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

A strain of Pseudomonas putida that can express a nitrate reductase that is located in the periplasmic compartment was isolated from freshwater. The enzyme was active in vivo during arginine fermentation and at the onset of oxygen limitation in batch cultures. The activity of the enzyme increased the yield of bacteria following fermentative growth under anoxic conditions with arginine, but nitrate reduction did not support growth on non-fermentable carbon substrates under anoxic conditions. Cells expressing the periplasmic nitrate reductase were capable of reducing nitrate in the presence of oxygen. Nitrate reduction under oxic conditions was clearly coupled to a respiratory electron transport chain because: (1) the process was sensitive to the respiratory inhibitors rote-none and 2-n-heptyl-4-hydroxyquinoline N-oxide, and (2) membrane-bound and periplasmic cytochromes were involved. This is the first report of the presence of a periplasmic nitrate reductase in a member of the gamma proteobacteria.
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PMID:Isolation and characterisation of a strain of Pseudomonas putida that can express a periplasmic nitrate reductase. 777 73

The structure of NADH-cytochrome b5 reductase from pig liver microsomes has been refined to a crystallographic R factor of 0.223 at 2.4 A resolution. A structural comparison between the flavin-binding beta barrel domain of NADH-cytochrome b5 reductase and those of the other flavin-dependent reductases, ferredoxin-NADP+ reductase, phthalate dioxygenase reductase and nitrate reductase, indicated that the overall barrel foldings are similar to each other and that the specific arrangement of three amino acid residues (Arg, Tyr and Ser/Thr) is usually necessary for flavin-binding. These conserved residues overlap each other in their three-dimensional structures and stabilize the flavin-binding site in the four flavin-dependent reductases.
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PMID:Specific arrangement of three amino acid residues for flavin-binding barrel structures in NADH-cytochrome b5 reductase and the other flavin-dependent reductases. 789 48

Nitric oxide (NO) is known to be synthesized by mammalian cells from L-arginine by a group of NO synthase enzymes. We now show that NO is generated from human skin and propose a different mechanism of production. Whereas enzymatic NO synthesis is inhibited by monomethyl L-arginine, this arginine analog, when infused into the brachial artery at concentrations sufficient to inhibit endothelial NO synthase activity, has little effect on hand skin NO production. Hand skin NO production is increased by topical acidification of the skin surface and greatly increased by the addition of nitrite solutions. We propose that NO generation from skin derives from sweat nitrite (the concentration of which was found to average 3.4 microM in six subjects) due to chemical reduction consequent to the acidic nature of sweat. Sweat contains nitrate in appreciable amounts, and skin commensal bacteria can synthesize nitrate reductase enzyme. Patients on long-term tetracycline antibiotics showed significantly reduced skin NO synthesis, although topical antiseptic and antibiotics had little effect on NO generation in the short-term. We propose that NO generation from skin is dependent on bacterial nitrate reduction to nitrite and subsequent reduction by acidification. We speculate that this has a physiologic role in the inhibition of infection by pathogenic fungi and other susceptible microorganisms and may affect cutaneous T-cell function, keratinocyte differentiation, and skin blood flow.
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PMID:Nitric oxide is generated on the skin surface by reduction of sweat nitrate. 875 65

This work presents the results on two different approaches of RNA-mediated transformation in A. nidulans: a) the receptor strain was an argB2 (III) mutant deficient in arginine (OTCase deficient), and b) the receptor was an A. nidulans mutant defective in nitrate reductase synthesis due to a deletion in the niaD gene (VIII). The analyses of the arg+ and the nia+ retrotransformants allowed an insight on the fate and inheritance of the newly acquired characteristics. The occurrence and the study of Gene Inactivation Mechanism (RIP-like) inactivating the expression of extra copies of genes ectopically scattered over the receptor genome, was a byproduct of this research. Retrotransformants were also used as RNA-donor for a second turn of retrotransformation of the argB and niaD receptor strains. Genetic analyses of the new retrotransformants proved that the retrotransformation ability is kept by the re-extracted RNA when used in a second round of transformation process. This is the best genetic evidence that the newly acquired genetic characteristics were cDNA inserted, precisely transcribed and expressed. These are the first in vivo evidences of genetic information transference mediated by homologous RNA in lower eukaryotes.
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PMID:RNA-mediated transformation in Aspergillus nidulans recovers gene functions lost by deletion or by point mutations. 889 57

We studied the effects of administrating the nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), or the nitric oxide precursor, L-arginine, on hemodynamic variables and serum nitrate concentrations in an anesthetized ovine model of endotoxemia to assess the effects on regional visceral blood flow and to determine whether L-arginine availability limits nitric oxide production. Animals received Escherichia coli endotoxin (2 micrograms/kg) followed 2 h later by L-NAME (25 mg/kg), L-arginine (0.575 g/kg), or saline administered over 1 h followed by an infusion of the same dose over 8 h (n = 6 per group). Renal and mesenteric blood flow were measured by placement of electromagnetic flow probes, and serum nitrate concentrations were determined using vanadium III chloride or nitrate reductase reduction to nitric oxide or nitrite, respectively. The results showed L-NAME significantly increased systemic vascular resistance (P < 0.01), decreased serum nitrate concentrations (P < 0.05), and caused a transient reduction in mesenteric blood flow (P < 0.05). L-Arginine caused a reduction in systemic vascular resistance (P < 0.01), increased mesenteric blood flow (P < 0.001) and conductance (P < 0.05). There were no significant changes in renal arterial blood flow in either group. We conclude that the availability of L-arginine limits nitric oxide production in endotoxemia and, furthermore, that L-arginine administration in this model causes significant mesenteric vasodilatation. L-NAME administration had only limited effect on visceral blood flow despite a marked increase in systemic vascular resistance and a reduction in nitric oxide production.
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PMID:L-arginine augments nitric oxide production and mesenteric blood flow in ovine endotoxemia. 889 20

1. Within vessels, the formation of nitric oxide (NO) or prostaglandins is normally catalysed in the endothelium by constitutive isoforms of NO synthase (eNOS) and cyclo-oxygenase (COX-1), respectively. However, during inflammatory conditions, the underlying smooth muscle acquires the ability to release NO and prostaglandins after the expression of inducible isoforms of NOS (iNOS) and COX (COX-2). The co-induction of iNOS and COX-2 has been studied over 24 h in isolated vascular smooth muscle cells in vitro. However, due to the limitation of using cultured cells, the relationship between the activities of iNOS and COX over longer periods has not been addressed. Moreover, the relative contribution of the endothelium to the production of NO and prostaglandins under inflammatory conditions is not completely understood. 2. Here using an organ culture system, we have determined the profile of COX (6-keto prostaglandin F1 alpha (6-keto PGF1 alpha), PGE2, thromboxane B2 (TXB2) and NOS (nitrite and nitrate) metabolites released over a period of 10 days from segments of rat aorta. In each case, segments from the same animal were left untreated or treated with bacterial lipopolysaccharide (LPS; 10 micrograms ml-1) in order to induce iNOS and COX-2. Prostaglandins were measured by radioimmunoassay whilst nitrite and nitrate were measured, respectively, by Greiss reaction alone, or following a nitrate reductase step. The isoforms of NOS and COX responsible for metabolite release were characterized pharmacologically by use of inhibitors and at the molecular level by reverse transcription polymerase chain reaction with specific primers for iNOS, eNOS, COX-1 and COX-2. In separate experiments the role of the endothelium in the release of nitrite, nitrate and prostaglandins and in the expression of iNOS, eNOS, COX-1 and COX-2 was determined by comparing responses in endothelium denuded and endothelium-intact segments of rat aorta. 3. Under control culture conditions vessels released prostaglandins in the following rank order 6-keto PGF1 alpha = PGE2 > > TXB2. LPS increased the release of 6-keto PGF1 alpha and PGE2 but not of TXB2, an effect that was inhibited by the protein synthesis inhibitor cycloheximide (1 microM), the anti-inflammatory steroid dexamethason (1 microM), the nonsteroidal anti-inflammatory drug indomethacin (30 microM) and, where tested, the selective COX-2 inhibitor NS-398 (30 microM). Similarly, segments of rat aorta released detectable levels of nitrite and nitrate, which were reduced by NG-nitro-L-arginine methyl ester (L-NAME, 1 mM), which inhibits all isoforms of NOS, and by dexamethasone (1 microM), which inhibits the induction of iNOS. The proportion of nitrate to nitrite released over the 10 day period varied greatly from approximately 1:1 on days 5 to 8 to 5:1 on day 9. However, the sum of nitrite and nitrate (NOx) as well as PGE2 remained elevated over the whole 10 day period. The formation of 6-keto PGF1 alpha peaked on days 1 and 2. 4. In freshly prepared tissue, mRNAs for eNOS, COX-1, iNOS and COX-2 were detected. After 24 h in culture, there was an apparent increase in the level of mRNAs for iNOS and COX-2 but not for eNOS or COX-1, an effect that was further enhanced when LPS was included in the culture medium. The expressions of mRNA for eNOS, COX-1, iNOS or COX-2 were not greatly different in vessels with intact or disrupted endothelium. Similarly the release of NOx or PGE2 by vessels after the 1st or 9th day in culture were not significantly different from vessels prepared with or without endothelium. 5. Thus, COX-2 and iNOS are co-induced in intact vessels in culture, with the vascular smooth muscle being the main site of mediator generation. In contrast to data from isolated cells in culture (observed usually over 1 day), both COX and NOS activities in cultured blood vessels were elevated for at least 10 days. Also, unlike isolated cells in culture, the COX and NOS pathways were active independently; L-NAME had little effect on the activity of COX and indomethacin had little effect on the activity of NOS.
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PMID:Characterization of the induction of nitric oxide synthase and cyclo-oxygenase in rat aorta in organ culture. 914 96

Optical spectroscopy and EPR studies confirm the existence of two b-type hemes in the NarI subunit (cytochrome bnr) of the membrane-bound nitrate reductase (NarGHI) of Escherichia coli. Replacement of His-56 by Arg and His-66 by Tyr results in the loss of the high-potential heme and of the low-potential heme, respectively. These data support the assignment of the axial ligands to the low-potential heme (His-66 and His-187) and to the high-potential heme (His-56 and His-205). This pairing is consistent with the model proposed for NarI of the nitrate reductase of Thiosphaera pantotropha (Berks, B. C., Page, M. D., Richardson, D. J. , Reilly, A., Cavill, A., Outen, F., and Ferguson, S. J. (1995) Mol. Microbiol. 15, 319-331) in which the two bis-histidine ligated hemes are coordinated by conserved His residues of helix II and V. EPR and optical studies suggest that the low-potential heme (Em,7 = +17 mV) and the high-potential heme (Em,7 = +122 mV) are located near the periplasmic side and the cytoplasmic side of the membrane, respectively. Moreover, correct insertion of both hemes into NarI requires anchoring to NarGH.
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PMID:Heme axial ligation by the highly conserved His residues in helix II of cytochrome b (NarI) of Escherichia coli nitrate reductase A. 932 88

We report the identification of the proteins encoded by the mttABC operon (formerly yigTUW), which mediate a novel Sec-independent membrane targeting and translocation system in Escherichia coli that interacts with cofactor-containing redox proteins having a S/TRRXFLK "twin arginine" leader motif. A pleiotropic-negative mutant in mttA prevents the periplasmic localization of twin arginine redox enzymes, including nitrate reductase (NapA) and trimethylamine N-oxide reductase (TorA). The mutation also prevents the correct localization of the integral membrane molybdoenzyme dimethylsulfoxide reductase (DmsABC). The DmsA subunit has a twin arginine leader. Proteins with a Sec-dependent leader or which assemble spontaneously in the membrane are not affected by this mutation. MttA, B, and C are members of a large family of related sequences extending from archaebacteria to higher eukaryotes.
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PMID:A novel and ubiquitous system for membrane targeting and secretion of cofactor-containing proteins. 954 95

The periplasmic nitrate reductase, NapA, from Escherichia coli was identified as a 90 kDa molybdoprotein which comigrated during polyacrylamide gel electrophoresis with the di-haem c-type cytochrome, NapB. The DNA sequence of the 5' end of the napA gene and the N-terminal amino acid sequences of both NapA and NapB were determined. The 36 residue leader peptide for NapA includes the double-arginine motif typical of proteins to which complex redox cofactors are attached in the cytoplasm prior to targeting to the periplasm. The pre-NapA leader sequence is both unexpectedly long and, unless two successive proteolysis steps are involved, is cleaved at the unprecedented sequence G-Q-Q-. Nap activity was suppressed during growth in the presence of tungstate and was absent from a mutant unable to synthesise the molybdopterin cofactor.
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PMID:The periplasmic nitrate reductase from Escherichia coli: a heterodimeric molybdoprotein with a double-arginine signal sequence and an unusual leader peptide cleavage site. 1023 35


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