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Query: EC:1.7.1.1 (nitrate reductase)
 3,728 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The influence of photosynthetic electron flow in chloroplasts on the expression and enzyme activity of the cytosolic nitrate reductase (NR) was studied. Using light sources that predominantly excite either photosystem I (PSI) or photosystem II (PSII), we modulated photosynthetic electron transport in tobacco, Arabidopsis, and Lemna sprouts. In all instances, oxidation of components of photosynthetic electron flow by PSI light correlated with an increase in NR activity and/or transcription. This is confirmed by experiments with electron transport inhibitors 3-(3',4'-dichlorophenyl)-1,1'-dimethyl urea and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone. In addition, a Lemna mutant deficient in the cytochrome b(6)/f complex failed to respond to the different light sources and exhibited a constitutively high level of NR activity. These data indicate that NR is activated by the oxidized state of an electron transport component located after the plastoquinone pool. An involvement of the cytoplasmic photoreceptor phytochrome A in this light regulation could be excluded, since an Arabidopsis phytochrome A mutant exhibited a wild-type like response. The observation that NR activity in the cytoplasm and the expression of its gene in the nucleus is controlled by signals from photosynthetic electron flow adds a new facet to the intracellular cross-talk between chloroplasts and the nucleus.
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PMID:Photosynthetic electron transport determines nitrate reductase gene expression and activity in higher plants. 1224 40

Erythrocyte NADH-cytochrome b(5) reductase reduces methaemoglobin to functional haemoglobin. In order to examine the function of the enzyme, the structure of NADH-cytochrome b(5) reductase from human erythrocytes has been determined and refined by X-ray crystallography. At 1.75 A resolution, the root-mean-square deviations (r.m.s.d.) from standard bond lengths and angles are 0.006 A and 1.03 degrees , respectively. The molecular structure was compared with those of rat NADH-cytochrome b(5) reductase and corn nitrate reductase. The human reductase resembles the rat reductase in overall structure as well as in many side chains. Nevertheless, there is a large main-chain shift from the human reductase to the rat reductase or the corn reductase caused by a single-residue replacement from proline to threonine. A model of the complex between cytochrome b(5) and the human reductase has been built and compared with that of the haem-containing domain of the nitrate reductase molecule. The interaction between cytochrome b(5) and the human reductase differs from that of the nitrate reductase because of differences in the amino-acid sequences. The structures around 15 mutation sites of the human reductase have been examined for the influence of residue substitutions using the program ROTAMER. Five mutations in the FAD-binding domain seem to be related to cytochrome b(5).
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PMID:Structure of human erythrocyte NADH-cytochrome b5 reductase. 1550 98

Rate-limiting processes of catalysis by eukaryotic molybdenum-containing nitrate reductase (NaR, EC 1.7.1.1-3) were investigated using two viscosogens (glycerol and sucrose) and observing their impact on NAD(P)H:NaR activity of corn leaf NaR and recombinant Arabidopsis and yeast NaR. Holo-NaR has two "hinge" sequences between stably folded regions housing its internal electron carriers: 1) Hinge 1 between the molybdenum-containing nitrate reducing module and cytochrome b domain containing heme and 2) Hinge 2 between cytochrome b and cytochrome b reductase (CbR) module containing FAD. Solution viscosity negatively impacted the activity of these holo-NaR forms, which suggests that the rate-limiting events in catalysis were likely to involve large conformational changes that restrict or "gate" internal electron-proton transfers (IET). Little effect of viscosity was observed on recombinant CbR module and methyl viologen nitrate reduction by holo-NaR, suggesting that these activities involved no large conformational changes. To determine whether Hinge 2 is involved in gating the first step in IET, the effects of viscosogen on cytochrome c and ferricyanide reductase activities of holo-NaR and ferricyanide reductase activity of the recombinant molybdenum reductase module (CbR, Hinge 2, and cytochrome b) were analyzed. Solution viscosity negatively impacted these partial activities, as if Hinge 2 were involved in gating IET in both enzyme forms. We concluded that both Hinges 1 and 2 appear to be involved in gating IET steps by restricting the movement of the cytochrome b domain relative to the larger nitrate-reducing and electron-donating modules of NaR.
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PMID:Viscosity effects on eukaryotic nitrate reductase activity. 1589 95

Excessive nitrate accumulated in plants affects vegetable quality severely and excessive nitrate ingestion would do harm to human health. Assimilatory NADH: nitrate reductase (NR, 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. Enhancing the activity of NR is conducive to reduce the concentration of nitrate in plants. The experiments were conducted to investigate the activity of nitrate reductase in different plant tissues and the relationship between external inducing solution concentration and NR activity (NRA) in plant leaves. Six plant seedlings growing in solution culture were deprived of an external nitrogen (N) supply for 2 weeks. On selected days, three of six plant seedlings were exposed to 50mmol/L NO3- for 0, 2, 5, 8, 11h, and four of the six plant seedlings were exposed to 0, 10, 30, 50mmol/L NO3- for 2h. The NRA was determined in vivo at 538nm using spectrophotometer. The results showed that NRA increased when those plant seedlings were induced by nitrate solution. The change trends of NRA in roots and in leaves of cole, pea and tomato were different during treating time. The NRA in cole leaves was higher than that in its root and in other two plants and increased along with inducing time, but the NRA in bea and tomato was highest when the treating time was 8h and 2h, respectively. The highest NRA in leaves of three kinds of Chinese cabbages and tomato was induced by different concentrations of KNO3 solution. In tomato leaves, the highest NRA was induced by 10 - 30mmol/L KNO3 solution. In three Chinese cabbages, Brassica chinensis L. cv. AJH, XBC and KR-605, the highest NRA was induced by 10, 30, 10mmol/L KNO3 solution, respectively. The results indicated that the response manners of NRA in plants to external nitrate solutions were different. According to these results, the level of NR mRNA in plants could be enhanced by nitrate inducement. The total RNA was isolated from tomato leaves and root which induced by 30mmol/L KNO3 solution for 2h, and NR cDNA was obtained by RT-PCR using the specific primers. The fragments of PCR products were cloned and sequenced. There are 2736 base pairs in the whole cDNA fragment. The deduced protein sequence contains 911 amino acids. The NR gene can be fused to the CaMV 35S promoter, then introduced to higher plants, such as vegetables. It is hoped to decrease drastically the nitrate content of the transgenic plants.
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PMID:[Induced activity of nitrate reductase by nitrate and cloning of nitrate reductase gene]. 1596 98

Xanthomonas maltophilia ATCC 17666 is an obligate aerobe that accumulates nitrite when grown on nitrate. Spectra of membranes from nitrate-grown cells exhibited b-type cytochrome peaks and A(615-630) indicative of d-type cytochrome but no absorption peaks corresponding to c-type cytochromes. The nitrate reductase (NR) activity was located in the membrane fraction. Triton X-100-extracted reduced methyl viologen-NRs were purified on DE-52, hydroxylapatite, and Sephacryl S-300 columns to specific activities of 52 to 67 mumol of nitrite formed per min per mg of protein. The cytochrome-containing NR(I) separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis into a 135-kDa alpha-subunit, a 64-kDa beta-subunit, and a 23-kDa gamma-subunit with relative band intensities indicative of a 1:1:1 alpha/beta/gamma subunit ratio and a M(r) of 222,000. The electronic spectrum of dithionite-reduced purified NR displayed peaks at 425, 528, and 558 nm, indicative of the presence of a cytochrome b, an interpretation consistent with the pyridine hemochrome spectrum formed. The cytochrome b of the NR was reduced under anaerobic conditions by menadiol and oxidized by nitrate with the production of nitrite. This NR contained 0.96 Mo, 12.5 nonheme iron, and 1 heme per 222 kDa: molybdopterin was detected with the Neurospora crassa nit-1 assay. A smaller reduced methyl viologen-NR (169 kDa), present in various concentrations in the Triton X-100 preparations, lacked a cytochrome spectrum and did not oxidize menadiol. The characteristics of the NRs and the absence of c-type cytochromes provide insights into why X. maltophilia accumulates nitrite.
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PMID:Purification of Two Nitrate Reductases from Xanthomonas maltophilia Grown in Aerobic Cultures. 1634 5

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

A study of the growth-inhibiting effect of chlorate on the Berlin strain of Chlorella vulgaris Beijerinck provided complete confirmation of the theory of chlorate toxicity first proposed by Aberg in 1947. Chlorate was toxic to the cells growing on nitrate, and relatively nontoxic to the cells growing on ammonium. The latter cells contained only 0.01 as much NADH-nitrate reductase as the nitrate-grown cells. Chlorate could substitute for nitrate as a substrate of the purified nitrate reductase with Km = 1.2 mm, and V(max) = 0.9V(max) for nitrate. Bromate, and to a much smaller extent, iodate, also served as alternate substrates. Nitrate is a reversible competitive inhibitor of chlorate reduction, which accounts for the partial reversal, by high nitrate concentrations, of the observed inhibition of cell growth by chlorate. During the reduction of chlorate by NADH in the presence of purified nitrate reductase, there was a progressive, irreversible inhibition of the enzyme activity, presumably brought about by the reduction product, chlorite. Both the NADH-nitrate reductase activity and the associated NADH-cytochrome c reductase activity were inactivated to the same extent by added chlorite. The spectral properties of the cytochrome b(557) associated with the purified enzyme were not affected by chlorite. The inactivation of the nitrate reductase by chlorite could account for the toxicity of chlorate to cells grown on nitrate, though the destruction of other cell components by chlorite or its decomposition products cannot be excluded.
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PMID:Nitrate Reductase and Chlorate Toxicity in Chlorella vulgaris Beijerinck. 1665 89

We have assayed absorbance changes generated by blue light in plasma membranes, endoplasmic reticulum, and mitochondrial membranes from Neurospora crassa. Light minus dark difference spectra, obtained anaerobically in the presence of ethylenediaminetetraacetate, indicated that b-type cytochromes could be photoreduced in all three membranes. In plasma membranes, a b-type cytochrome with a distinct difference spectrum was photoreducible without addition of exogenous flavin. Addition of riboflavin greatly stimulated the photoreduction of cytochromes in endoplasmic reticulum and mitochondrial membranes. In its spectral characteristics the cytochrome on the endoplasmic reticulum resembled cytochrome b(5) or nitrate reductase, while the cytochrome in mitochondrial membranes had the same spectrum as cytochrome b of the mitochondrial respiratory chain.Cytochromes in the three membrane fractions reacted differently to blue light in the presence of various inhibitors. Potassium azide inhibited reduction of plasma membrane cytochrome b, with 50% inhibition at 1.0 millimolar. The same concentration of azide stimulated photoreduction of cytochromes in both endoplasmic reticulum and mitochondria. Although photoreduction of cytochromes in all three membranes was inhibited by salicylhydroxamic acid, cytochromes in plasma membranes were more sensitive to this inhibitor than those in endoplasmic reticulum and mitochondria. Cells grown to induce nitrate reductase activity showed an elevated amount of blue light-reducible cytochrome b in the endoplasmic reticulum.
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PMID:Blue Light-Reducible Cytochromes in Membrane Fractions from Neurospora crassa. 1666 61

Plasma membranes obtained by two-phase partitioning of microsomal fractions from spinach (Spinacea oleracea L. cv Medania) and sugar beet leaves (Beta vulgaris L.) contained relatively high NADH-ferricyanide reductase and NADH-nitrate reductase (NR; EC 1.6.6.1) activities. Both of these activities were latent. To investigate whether these activities were due to the same enzyme, plasma membrane polypeptides were separated with SDS-PAGE and analyzed with immunoblotting methods. Antibodies raised against microsomal NADH-ferricyanide reductase (tentatively identified as NADH-cytochrome b(5) reductase, EC 1.6.2.2), purified from potato (Solanum tuberosum L. cv Bintje) tuber microsomes, displayed one single band at 43 kilodaltons when reacted with spinach plasma membranes, whereas lgG produced against NR from spinach leaves gave a major band at 110 kilodaltons together with a few fainter bands of lower molecular mass. Immunoblotting analysis using inside-out and right-side-out plasma membrane vesicles strongly indicated that NR was not an integral protein but probably trapped inside the plasma membrane vesicles during homogenization. Proteins from spinach plasma membranes were solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio] 1-propane-sulfonate and separated on a Mono Q anion exchange column at pH 5.6 with fast protein liquid chromatography. One major peak of NADH-ferricyanide reductase activity was found after separation. The peak fraction was enriched about 70-fold in this activity compared to the plasma membrane. When the peak fractions were analyzed with SDS-PAGE the NADH-ferricyanide reductase activity strongly correlated with a 43 kilodalton polypeptide which reacted with the antibodies against potato microsomal NADH-ferricyanide reductase. Thus, our data indicate that most, if not all, of the truly membrane-bound NADH-ferricyanide reductase activity of leaf plasma membranes is due to an enzyme very similar to potato tuber microsomal NADH-ferricyanide reductase (NADH-cytochrome b(5) reductase).
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PMID:NADH-Ferricyanide Reductase of Leaf Plasma Membranes : Partial Purification and Immunological Relation to Potato Tuber Microsomal NADH-Ferricyanide Reductase and Spinach Leaf NADH-Nitrate Reductase. 1666 82

A cDNA clone was isolated from a maize (Zea mays L. cv W64AxW183E) scutellum lambdagt11 library using maize leaf NADH:nitrate reductase Zmnr1 cDNA clone as a hybridization probe; it was designated Zmnr1S. Zmnr1S was shown to be an NADH:nitrate reductase clone by nucleotide sequencing and comparison of its deduced amino acid sequence to Zmnr1. Zmnr1S, which is 1.8 kilobases in length and contains the code for both the cytochrome b and flavin adenine dinucleotide domains of nitrate reductase, was cloned into the EcoRI site of the Escherichia coli expression vector pET5b and expressed. The cell lysate contained NADH:cytochrome c reductase activity, which is a characteristic partial activity of NADH:nitrate reductase dependent on the cytochrome b and flavin adenine dinucleotide domains. Recombinant cytochrome c reductase was purified by immunoaffinity chromatography on monoclonal antibody Zm2(69) Sepharose. The purified cytochrome c reductase, which had a major size of 43 kilodaltons, was inhibited by polyclonal antibodies for maize leaf NADH:nitrate reductase and bound these antibodies when blotted to nitrocellulose. Ultraviolet and visible spectra of oxidized and NADH-reduced recombinant cytochrome c reductase were nearly identical with those of maize leaf NADH:nitrate reductase. These two enzyme forms also had very similar kinetic properties with respect to NADH-dependent cytochrome c and ferricyanide reduction.
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PMID:Expression in Escherichia coli of Cytochrome c Reductase Activity from a Maize NADH:Nitrate Reductase Complementary DNA. 1666 41


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