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PubMed1975

Purification, structure and properties of the respiratory nitrate reductase of Klebsiella aerogenes.

1. The respiratory nitrate reductase of Klebsiella aerogenes was solubilized from the bacterial membranes by deoxycholate and purified further by means of gel chromatography in the presence of deoxycholate, and anion-exchange chromatography. 2. Dependent on the isolation procedure two different homogeneous forms of the enzyme, having different subunit compositions, can be obtained. These forms are designated nitrate reductase I and nitrate reductase II. Both enzyme preparations are isolated as tetramers having sedimentation constants (s20,w) of 22.1 S and 21.7 S for nitrate reductase I and II, respectively. The nitrate reductase I tetramer has a molecular weight of about 106. 3. In the presence of deoxycholate both enzyme preparations dissociate reversibly into their respective monomeric forms. The monomeric form of nitrate reductase I has a molecular weight of about 260 000 and a sedimentation constant of 9.8 S. For nitrate reductase II these values are 180 000 and 8.5 S, respectively. 4. Nitrate reductase I consists of three different subunits, having molecular weights of 117 000; 57 000 and 52 000, which are present in a 1:1:2 molar ratio, respectively. Nitrate reductase II contains only the subunits with a molecular weight of 117 000 and 57 000 in a equimolar ratio. 5. Treatment at pH 9.5 in the presence of deoxycholate and 0.05 M NaCl or ageing removes the 52 000 Mr subunit from nitrate reductase I. This smallest subunit, in contrast to the other subunits, is a basic protein. 6. The 52 000 Mr subunit has no catalytic function in the intramolecular electron transfer from reduced benzylviologen to nitrate. However, it appears to have a structural function since nitrate reductase II, which lacks this subunit, is much more labile than nitrate reductase I. Inactivation of nitrate reductase II can be prevented by the presence of deoxycholate. 7. The spectrum of the enzyme resembles that of iron-sulfur proteins. No cytochromes or contaminating enzyme activities are present in the purified enzyme. Only reduced benzylviologen was found to be capable of acting as an electron donor. 8. p-Chlormercuribenzoate enhances the enzymatic activity at concentrations of 0.1 mM and lower. At higher p-chlormercuribenzoate concentrations the enzymatic activity is inhibited non-competitively with either nitrate or benzylviologen as a substrate. The inhibition is not counteracted by cysteine.

Cell Membrane↗
PubMed1974

Control of Diurnal Variations in Photosynthetic Products: II. Nitrate Reductase Activity.

Nitrate accumulates in the leaves of Capsicum annuum L. cv. California Wonder and the leaf content is dependent on the nitrate level supplied to the roots. There is no consistent diurnal periodicity in the leaf nitrate levels.Nitrate reductase activity exhibits three distinct peaks in the leaves. One in the dark period, a second at the time of the prevailing dawn, and a third 6 hours after the beginning of the photoperiod. The third peak of activity can be induced by a short period of illumination (20 minutes) which is also sufficient to induce the labeling of the amino acid fraction by (14)CO(2).

Journal Article↗
PubMed1976

Nitrate Absorption by Barley: II. Influence of Nitrate Reductase Activity.

The influence of protein synthesis and nitrate reductase activity on nitrate absorption by barley (Hordeum vulgare L.) was investigated. Cycloheximide decreased nitrate absorption. Pretreatment studies showed that cycloheximide affects either energy transfer or nitrate reductase activity or both.Illumination increased plant capacity for nitrate absorption, possibly through increased energy supply and/or increased nitrate reductase activity. There was a positive correlation between nitrate reductase activity and light. Inhibiting the development of nitrate reductase activity by tungstate decreased nitrate absorption.AT LEAST TWO NITRATE TRANSPORT SYSTEMS ARE THUS PROPOSED IN BARLEY: one operating in the dark, with little nitrate reductase activity detectable; and one closely correlated with nitrate reductase activity. Total absorption is the sum of dark absorption and absorption facilitated by nitrate reductase.

Journal Article↗
PubMed1980

The location of dissimilatory nitrite reductase and the control of dissimilatory nitrate reductase by oxygen in Paracoccus denitrificans.

1. A method is described for preparing spheroplasts from Paracoccus denitrificans that are substantially depleted of dissimilatory nitrate reductase (cytochrome cd) activity. Treatment of cells with lysozyme + EDTA together with a mild osmotic shock, followed by centrifugation, yielded a pellet of spheroplasts and a supernatant that contained d-type cytochrome. The spheroplasts were judged to have retained an intact plasma membrane on the basis that less than 1% of the activity of a cytoplasmic marker protein, malate dehydrogenase, was released from the spheroplasts. In addition to a low activity towards added nitrite, the suspension of spheroplasts accumulated the nitrite that was produced by respiratory chain-linked reduction of nitrate. It is concluded that nitrate reduction occurs at the periplasmic side of the plasma membrane irrespective of whether nitrite is generated by nitrate reduction or is added exogenously. 2. Further evidence for the integrity of the spheroplasts was that nitrate reduction was inhibited by O2, and that chlorate was reduced at a markedly lower rate than nitrate. These data are taken as evidence for an intact plasma membrane because it was shown that cells acquire the capability to reduce nitrate under aerobic conditions after addition of low amounts of Triton X-100 which, with the same titre, also overcame the permeability barrier to chlorate reduction by intact cells. The close relationship between the appearance of chlorate reduction and the loss of the inhibitory effect of O2 on nitrate reduction also suggests that the later feature of nitrate respiration is due to a control on the accessibility of nitrate to its reductase rather than on the flow of electrons to nitrate reductase.

Chlorates↗
PubMed1984

Siderophore reduction catalyzed by higher plant NADH:nitrate reductase.

Squash cotyledon NADH:nitrate reductase catalyzes the reduction of the siderophore ferrioxamine B. The enzyme also reduced ferric ion in a buffer system containing the chelators oxalate and maleate. Ferrioxamine B reduction was maximal at pH 4; ferric ion reduction was maximal at pH 8. The present study indicates that iron assimilation by higher plants may occur with microbial siderophores serving as ferric ion sources and nitrate reductase functioning as the siderophore reductase.

Antibodies↗
PubMed2002

Tobacco Nia2 cDNA functionally complements a Hansenula polymorpha yeast mutant lacking nitrate reductase. A new expression system for the study of plant proteins involved in nitrate assimilation.

An integrative expression vector based on promoter and terminator transcriptional sequences from the Hansenula polymorpha nitrate reductase gene (YNR1) has been developed to express nitrate assimilation plant genes in the nitrate assimilatory yeast H. polymorpha. Using this vector a plant nitrate reductase cDNA (tobacco Nia2) was expressed for the first time in a nitrate assimilatory yeast. The heterologous nitrate reductase produced retained its biochemical and physiological properties such as its NADH-dependent nitrate reductase activity, and allowed growth in nitrate containing media in a strain lacking endogenous nitrate reductase activity. In the transgenic strain, maximum tobacco nitrate reductase activity was about 70% of that presented in the wild-type. On the other hand, the disappearance of nitrate reductase activity correlated with that of the enzyme protein in response to the addition of ammonium to the medium and took place more rapidly in the transgenic strain than in the wild-type. Nitrate reductase activity of the recombinant strain assayed in the presence of Mg2+ was about 30% of that observed when assayed with EDTA. This result, together with a decreased growth rate in nitrate, suggests that tobacco nitrate reductase could be partially inactivated in H. polymorpha by phosphorylation and binding of 14-3-3-like proteins. These results show that H. polymorpha is a useful yeast heterologous expression system for studying plant proteins involved in nitrate assimilation.

Cell Division↗
PubMed1989

Immunological approach to the regulation of nitrate reductase in Monoraphidium braunii.

The effects of different culture conditions on nitrate reductase activity and nitrate reductase protein from Monoraphidium braunii have been studied, using two different immunological techniques, rocket immunoelectrophoresis and an enzyme-linked immunosorbent assay, to determine nitrate reductase protein. The nitrogen sources ammonium and glutamine repressed nitrate reductase synthesis, while nitrite, alanine, and glutamate acted as derepressors. There was a four- to eightfold increase of nitrate reductase activity and a twofold increase of nitrate reductase protein under conditions of nitrogen starvation versus growth on nitrate. Nitrate reductase synthesis was repressed in darkness. However, when Monoraphidium was grown under heterotrophic conditions with glucose as the carbon and energy source, the synthesis of nitrate reductase was maintained. With ammonium or darkness, changes in nitrate reductase activity correlated fairly well with changes in nitrate reductase protein, indicating that in both cases loss of activity was due to repression and not to inactivation of the enzyme. Experiments using methionine sulfoximine, to inhibit ammonium assimilation, showed that ammonium per se and not a product of its metabolism was the corepressor of the enzyme. The appearance of nitrate reductase activity after transferring the cells to induction media was prevented by cycloheximide and by 6-methylpurine, although in this latter case the effect was observed only in cells preincubated with the inhibitor for 1 h before the induction period.

Ammonia↗
PubMed1982

Purification and characterization of the assimilatory NADPH-nitrate reductase of Aspergillus nidulans.

NADPH-nitrate reductase [NADPH : nitrate oxidoreductase, EC 1.6.6.3] was purified 500-fold from Aspergillus nidulans with an overall yield of about 20%. The purified enzyme catalyzed NADPH-nitrate, NADPH-cytochrome c, FADH2-nitrate and reduced methyl viologen-nitrate reductase activities. Its molecular weight was estimated to be 180,000 from the Stokes radius and sedimentation coefficient. The oxidized enzyme exhibited an absorption spectrum having a peak at 412 nm and a broad shoulder at about 450 nm. When reduced with NADPH, absorption peaks appeared at 423 (Soret), 527 (beta) and 557 (alpha) nm, and absorption in the 450 nm region decreased. Upon treatment of the reduced enzyme with KNO3, the spectrum returned to that of the oxidized enzyme. The presence of protoheme in the enzyme was confirmed by the absorption spectrum of reduced pyridine hemochromogen. It was concluded that a b-type cytochrome ("cytochrome b-557") is present in the enzyme and is involved in the intramolecular electron transport from NADPH to nitrate. The NADPH-nitrate and NADPH-cytochrome c reductase activities, but not the other two activities, were significantly decreased by incubation of the enzyme at 37 degrees C in the absence of FAD. Analysis by SDS slab gel electrophoresis suggested that the nitrate reductase consists of two each of two subunits of 59,000 and 38,000 daltons and that a dissociation of 38,000 subunits from the native enzyme occurs during heat treatment, resulting in alteration of the catalytic activity.

Aspergillus nidulans↗
PubMed1988

Roles of the narJ and narI gene products in the expression of nitrate reductase in Escherichia coli.

Nitrate reductase, released and purified from membrane fractions of Escherichia coli, is composed of three subunits. Formation of the enzyme depends on induction of the nar operon, narGHJI, which is composed of four open reading frames (ORF). Previous studies established that the first two genes in the operon narG and narH encode the alpha and beta subunits, respectively, while formation of the gamma subunit, cytochrome bNR, depends on expression of the promoter distal genes. The narJ and narI genes were subcloned separately into plasmids where each was under the control of the nar promoter. Expression of these plasmids in a mutant which forms only alpha and beta subunits revealed that expression of the narI gene is sufficient to restore normal levels of cytochrome bNR, but expression of both genes is required for assembly of fully active, membrane-bound nitrate reductase. The amino acid composition, the N-terminal sequence, and the sequence of cyanogen bromide fragments derived from the isolated gamma subunit corresponds to that expected for a protein produced by the narI ORF. A protein corresponding to the narJ ORF did not appear to be associated with the purified nitrate reductase complex or with the complex immunoprecipitated from Triton X-100-solubilized membrane preparations. We conclude that narI encodes the gamma subunit (cytochrome bNR) and that while the product of the narJ gene is required for assembly of fully active membrane-bound enzyme it is not tightly associated with the active enzyme.

Amino Acid Sequence↗
PubMed1974

Purification and Characterization of the Nitrate Reductase from the Diatom Thalassiosira pseudonana.

THE ASSIMILATORY NITRATE REDUCTASE (NADH: nitrate oxidoreductase, E.C. 1.6.6.2.) from the marine diatom Thalassiosira pseudonana, Hasle and Heimdal, has been purified 200-fold and characterized. The regulation of nitrate reductase in response to various conditions of nitrogen nutrition has been investigated.Nitrate reductase activity is repressed by the presence of ammonium in vivo, and its synthesis is derepressed when ammonium is absent. The derepression process is sensitive to cycloheximide and apparently requires protein synthesis. Repression of enzyme activity by ammonium is neither inhibited nor delayed by the presence of cycloheximide. In vitro, ammonium does not inhibit enzyme activity.NADH is the physiological electron donor for the enzyme in a flavin-dependent reaction. Spectral studies have indicated the presence of a b-type cytochrome associated with the enzyme. It is possible to observe enzymatic oxidation-reduction reactions which represent partial functions of the over-all electron transport capacity of this enzyme. Nitrate reductase will accept electrons from artificial electron donors such as reduced methyl viologen in a flavin-independent reaction. Further, dithionitereduced flavin adenine dinucleotide can donate electrons to the enzyme to reduce nitrate to nitrite. Finally, the nitrate reductase will exhibit a diaphorase activity and reduce the artificial electron acceptor mammalian cytochrome c in flavin-adeninedinucleotide-dependent reaction.Inhibition studies with potassium cyanide, sodium azide, and o-phenanthroline have yielded indirect evidence for metal component (s) of the enzyme.The inhibition of the NADH-requiring enzyme activities by p-hydroxymercuribenzoate has shown that an essential sulfhydryl group is involved in the initial portion of the electron transport. Heat treatment exerts an effect similar to the p-hydroxymercuribenzoate inhibition; namely, the NADH-requiring activities are rapidly inactivated, whereas the terminal nitrate-reducing activities are relatively stable to heat.The T. pseudonana nitrate reductase molecule has the hydrodynamic properties of an ellipsoid with a frictional coefficient of 1.69 and a molecular weight of 330,000.

Journal Article↗
PubMed1992

Nitrate reductase transcript is expressed in the primary response of maize to environmental nitrate.

The nitrate induction of NADH:nitrate reductase mRNA in maize roots, scutella and leaves was investigated in the presence and absence of inhibitors of protein synthesis. In the absence of inhibitors, nitrate treatment caused a fairly rapid (2 to 3 h) increase in the level of the nitrate reductase transcript in all tissues. When cytoplasmic protein synthesis was inhibited by cycloheximide, nitrate reductase mRNA was induced by nitrate in all tissues to levels equal to or greater than those found with nitrate treatment alone. Treatment of maize tissues with cycloheximide in the absence of nitrate had only a small effect on the accumulation of the nitrate reductase mRNA. Inhibition of organellar protein synthesis with chloramphenicol also had little or no effect on nitrate-induced nitrate reductase mRNA accumulation in roots and scutella, but did appear to partially inhibit appearance of transcript in leaves. Excision of scutella in the absence of nitrate was sufficient to cause some accumulation of the nitrate reductase transcript. Since cytoplasmic protein synthesis was not required for expression of nitrate reductase transcripts, induction of these transcripts by nitrate is a primary response of maize to this environmental signal. Thus, it appears that the signal transduction system mediating this response is constitutively expressed in roots, scutella and leaves of maize.

Blotting, Northern↗
PubMed1994

Purification and characterization of the periplasmic nitrate reductase from Thiosphaera pantotropha.

The periplasmic nitrate reductase of Thiosphaera pantotropha has been purified from a mutant strain (M-6) that overproduces the enzyme activity under anaerobic growth conditions. The enzyme is a complex of a 93-kDa polypeptide and a 16-kDa nitrate-oxidizable cytochrome c552. The complex contains molybdenum; a fluorescent compound with spectral features of a pterin derivative can be extracted. In contrast to the dissimilatory membrane-bound nitrate reductases, the periplasmic nitrate reductase shows high specificity for nitrate as a substrate and is insensitive to inhibition by azide. The 93-kDa subunit exhibits immunological cross-reactivity with the catalytic subunit of Rhodobacter capsulatus N22DNAR+ periplasmic nitrate reductase. Mass spectrometric comparisons of holo-cytochrome c552 and apo-cytochrome c552 demonstrated that the polypeptide bound two haem groups. Mediated redox potentiometry of the cytochrome indicated that the haem groups have reduction potentials (pH = 7.0) of approximately -15 mV and + 80 mV. The functional significance of these potentials is discussed in relation to the proposed physiological role of the enzyme as a redox valve.

Cell Membrane↗
PubMed2004

Nitrate reductase regulation in tomato roots by exogenous nitrate: a possible role in tolerance to long-term root anoxia.

The mechanism of nitrate reductase (NR) regulation under long-term anoxia in roots of whole plants and the putative role of nitrate in anoxia tolerance have been addressed. NR activity in tomato roots increased significantly after 24 h of anaerobiosis and increased further by 48 h, with a concomitant release of nitrite into the culture medium. Anoxia promoted NR activation through dissociation of the 14-3-3 protein inhibitor and NR dephosphorylation. After 24 h of anoxia, the total amount of NR increased slightly up to 48 h. However, NR-mRNA levels remained constant between 0 h and 24 h of root anoxia and decreased after 48 h. This is probably due to the inhibition of NR degradation and the accumulation of its native form. NR was slightly dephosphorylated in the absence of oxygen and nitrate. Under anoxia, NR dephosphorylation was modulated by nitrate-controlled NR activity. In addition, the presence of nitrate prevents anoxic symptoms on leaves and delays wilting by 48 h during root anoxia. In the absence of nitrate, plants withered within 24 h, as they did with tungstate treatment, an inhibitor of NR activity. Thus, anoxia tolerance of tomato roots could be enhanced by nitrate reduction.

Adaptation, Physiological↗
PubMed1983

Reduction of ferric citrate catalyzed by NADH:nitrate reductase.

We show that NADH:nitrate reductase from squash cotyledons can catalyze the reduction of ferric citrate. When nitrate reductase was purified to homogeneity using a two-step affinity chromatography procedure, an NADH:Fe(III)-citrate reductase activity copurified with it and had identical electrophoretic mobility to it. The iron reductase activity was optimum near pH 6.3, had an apparent Km for Fe(III)-citrate of 0.02 mM, and was inhibited by monospecific anti-nitrate reductase rabbit sera. Differential inhibition of the enzyme's activities indicated iron and nitrate were reduced at different sites. In addition to its role in nitrogen assimilation, nitrate reductase catalyzes ferric citrate reduction and could have a role in iron assimilation.

Antigen-Antibody Complex↗
PubMed2001

Nitrate reductase activity, distribution, and response to nitrate in two contrasting Phaseolus species inoculated with Rhizobium spp.

The nitrate reductase activity distribution and response of two nodulated species of Phaseolus (Phaseolus vulgaris-common bean, and Phaseolus lunatus-lima bean) to different exogenous nitrate levels were studied during the vegetative period. These Phaseolus species showed to be very contrasting in respect to the pattern of nitrate reductase (NR) activity distribution thought the plant. The highest level of NR activity in P. vulgaris was clearly shown to occur in leaves in contrast with the lowest one detected in roots and nodules as widely seen for other tropical species of the Phaseoleae tribe. Conversely, P. lunatus had higher NR activity in the nodules, whereas its leaves exhibited a steadily decrease during the plant development. Indeed, at 32 days after emergence (pre-flowering stage), the nodulated P. vulgaris had approximately 95% of the total NR activity localized in its leaves, whereas in P. lunatus it was equally distributed in the nodules and in the leaves. Under long-term exposure to increasing exogenous level of nitrate, the leaf-NR activity of nodulated P. vulgaris presented a positive response, whereas the enzyme activity was very low and unresponsive in P. lunatus. In contrast, the nodule-NR activity showed a reverse response to the increasing NO(3)(-) level. The nodule-NR activity of P. lunatus significantly increased whereas in the P. vulgaris nodules it was very low and unresponsive. This present study suggests that P. lunatus inoculated with Rhizobium tropici presents a singular pattern of nitrate reduction distribution among leaves and nodules during the vegetative development. It is speculated that the nodulated Phaseolus lunatus may have different NR isoforms in their leaves (at least a constitutive type) and an inducible form in their nodules, responsive to long-term exposure to nitrate.

Journal Article↗
PubMed2004

Indirect determination of nitric oxide production by reduction of nitrate with a freeze-thawing-resistant nitrate reductase from Escherichia coli MC1061.

Preparation of a nitrate reductase lysate of Escherichia coli MC1061 to measure nitrate and nitrite in biologic fluids is described. To obtain the crude bacterial lysate containing nitrate reductase activity, E. coli MC1061 was subjected to 16-20 freeze-thawing cycles, from -70 to 60 degrees C, until nitrite reductase activity was < or = 25%. Nitrate reductase activity was detected mainly in the crude preparation. To validate the nitrate reduction procedure, standard nitrate solutions (1.6-100 microM) were incubated with the nitrate reductase preparation for 3 h at 37 degrees C, and nitrite was estimated by the Griess reaction in a microassay. Nitrate solutions were reduced to nitrite in a range of 60-70%. Importantly, no cofactors were necessary to perform nitrate reduction. The biological samples were first reduced with the nitrate reductase preparation. After centrifugation, samples were deproteinized with either methanol/ether or zinc sulfate and nitrite was quantified. The utility of the nitrate reductase preparation was assessed by nitrate+nitrite determination in serum of animals infected with the protozoan Entamoeba histolytica or the bacteria E. coli and in the supernatant of cultured lipopolysaccharide-stimulated RAW 264.7 mouse macrophages. Our results indicate that the nitrate reductase-containing lysate provides a convenient tool for the reduction of nitrate to determine nitrate+nitrite in biological fluids by spectrophotometric methods.

Animals↗