipitation, Sephadex G-100 filtration, DEAE-cellulose chromatography, and Sephadex G-150 filtration. No accurate assessment could be made as to the degree of purification relative to crude extract as the inactivating factor could not be detected in crude extract. However a 2,446-fold purification was achieved from the ammonium sulfate fraction to the pooled enzyme from the Sephadex G-150 step.The inactivating factor was heat-labile and had a molecular weight of 37,500. The inactivating factor was particularly sensitive to the divalent metal chelators, 1,10-phenanthroline and bathophenanthroline. Evidence indicated that Fe(2+) may be the functional metal. The trypsin inhibitors N-alpha-p-tosyl-l-lysine chloromethyl ketone and alpha-N-benzoyl-l-arginine were inhibitory. However, phenylmethyl sulfonyl fluoride, an inhibitor of serine peptide hydrolases, was not inhibitory. Neither casein nor hemoglobin nor a range of artificial substrates were hydrolyzed by the inactivating factor. Highly purified wheat leaf nitrite reductase (EC 1.7.99.3) and ribulose 1,5-bisphosphate carboxylase:oxygenase (EC 4.1.1.39) were not affected by the nitrate reductase-inactivating factor.The inactivating factor was more active toward the NADH-nitrate reductase compared to either of the component enzymic activities flavin adenine mononucleotide-nitrate reductase and methyl viologen-nitrate reductase. The NADH-ferricyanide reductase (diaphorase) component was the least sensitive.
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PMID:In Vitro Stability of Nitrate Reductase from Wheat Leaves: III. Isolation and Partial Characterization of a Nitrate Reductase-inactivating Factor. 1666 Oct 24

Nitric oxide (NO) has diverse biological functions. Numerous studies have documented NO's biosynthetic pathway in a wide variety of organisms. Little is known, however, about NO production in intraerythrocytic Plasmodium falciparum. Using diaminorhodamine-4-methyl acetoxymethylester (DAR-4M AM), a fluorescent indicator, we obtained direct evidence of NO and NO-derived reactive nitrogen species (RNS) production in intraerythrocytic P. falciparum parasites, as well as in isolated food vacuoles from trophozoite stage parasites. We preliminarily identified two gene sequences that might be implicated in NO synthesis in intraerythrocytic P. falciparum. We showed localization of the protein product of one of these two genes, a molecule that is structurally similar to a plant nitrate reductase, in trophozoite food vacuole membranes. We confirmed previous reports on the antiproliferative effect of NOS (nitric oxide synthase) inhibitors in P. falciparum cultures; however, we did not obtain evidence that NOS inhibitors had the ability to inhibit RNS production or that there is an active NOS in mature forms of the parasite. We concluded that a nitrate reductase activity produce NO and NO-derived RNS in or around the food vacuole in P. falciparum parasites. The food vacuole is a critical parasitic compartment involved in hemoglobin degradation, heme detoxification and a target for antimalarial drug action. Characterization of this relatively unexplored synthetic activity could provide important clues into poorly understood metabolic processes of the malaria parasite.
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PMID:Plasmodium falciparum: food vacuole localization of nitric oxide-derived species in intraerythrocytic stages of the malaria parasite. 1850 40

At sufficiently low oxygen concentrations, hemeproteins are deoxygenated and become capable of reducing nitrite to nitric oxide (NO), in a reversal of the reaction in which NO is converted to nitrate or nitrite by oxygenated hemeproteins. The maximum rates of NO production depend on the oxygen avidity. The hemeproteins with the highest avidity, such as hexacoordinate hemoglobins, retain oxygen even under anoxic conditions resulting in their being extremely effective NO scavengers but essentially incapable of producing NO. Deoxyhemeprotein-related NO production can be observed in mitochondria (at the levels of cytochrome c oxidase, cytochrome c, complex III and possibly other sites), in plasma membrane, cytosol, endoplasmic reticulum and peroxisomes. In mitochondria, the use of nitrite as an alternative electron acceptor can contribute to a limited rate of ATP synthesis. Non-heme metal-containing proteins such as nitrate reductase and xanthine oxidase can also be involved in NO production. This will result in a strong anoxic redox flux of nitrogen through the hemoglobin-NO cycle involving nitrate reductase, nitrite: NO reductase, and NO dioxygenase. In normoxic conditions, NO is produced in very low quantities, mainly for signaling purposes and this nitrogen cycling is inoperative.
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PMID:Anoxic nitric oxide cycling in plants: participating reactions and possible mechanisms. 1992 98


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