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)

Flavodoxins synthesized by Azotobacter vinelandii strain UW 36 during growth on nitrate as nitrogen source were separated by FPLC on a Mono Q column into two species, flavodoxin 1 (AvFld 1) and flavodoxin 2 (AvFld 2). Both proteins migrated as single bands on SDS/PAGE. AvFld 1 was approx. 5-fold more abundant than AvFld 2 in the unresolved flavodoxin mixture. N-terminal amino acid analysis showed the sequence of AvFld 2 to correspond to the nif F gene product, an electron donor to nitrogenase. The sequences also show that these species corresponded to the flavodoxins Fld A and Fld B isolated from N2-grown cultures of the closely related organism Azotobacter throococcum [Bagby, Barker, Hill, Eady and Thorneley (1991) Biochem.J.277, 313-319]. Electrospray mass spectrometry gave M, values for the polypeptides of 19430 +/- 3 and 19533 +/- 5 respectively. 31P-NMR measurements showed that in addition to the phosphate associated with the FMN (delta = -136.3 p.p.m. and -135.48 p.p.m.), AvFld 1 had a signal at delta = -142.1 p.p.m. and AvFld 2 at delta = -138.59 p.p.m. present in substoichiometric amounts with FMN. These appeared to arise from unstable species since they were readily lost on further manipulation of the proteins. The mid-point potentials of the semiquinone hydroquinone redox couples were -330 mV and -493 mV for AvFld 1 and AvFld 2 respectively, but only AvFld 1 was competent in donating electrons to the purified assimilatory nitrate reductase of A. vinelandii to catalyse the reduction of nitrate to nitrite. Flavodoxin isolated from NH4(+)-grown cells (Fld 3) also functioned as electron donor at half the rate of AvFld 1, but ferredoxin 1 from A. chroococcum did not.
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PMID:Flavodoxin 1 of Azotobacter vinelandii: characterization and role in electron donation to purified assimilatory nitrate reductase. 869 50

The distribution of the Mo-enzymes aldehyde oxidase (AO; EC 1.2.3.1) xanthine dehydrogenase (XDH; EC 1.2.1.37) and nitrate reductase (NAD(P)H NR; EC 1.6.6.1-2) was studied along the longitudinal and transversal axes of maize (Zea mays L. cv. Jubily) nodal roots as affected by nitrogen sources and salinity. Activities of the Mo-enzymes were considerably enhanced under mild saline conditions. The activities of AO and XDH increased following addition of ammonium to the nutrient solution. Immunoblot analysis with antibodies raised against maize AO protein revealed increased levels of AO proteins in root tips of ammonium fed plants. Application of salinity to nitrate fed plants did not affect the enzyme protein level, although it enhanced the activity of the Mo-hydroxylases. The specific activities of the Mo-enzymes were the highest in root tips (0-1 cm segments) while on the transversal axis maximal activity was observed in the stele or vascular cylinder. Activity staining of AO after native PAGE of root extracts revealed four bands of AO proteins (AO1-4) capable of oxidizing a number of aliphatic and aromatic aldehydes. Increased AO activity in maize nodal roots grown with ammonium, and salinity were observed mainly at the AO3 and AO4 bands. Tips and stele contained primarily AO3 and AO4, and only traces of AO1 and AO2. SDS-PAGE of root extracts followed by Western blots revealed, besides the major 150 kD subunit of AO, two polypeptides with molecular masses of 72 and 85 kD located specifically in the cortex. Part of the polymorphism of AO in plant roots may be related to the allocation of distinct isoforms to different regions of the root, although the specific metabolic roles of the different bands have not been established.
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PMID:Distribution of the Mo-enzymes aldehyde oxidase, xanthine dehydrogenase and nitrate reductase in maize (Zea mays L.) nodal roots as affected by nitrogen and salinity. 1077 39

Assimilatory NADH:nitrate reductase (EC 1.6.6.1), a complex Mo-pterin-, cytochrome b(557)-, and FAD-containing protein, catalyzes the regulated and rate-limiting step in the utilization of inorganic nitrogen by higher plants. A codon-optimized gene has been synthesized for expression of the central cytochrome b(557)-containing fragment, corresponding to residues A542-E658, of spinach assimilatory nitrate reductase. While expression of the full-length synthetic gene in Escherichia coli did not result in significant heme domain production, expression of a Y647* truncated form resulted in substantial heme domain production as evidenced by the generation of "pink" cells. The histidine-tagged heme domain was purified to homogeneity using a combination of NTA-agarose and size-exclusion FPLC, resulting in a single protein band following SDS-PAGE analysis with a molecular mass of approximately 13 kDa. MALDI-TOF mass spectrometry yielded an m/z ratio of 12,435 and confirmed the presence of the heme prosthetic group (m/z=622) while cofactor analysis indicated a 1:1 heme to protein stoichiometry. The oxidized heme domain exhibited spectroscopic properties typical of a b-type cytochrome with a visible Soret maximum at 413 nm together with epr g-values of 2.98, 2.26, and 1.49, consistent with low-spin bis-histidyl coordination. Oxidation-reduction titrations of the heme domain indicated a standard midpoint potential (E(o)') of -118 mV. The isolated heme domain formed a 1:1 complex with cytochrome c with a K(A) of 7 microM (micro=0.007) and reconstituted NADH:cytochrome c reductase activity in the presence of a recombinant form of the spinach nitrate reductase flavin domain, yielding a k(cat) of 1.4 s(-1) and a K(m app) for cytochrome c of 9 microM. These results indicate the efficient expression of a recombinant form of the heme domain of spinach nitrate reductase that retained the spectroscopic and thermodynamic properties characteristic of the corresponding domain in the native spinach enzyme.
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PMID:Synthesis and bacterial expression of a gene encoding the heme domain of assimilatory nitrate reductase. 1205 81

A daily rhythm in the activity of nitrate reductase (NR: EC 1.6.6.1) isolated from the marine red algae Gracilaria tenuistipitata is shown to be attributable to changes in amounts of the protein. The enzyme was purified in four steps: ion exchange Q-Sepharose separation, ammonium sulfate precipitation, gel filtration on Sephacryl S-300, and affinity chromatography on Affigel-blue resin. This purification procedure yielded an active purified NR of about 500-fold with a recovery of 85%. The SDS-PAGE silver staining of purified NR revealed a 110 kDa single band. Non-denaturated protein showed a molecular mass of 440 kDa on gel filtration comparing with SDS-PAGE, the enzyme is apparently composed of four identical subunits. In extracts of algae grown under either constant dim light or a light-dark cycle, the activity of NR exhibited a daily rhythm, peaking at midday phase as does photosynthesis. Staining with monoclonal antibodies, raised against NR from Porphyra yezoensis, showed that the amount of protein changes by a factor of about 12, with a maximum occurring in the midday phase.
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PMID:Characterization and daily variation of nitrate reductase in Gracilaria tenuistipitata (Rhodophyta). 1208 65

The Haloferax mediterranei nar operon has been sequenced and its regulation has been characterized at transcriptional level. The nar operon encodes seven open reading frames(ORFs) (ORF1 narB, narC, ORF4, narG, narH, ORF7 and narJ). ORF1, ORF4 and ORF7 are open reading frames with no assigned function, however the rest of them encoded different proteins. narB codes for a 219-amino-acid-residue iron Rieske protein. narC encodes a protein of 486 amino acid residues identified by databases searches as cytochrome-b (narC). The narG gene encodes a protein with 983 amino acid residues and is identified as a respiratory nitrate reductase catalytic subunit (narG). NarH protein has been identified as an electron transfer respiratory nitrate reductase subunit (narH). The last ORF encodes a chaperonin-like protein (narJ) of 242 amino acid residues. The respiratory nitrate reductase was purified 21-fold from H. mediterranei membranes. Based on SDS-PAGE and gel-filtration chromatography under native conditions, the enzyme complex consists of two subunits of 112 and 61 kDa. The optimum temperature for activity was 70 degrees C at 3.4 M NaCl and the stability did not show a direct dependence on salt concentration. Respiratory nitrate reductase showed maximum activity at pH 7.9 and pH 8.2 when assays were carried out at 40 and 60 degrees C, respectively. The absorption spectrum indicated that Nar contains Fe-S clusters. Reverse transcriptase (RT-PCR) shows that regulation of nar genes occurs at transcriptional level induced by oxygen-limiting conditions and the presence of nitrate.
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PMID:Respiratory nitrate reductase from haloarchaeon Haloferax mediterranei: biochemical and genetic analysis. 1534 13

Assimilatory nitrate reductase has been purified with 55% recovery from a Neurospora crassa nmr-1 nit-6 mutant, using a modification of a published procedure. It possesses one heme per 240 000 g, and subunits of mol. wt. 68 000. Upon digestion with chymotrypsin, a heme-binding domain was isolated by gel filtration; its visible spectrum was highly similar to that of cytochrome b(5). On SDS gels, the fraction showed two heme-containing bands of 10 000 and 12 5000 daltons; their amino acid composition was not very different, suggesting that they originated from the same region of the polypeptide chain. After S-carboxymethylation, the mixture of bands was submitted to cyanogen bromide cleavage, and the fragments were separated by h.p.l.c. The two largest fragments yielded an identical sequence upon automated degradation. This sequence (39 residues with some gaps) could be easily aligned with that of cytochrome b(5) starting close to the N terminus. These results are discussed in terms of the possible quaternary structure of N. crassa nitrate reductase, whose heme-binding domain proves to be another member of the family of b(5)-like cytochromes.
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PMID:On the presence of a heme-binding domain homologous to cytochrome b(5) in Neurospora crassa assimilatory nitrate reductase. 1645 80

The binding of 14-3-3omega to phosphorylated nitrate reductase (pNR) is stimulated by cations such as Mg(2+) or spermine, and decreased by 5'-AMP. In order to determine whether binding to other cellular proteins is affected similarly, far-Western overlays of extracts prepared from light- or dark-treated spinach (Spinacia oleracea) leaves were performed using digoxigenin (DIG)-labeled Arabidopsis 14-3-3omega. When separated by SDS-PAGE, approximately 25 proteins of >35 kDa could be resolved that interacted with DIG-labeled 14-3-3omega in the absence of added cations. The presence of 5 mM Mg(2+) or 0.5 mM spermine enhanced binding to most of the target proteins to a maximum of about a doubling of the observed binding. In most cases, the binding was dependent on phosphorylation of the target protein, whereas that was not necessarily the case for binding to target proteins that were unaffected by polycations. The extent of stimulation varied among the target proteins, but there was no indication that the nature of the cation activator (e.g. Mg(2+) vs. spermine(4+)) altered the specificity for target proteins. In addition, binding of DIG-labeled 14-3-3omega to some, but not all, target proteins was reduced by 5 mM 5'-AMP. Interestingly, light/dark treatment of spinach leaves affected the subsequent binding of DIG-labeled 14-3-3omega in the overlay assay to only a few of the target proteins, one of which was identified as NADH:nitrate reductase. Overall, the results suggest that the binding of 14-3-3s to targets in addition to pNR may also be regulated by polycations and 5'-AMP.
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PMID:Polycations globally enhance binding of 14-3-3omega to target proteins in spinach leaves. 1662 45

Nitrate reductase was purified about 3,000-fold from spinach leaves by chromatography on butyl Toyopearl 650-M, hydroxyapatite-brushite, and blue Sepharose CL-6B columns. The purified enzyme yielded a single protein band upon polyacrylamide gel electrophoresis under nondenaturing conditions. This band also gave a positive stain for reduced methylviologen-nitrate reductase activity. The specific NADH-nitrate reductase activities of the purified preparations varied from 80 to 130 units per milligram protein. Sucrose density gradient centrifugation and gel filtration experiments gave a sedimentation coefficient of 10.5 S and a Stokes radius of 6.3 nanometers, respectively. From these values, a molecular weight of 270,000 +/- 40,000 was estimated for the native reductase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the denatured enzyme yielded a subunit band having a molecular weight of 114,000 together with a very faint band possessing a somewhat smaller molecular weight. It is concluded that spinach nitrate reductase is composed of two identical subunits possessing a molecular weight of 110,000 to 120,000.
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PMID:Spinach nitrate reductase: purification, molecular weight, and subunit composition. 1666 94

Corn seedlings (Zea mays cv W64A x W182E) were grown hydroponically, in the presence or absence of NO(3) (-), with or without light and with NH(4)Cl as the only N source. In agreement with earlier results nitrate reductase (NR) activity was found only in plants treated with both light and NO(3) (-). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transfer of the proteins to nitrocellulose paper and reaction with antibodies prepared against a pure NR showed that crude extracts prepared from light-grown plants had a polypeptide of approximately 116 kilodaltons (the subunit size for NR) when NO(3) (-) was present in the growth medium. Crude extracts from plants grown in the dark did not have the 116 kilodalton polypeptide, although smaller polypeptides, which reacted with NR-immunoglobulin G, were sometimes found at the gel front. When seedlings were grown on Kimpack paper or well washed sand, NR activity was again found only when the seedlings were exposed to light and NO(3) (-). Under these conditions, however, a protein of about 116 kilodaltons, which reacted with the NR antibody was present in light-grown plants whether NO(3) (-) was added to the system or not. The NR antibody cross-reacting protein was also seen in hydroponically grown plants when NH(4)Cl(-) was the only added form of nitrogen. These results indicate that the induction of an inactive NR-protein precursor in corn is mediated either by extremely low levels of NO(3) (-) or by some other unidentified factor, and that higher levels of NO(3) (-) are necessary for converting the inactive NR cross-reacting protein to a form of the enzyme capable of reducing NO(3) (-) to NO(2) (-).
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PMID:The role of nitrate and ammonium ions and light on the induction of nitrate reductase in maize leaves. 1666 23

Respiratory nitrate reductase (NR) from Bradyrhizobium sp. (Lupinus) USDA 3045 has biochemical properties of the membrane-bound NR type. However, in the completely sequenced rhizobium genomes only genes for the periplasmic type of dissimilatory NR were found. Therefore purification and identification of the enzyme by tandem mass spectrometry (MS/MS) was undertaken. MS/MS spectra representing 149 unique tryptic peptides derived from purified 137-kDa subunit matched the NCBInr-deposited NarG sequences. MS/MS sequencing of two other SDS/PAGE bands (65- and 59-kDa) identified them as derivatives of the NarH-type protein. Applying additional validation criteria, 73% of the sequence of the NarG subunit (902 aa) and 52% of NarH sequence (266 aa) was assembled (UniProt KB acc. no. P85097 and P85098). This is the first unambiguous identification of an active NarGH-like NR in rhizobia. Moreover, arguments are provided here for the existence of a functional enzyme of this type also among other rhizobial species, basing on immunoblot screening and the presence of membrane-associated NR-active electrophoretic forms.
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PMID:Mass spectrometry identification of membrane-bound respiratory nitrate reductase from Bradyrhizobium sp. (Lupinus). 1901 74


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