NADH-nitrate Reductase Activity Research Articles

The occurence of a nitrate reductase inactivating factor in extracts of Spirodela polyrrhiza

NADH-nitrate reductase (NR) had a low activity immediately after extraction from 14 day-old cultures of <em>Spirodela polyrrhiza</em> with the basic extraction solution (50 mM K-phosphate buffer, pH 7.5, 5 mM cysteine and 3 mM EDTA) whereas nitrite reductase (NiR) activity in the same extract was very high. Increasing the EDTA or cysteine concentration or replacing cysteine by dithiothreitol had no effect on the level of NR activity. The addition of 3% bovine serum albumine (BSA) and 0.4 mM phenylmethyl sulfonyfluoride (PMSF) or 3% BSA and 1.5% polyvinylpolypyrrolidone (PVPP) to the extraction solution greatly increased the NR activity. The enzyme activity increased 40 times in the presence of 1.5% PVPP with 3% BSA and 0.4 mM PMSF. The highest NR activity (over 100-fold) was found in the presence of 3% caseine. The probable nature of the factor inactivating nitrate reductase in <em>S. polyrrhiza</em> extracts is discussed.

Influence of plant age and growth conditions on nitrate assimilation in roots of Lotus japonicus plants.

In roots of Lotus japonicus (Regel) Larsen cv. Gifu, the level of NADH-nitrate reductase (NR) activity and protein, as well as ferredoxin-nitrite reductase activity and nitrate accumulation, were higher in roots of young plants, and decreased in mature plants grown in seed trays. When plants were grown in larger pots, the decrease in NR activity and nitrate accumulation took place at a later stage of growth, suggesting that the cessation of nitrate assimilation in mature plants could be related to both ageing and a lower availability of space for roots. Low NR activity was detected in leaves, whereas nitrate accumulation in this tissue could reach relatively high levels. NR activity and protein, and nitrate accumulation, also decreased in leaves of mature plants, indicating that the diminution in nitrate accumulation and NR in roots from mature plants is not related to a shift of the nitrate assimilation process from roots to leaves. Measurement of the extent of total and active NR according to the phosphorylation/dephosphorylation inactivating mechanism described for spinach leaf NR, indicates that this mechanism is operative on NR from L. japonicus roots, being responsible for a great proportion of inactive NR protein. The amount of inactive NR protein in roots due to the aforementioned phosphorylation mechanism also increased in mature plants.

Nitrate reductase in Zea mays L. under salinity

ABSTRACTYoung maize seedlings (Zea mays L. cv.Giza 2) were exposed to moderate salinity in hydroponic culture. NADH‐nitrate reductase (NR) activity (E.C. 1·6.6·1), NR activation state, NR‐mRNA‐steady state levels and major solute contents in leaves and roots were investigated. With increasing external salt concentration, Na+, Cl−, sugars, amino acids and quarternary ammonium compounds accumulated in leaves and roots, with concentrations in leaves exceeding those in roots. The nitrate content of leaves decreased, but increased in roots. The diurnal pattern of NR activity and of NR‐mRNA was also changed under salinity, but the NR activation state was not affected. In the first light phase, maximum NR activity increased rapidly in leaves of control plants, but was much slower in leaves from salinized plants. Thus, integrated over the whole day, the NR activity of salt‐stressed plants was lower than in control plants. NR transcript levels of control plants were low in the early night phase, started to increase in the second night phase, followed by a distinct peak at 2 h into the light period. This large ‘early morning peak’ of NR‐mRNA was hardly affected by salinity, whereas the initial slow increase of m‐RNA levels in the early night phase was almost absent in salinized plants. This is considered as one reason for the low NR activity of salinized plants in the first half of the day. It is also suggested that nitrate is a major signal affecting NR expression and activity under salinity. Sugars and amino acids appeared less important.

Nitrate reductase from rice seedlings: Partial purification, characterization and the effects of in situ and in vitro NaCl salinity

Summary The effect of NaCl salinity in situ on NADH-nitrate reductase (EC 1.6.6.1) activity was studied in rice seedlings grown in sand cultures. NaCl salinity levels of 7 dS m−1 and 14 dS m−1 caused significant inhibition in NR activity in 10- and 20-day-grown seedlings. The enzyme was partially purified from leaves of 15-day-grown non-salinized seedlings using ammonium sulphate (25–45 percent) precipitation and affinity chromatography on a Blue-Sepharose CL-6B column. The purification achieved was 303-fold with 53 percent yield and the purified enzyme showed a specific activity of 3.6 μmoles NO2− min−1 mg−1 protein. NR activity was inhibited in vitro with an increase in NaCl in the assay medium. With 1000 mmol/L NaCl in the assay medium almost 85 percent NR activity was lost. The purified enzyme showed optimum activity at pH 7.5 and temperature optima of 30°C for 30 min. Preincubation of NR with 0.4 mmol/L NADH caused a significant increase in its activity. Apparent Km values of the enzyme for nitrate and NADH were 0.286 mmol/L and 4 mmol/L, respectively. The enzyme NR thus appears to be fairly sensitive to in situ as well as in vitro effects of NaCl.

The Impact of Ozone on the15N Incorporation and Nitrogen Assimilation of Wheat and Maize

Abstract Young wheat (C3) and maize (C4) plants were exposed to near-ambient concentrations of ozone in open-top chambers in order to investigate the possible effects of ozone on nitrogen metabolism. Nitrogen was supplied to the plants by adding (15)N-labelled tracer substances via the soil substrate. Enzyme activities (NADH nitrate reductase, nitrite reductase, glutamine synthetase and NADH glutamate dehydrogenase) and the incorporation of (15)N were determined. The findings show that nitrogen metabolism was affected by O(3), however, there were distinct differences between the two species. In plants treated with O(3), NADH nitrate reductase activity in maize leaves was reduced, while NR activity in wheat leaves only slightly declined. Only minor changes were observed with respect to the activities of nitrite reductase, glutamine synthetase and NADH glutamate dehydrogenase. Feeding experiments using (15)NO(3) (-) showed that the incorporation of nitrate nitrogen in wheat plants exposed to ozone remains virtually unchanged, whereas in maize plants reduced incorporation rates were observed for nitrate nitrogen. The incorporation of ammonium nitrogen was distinctly increased in wheat and maize by the impact of ozone. When investigating pigment contents, reduced levels of chlorophyll a and b and carotenoids were observed, whereas the pigment content of wheat leaves remained unchanged. These results indicate that young maize plants are more susceptible than wheat plants to short-term ozone exposure.

Nir1, a conditional-lethal mutation in barley causing a defect in nitrite reduction.

Eleven green individuals were isolated when 95000 M2 plants of barley (Hordeum vulgare L.), mutagenised with azide in the M1, were screened for nitrite accumulation in their leaves after nitrate treatment in the light. The selected plants were maintained in aerated liquid culture solution containing glutamine as sole nitrogen source. Not all plants survived to flowering and some others that did were not fertile. One of the selected plants, STA3999, from the cultivar Tweed could be crossed to the wild-type cultivar and analysis of the F2 progeny showed that leaf nitrite accumulation was due to a recessive mutation in a single nuclear gene, which has been designated Nir1. The homozygous nir1 mutant could be maintained to flowering in liquid culture with either glutamine or ammonium as sole nitrogen source, but died within 14 days after transfer to compost. The nitrite reductase cross-reacting material seen in nitrate-treated wild-type plants could not be detected in either the leaf or the root of the homozygous nir1 mutant. Nitrite reductase activity, measured with dithionite-reduced methyl viologen as electron donor, of the nitrate-treated homozygous nir1 mutant was much reduced but NADH-nitrate reductase activity was elevated compared to wild-type plants. We conclude that the Nir1 locus determines the formation of nitrite reductase apoprotein in both the leaf and root of barley and speculate that it represents either the nitrite reductase apoprotein gene locus or, less likely, a regulatory locus whose product is required for the synthesis of nitrite reductase, but not nitrate reductase.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic and biochemical analysis of intragenic complementation events among nitrate reductase apoenzyme-deficient mutants of Nicotiana plumbaginifolia.

Intragenic complementation has been observed between apoenzyme nitrate reductase-deficient mutants (nia) of Nicotiana plumbaginifolia. In vivo as in vitro, the NADH-nitrate reductase (NR) activity in plants heterozygous for two different nia alleles was lower than in the wild type plant, but the plants were able to grow on nitrate as a sole nitrogen source. NR activity, absent in extracts of homozygous nia mutants was restored by mixing extracts from two complementing nia mutants. These observations suggest that NR intragenic complementation results from either the formation of heteromeric NR or from the interaction between two modified enzymes. Complementation was only observed between mutants retaining different partial catalytic activities of the enzyme. Results are in agreement with molecular data suggesting the presence of three catalytic domains in the subunit of the enzyme.

NADH-Ferricyanide Reductase of Leaf Plasma Membranes

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).

Characterization of Nitrate Reductase Deficient Mutants of Chlorella sorokiniana

After x-ray irradiation, 13 mutants of Chlorella sorokiniana incapable of using NO(3) (-) as N source were isolated using a pinpoint method. Using immunoprecipitation and Western blot assays, no nitrate reductase was found in five strains while in eight mutants the enzyme was detected. The latter strains contained different patterns of nitrate reductase partial reactions. All isolates were of the nia-type as indicated by the inducibility of purine hydroxylase I and by complementation of nitrate reductase activity in the Neurospora crassa mutant Nit-1. A restoration of NADP-nitrate reductase in Nit-1 was also obtained with NH(4) (+)-grown cells indicating that Mo-cofactor is constitutive in Chlorella. Complementation experiments among the Chlorella mutants resulted in restoration of NADH-nitrate reductase activity. The characteristics of some of the Chlorella mutants are discussed in view of an improper orientation of Mo-cofactor in the residual nitrate reductase protein.

Expression of Leaf Nitrate Reductase Genes from Tomato and Tobacco in Relation to Light-Dark Regimes and Nitrate Supply

The influence of light-dark cycles and nitrate supply on nitrate reductase (NR) mRNA levels was studied in two plant species, tobacco (Nicotiana tabacum) and tomato (Lycopersicon esculentum) using specific NR DNA probes. In the same series of experiments, changes in the levels of NR protein (NRP) by enzyme-linked immunosorbent assay and changes in the level of NADH-nitrate reductase activity (NRA) were also followed. During a light-dark cycle, it was found that in both tomato and tobacco, NR mRNA accumulation increased rapidly during the dark period and reached a maximum at the beginning of the day, while NRP reached a peak 2 and 4 hours after mRNA peaked, for tomato and tobacco, respectively. At the end of the day, the amount of mRNA was decreased by a factor of at least 100 compared to sunrise in both species. These results demonstrate that light is involved, although probably not directly, in the regulation of the NR gene expression at the mRNA level. The peak of NRA in tobacco coincided with the peak in NR mRNA accumulation (i.e. sunrise), whereas in tomato the peak of NRA was approximately 5 to 6 hours after sunrise. There is no obvious correlation between NRP and NRA levels during the day. In nitrogen starvation experiments, a rapid decrease of NRP and NRA was detected, while NR mRNA levels were not significantly altered. Upon nitrate replenishment, nitrogen-starved plants accumulated NR mRNA rapidly. These results suggest that the availability of nitrogen affects the expression of NR activity at the transcriptional as well as at the post-transcriptional levels.

Nitrogen Utilization in Lemna

Cultures of Lemna gibba L. G3 were maintained at a constant, N-limited growth rate by adding nitrate daily in amounts calculated to sustain a rate of culture N increment of 0.20 day(-1). Nitrate added to the culture was consumed within 8 to 10 hours and the partitioning to reduction and accumulation during this phase corresponded to, on the average, 75 and 25% of net uptake, respectively. The calculated rate of nitrate reduction was stimulated by onset of net uptake without delay and decreased when net uptake ceased. NADH-nitrate reductase (NR) activity measured in vitro without inclusion of antiproteolytic agents more than doubled during the first hour after nitrate addition and then gradually fell to its original level over the rest of the 24 hour interval. In the presence of the proteinase inhibitor leupeptin during extraction, however, NR activity was in general much higher and without any apparent cycles. The relative stabilizing effect of leupeptin was greatest on NADH-NR and reduced flavin adenine mononucleotide-NR activities whereas the effect was less on NADH-cytochrome c reductase activity (diaphorase) and reduced methylviologen-NR activity. The constant nitrate reductase activity measured in the presence of proteinase inhibitors is assumed to reflect the physiological situation. It thus appeares that short-term changes in nitrate assimilation by N-limited Lemna is related to the flux of nitrate to the reducing site and not to changes in nitrate reductase activity.

Inheritance and expression of NAD(P)H nitrate reductase in barley

NADH-specific and NAD(P)H bispecific nitrate reductases are present in barley (Hordeum vulgare L.). Wild-type leaves have only the NADH-specific enzyme while mutants with defects in the NADH nitrate reductase structural gene (nar1) have the NAD(P)H bispecific enzyme. A mutant deficient in the NAD(P)H nitrate reductase was isolated in a line (nar1a) deficient in the NADH nitrate reductase structural gene. The double mutant (nar1a;nar7w) lacks NAD(P)H nitrate reductase activity and has xanthine dehydrogenase and nitrite reductase activities similar to nar1a. NAD(P)H nitrate reductase activity in this mutant is controlled by a single codominant gene designated nar7. The nar7 locus appears to be the NAD(P)H nitrate reductase structural gene and is not closely linked to nar1. From segregating progeny of a cross between the wild type and nar1a;nar7w, a line was obtained which has the same NADH nitrate reductase activity as the wild type in both the roots and leaves but lacks NADPH nitrate reductase activity in the roots. This line is assumed to have the genotype Nar1Nar1nar7nar7. Roots of wild type seedlings have both nitrate reductases as shown by differential inactivation of the NADH and NAD(P)H nitrate reductases by a monospecific NADH-nitrate reductase antiserum. Thus, nar7 controls the NAD(P)H nitrate reductase in roots and in leaves of barley.

NADH Nitrate Reductase and NAD(P)H Nitrate Reductase in Genetic Variants and Regenerating Callus of Maize

Different organs of maize seedlings are known to contain different complements of NADH and NAD(P)H nitrate reductase (NR) activity. The study of the genetic programming that gives rise to such differences can be initiated by looking for genetic variants exhibiting different patterns of distribution of the above enzymes. We demonstrate in this work that scutella of very young maize seedlings contain NADH NR almost exclusively and that this activity is gradually replaced, as the seedling ages, with NAD(P)H NR. Leaves in the seedlings contain exclusively the NADH NR activity. A genetic variant is described that contains much reduced levels of NAD(P)H NR activity but not of NADH NR activity in the scutellum. This same variant exhibits a relatively low level of NAD(P)H NR but normal NADH NR activity in seedling root tips. These observations suggest that the genetic program used to specify the scutellar complement of NR activity shares some common components with the genetic program used to determine the young root tip complement of NR activities. Parts of regenerating callus at different stages of differentiation were examined to determine when the differences in NR complement begin to appear. The same pattern of NADH NR and NAD(P)H NR activities was found in unorganized as well as in organized callus, in recognizable root-like and even in green shoot-like material, both activities being present in all these tissues. An examination of the NR complement in different organs of a number of siblings originating from a cross involving transposon Mu-containing parents and having different levels of leaf NADH NR activity shows that the leaf NADH NR activity content and the scutellum NAD(P)H NR activity content are relatively independent of each other, indicating that the genetic programs specifying the NR content of these organs are not tightly coupled, if at all.

Purification of the molybdoenzyme nitrate reductase: production and characterization of monoclonal antibodies

The molybdoenzyme NADH-nitrate reductase (NADH-NR) (EC 1.6.61) has been purified 3400-fold, by a multi-stage procedure,to homogeneity from spinach ( Spinacea oleracea L.). The enzyme had a specific activity of 102 μmol NO − 2 produced min −1 mg −1. Denaturing electrophoresis revealed two subunits of molecular weight 110,000 and 120,000 and was substantially free of proteolytic degradation products. Extinction coefficients of the functional haem were 127 mM at 412 nm (oxidized) and 172 mM at 423 nm (NADH-reduced). A set of monoclonal antibodies was raised to the pure NR. Six selected for further cloning were all γG type and bound to five distinct and one overlapping site on the enzyme. The various activities of the NR (NADH-oxidizing and nitrate-reducing) were differentially inhibited by the monoclonal antibodies. NADH-NR activities of extracts of roots and leaves of a variety of species were inhibited to different extents.

Regulation of nitrate reductase in suspension culture of Silene alba Immunochemical approach

Silene alba cells grown on nitrate, usually develop NADH-nitrate reductase activity only at the beginning of their growth cycle. Immunodiffusion assays, with a specific nitrate reductase antiserum, revealed the presence of cross-reacting material in cells harvested at any time during their culture. Cells grown on ammonium lacked NADH-nitrate reductase activity but contained cross-reacting material. It is suggested that S. alba cells contain an enzymically inactive, antigenic form of nitrate reductase regardless of the nitrogen source.

Repair in vitro of nitrate reductase-deficient tobacco mutants (cnxA) by molybdate and by molybdenum cofactor

Two nitrate reductase-deficient mutant cell lines (CnxA68/2, CnxA101) of Nicotiana tabacum are shown to be repairable under in-vitro conditions by (i) molybdate or (ii) by preparations of active molybdenum cofactor of homologous or heterologous origin, thereby yielding about 20% and 80%, respectively, of the corresponding wild-type NADH-nitrate reductase (EC 1.6.6.1) activity. In-vitro repair of nitrate reductase activity is dependent on sulphydryl-group protecting reagents and ethylenediaminetetraacetic acid (EDTA) in the extraction medium, the nitrogen source in the growth medium and the age of the cells. The results support the conclusion that the cnxA gene controls the insertion of molybdenum into the molybdenum cofactor. They are consistent with the idea of two interlinked pathways for the metabolic processing of molybdenum acquisition, one involving the synthesis of the structural moiety of the molybdenum cofactor and the other involving processing of the molybdate anion.

Spinach Nitrate Reductase

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.

Effects of electron transport inhibitors and uncouplers on denitrification inParacoccus denitrificans

Gaschromatographic analysis shows that whole cells of Paracoccus denitrificans produce dinitrogen in the absence and nitrous oxide in the presence of thiocyanate during nitrate reduction. NADH nitrate reductase activity in vesicles is much more sensitive to thiocyanate than either NADH oxidase activity in vesicles or reduction of nitrate by endogenous substrates in whole cells. NADH nitrate reductase activity is not inhibited and NADH oxidase activity is partially inhibited by antimycin A in vesicles. Production of nitrous oxide from nitrate in cells is completely inhibited by the simultaneous presence of thiocyanate and Triton X-100. Carbonylcyanide m-chlorophenylhydrazone does not cause a lag phase in reduction of nitrate by NADH in vesicles, in contrast to the situation in cells.

A conditional-lethal cnx-type nitrate reductase-deficient barley mutant

Selection for resistance to chlorate in an M2 population of barley (Hordeum vulgare cv. Maris Mink, M1 seeds treated with sodium azide) yielded nine green plants with zero or very low in vivo nitrate reductase activity. One of these plants, R9401, was crossed with the wild-type cv. Golden Promise and analysis of the F2 progeny showed that both loss of nitrate reductase activity and resistance to chlorate was due to a recessive mutation in a single nuclear gene. Homozygous mutant plants did not grow on nitrate as sole nitrogen source, in vermiculite or in sterile culture. They grew aseptically on ammonium nitrate, ammonium citrate or urea, although glutamine was the best nitrogen source. Homozygous mutant plants possessd <1% of wild-type in vitro NADH-nitrate reductase activity and lacked detectable xanthine dehydrogenase activity. The plants possessed normal nitrite reductase activity and an inducible NADH-cytochrome c reductase activity. We conclude that loss of nitrate reductase activity is due to a cnx-type mutation affecting the formation of a functional cofactor.

Assimilatory nitrate reductase from the green alga Ankistrodesmus braunii.

Assimilatory nitrate reductase (NAD(P)H-nitrate oxidoreductase, EC 1.6.6.2) from the green alga Ankistrodesmus braunii can be purified to homogeneity by dye-ligand chromatography on blue-Sepharose. The purified enzyme, whose turnover number is 623 s-1, presents an optimum pH of 7.5 and Km values of 13 microM, 23 microM and 0.15 mM for NADH, NADPH and nitrate, respectively. The NADH-nitrate reductase activity exhibits an iso ping pong bi bi kinetic mechanism. The molecular weight of the native nitrate reductase is 467 400, while that of its subunits is 58 750. These values suggest an octameric structure for the enzyme, which has been confirmed by electron microscopy. As deduced from spectrophotometric and fluorimetric studies, the enzyme contains FAD and cytochrome b-557 as prosthetic groups. FAD is not covalently bound to the protein and is easily dissociated in diluted solutions from the enzyme. Its apparent Km value is 4 nM, indicative of a high affinity of the enzyme for FAD. The results of the quantitative analyses of prosthetic groups indicate that nitrate reductase contains four molecules of flavin, four heme irons, and two atoms of molybdenum. The three components act sequentially transferring electrons from reduced pyridine nucleotides to nitrate, thus forming a short electron transport chain along the protein. A mechanism is proposed for the redox interconversion of the nitrate reductase activity. Inactivation seems to occur by formation of a stable complex of reduced enzyme with cyanide or superoxide, while reactivation is a consequence of reoxidation of the inactive enzyme. Both reactions imply the transfer of only one electron.