NADH-dependent Nitrate Reductase Research Articles

Decreasing nitrogen assimilation under drought stress by suppressing DST-mediated activation of Nitrate Reductase 1.2 in rice

Nitrogen is an essential nutrient for plant growth and development, and plays vital roles in crop yield. Assimilation of nitrogen is thus fine-tuned in response to heterogeneous environments. However, the regulatory mechanism underlying this essential process remains largely unknown. Here, we report that a zinc-finger transcription factor, drought and salt tolerance (DST), controls nitrate assimilation in rice by regulating the expression of OsNR1.2. We found that loss of function of DST results in a significant decrease of nitrogen use efficiency (NUE) in the presence of nitrate. Further study revealed that DST is required for full nitrate reductase activity in rice and directly regulates the expression of OsNR1.2, a gene showing sequence similarity to nitrate reductase. Reverse genetics and biochemistry studies revealed that OsNR1.2 encodes an NADH-dependent nitrate reductase that is required for high NUE of rice. Interestingly, the DST-OsNR1.2 regulatory module is involved in the suppression of nitrate assimilation under drought stress, which contributes to drought tolerance. Considering the negative role of DST in stomata closure, as revealed previously, the positive role of DST in nitrogen assimilation suggests a mechanism coupling nitrogen metabolism and stomata movement. The discovery of this coupling mechanism will aid the engineering of drought-tolerant crops with high NUE in the future.

Fusarium as a Novel Fungus for the Synthesis of Nanoparticles: Mechanism and Applications.

Nanotechnology is a new and developing branch that has revolutionized the world by its applications in various fields including medicine and agriculture. In nanotechnology, nanoparticles play an important role in diagnostics, drug delivery, and therapy. The synthesis of nanoparticles by fungi is a novel, cost-effective and eco-friendly approach. Among fungi, Fusarium spp. play an important role in the synthesis of nanoparticles and can be considered as a nanofactory for the fabrication of nanoparticles. The synthesis of silver nanoparticles (AgNPs) from Fusarium, its mechanism and applications are discussed in this review. The synthesis of nanoparticles from Fusarium is the biogenic and green approach. Fusaria are found to be a versatile biological system with the ability to synthesize nanoparticles extracellularly. Different species of Fusaria have the potential to synthesise nanoparticles. Among these, F. oxysporum has demonstrated a high potential for the synthesis of AgNPs. It is hypothesised that NADH-dependent nitrate reductase enzyme secreted by F. oxysporum is responsible for the reduction of aqueous silver ions into AgNPs. The toxicity of nanoparticles depends upon the shape, size, surface charge, and the concentration used. The nanoparticles synthesised by different species of Fusaria can be used in medicine and agriculture.

Immobilized enzyme mediated synthesis of silver nanoparticles using cross-linked enzyme aggregates (CLEAs) of NADH-dependent nitrate reductase

NADH-dependent nitrate reductase CLEAs catalyzed synthesis of silver nanoparticles from silver nitrate has been investigated. NADH-dependent nitrate reductase CLEAs catalyzed reduction of silver ions to silver nanoparticles at pH 7.2 using NADH as electron source and 8-hydroxyquinoline as electron shuttle. Fourier transform infrared spectroscopy (FTIR) and Energy dispersive spectroscopy (EDS) showed that 8-hydroxyquinoline also acts as capping agent of silver nanoparticles. The effects of reaction time, reaction temperature, silver nitrate concentration and amount of CLEAs on silver nanoparticles synthesis were studied with aid of UV–Vis spectrophotometer. Optimum silver nanoparticles synthesis occurred after 24 h reaction of 100 mg CLEAs with 1 mM silver nitrate at pH 7.2 and temperature 35 °C. Scanning electron microscopy (SEM), Transmission electron microscopy (TEM) and High resolution transmission electron microscopy (HRTEM) showed the silver nanoparticles were spherical in shape. The particle size calculated using Dynamic light scattering measurements (DLS) was found to be in the range of 5–7 nm. X-ray diffraction (XRD) and selected area electron diffraction (SAED) patterns confirmed formation of crystalline silver nanoparticles with face-centred-cubic (fcc) lattice structure. Notably, nitrate reductase CLEAs showed 80% catalytic yield even after 5 cycles of repeated use in silver nanoparticle synthesis at pH 7.2 and temperature 35 °C. Moreover, silver nanoparticles of same morphology and dimensions were formed in each cycle.

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Nanomaterials have strikingly different physicochemical and optoelectronic properties which are now being widely investigated for a broad spectrum of applications in agriculture, textile, medicine, drug delivery, biochemical sensors, and allied areas. Hereby, the growing need for the fabrication of exotic nanostructures, surface manipulation, and functionalization has inspired us to explore a biological route apart from several available chemical and physical approaches. Recently, mycogenic synthesis has gained considerable attention due to the promising ability of a diverse group of yeast and fungus to produce an array of nanoparticles composed of gold, silver, copper, aluminum, zinc, platinum, cadmium, and even metal composites and oxides. Fungi-assisted synthesis of nanostructures with desirable shapes and sizes is either intracellular or extracellular, where NADH-dependent nitrate reductase enzyme plays an important role in the transformation of metal ions into metal nanoparticles. Morphological features like the size and shape of the nanoparticles depend on the microorganism utilized and the reaction conditions employed during the synthesis process. This mycogenic green synthesis of nanoparticles is safe, environmentally benign, rapid, efficient, and biocompatible. In this chapter, we provide an elaborate description of the recent advances of nanobiotechnological applications of fungi that include synthesis, possible mechanisms, and their potential applications.

In vivo synthesis of selenium nanoparticles by Halococcus salifodinae BK18 and their anti-proliferative properties against HeLa cell line.

Nanoparticles synthesis by bacteria and yeasts has been widely reported, however, synthesis using halophilic archaea is still in a nascent stage. This study aimed at the intracellular synthesis of selenium nanoparticles (SeNPs) by the haloarchaeon Halococcus salifodinae BK18 when grown in the presence of sodium selenite. Crystallographic characterization of SeNPs by X-ray diffraction, Selected area electron diffraction, and transmission electron microscopy exhibited rod shaped nanoparticles with hexagonal crystal lattice, a crystallite domain size of 28 nm and an aspect ratio (length:diameter) of 13:1. Energy disruptive analysis of X-ray analysis confirmed the presence of selenium in the nano-preparation. The nitrate reductase enzyme assay and the inhibitor studies indicated the involvement of NADH-dependent nitrate reductase in SeNPs synthesis and metal tolerance. The SeNPs exhibited good anti-proliferative properties against HeLa cell lines while being non-cytotoxic to normal cell line model HaCat, suggesting the use of these SeNPs as cancer chemotherapeutic agent. This is the first study on selenium nanoparticles synthesis by haloarchaea.

Green Synthesis of Silver Nanoparticles by Haloarchaeon <i>Halococcus salifodinae </i>BK6

Nanobiotechnology is a multidisciplinary branch of nanotechnology which includes fabrication of nanosized materials using biological approaches. Highly structured metallic and metal sulfide nanoparticles have been reported to be synthesized by numerous bacteria, fungi, yeasts and viruses. However, biosynthesis of nanoparticles by Haloarchaea (salt-loving archaea) of the third domain of life, Archaea, is in its nascent stages. In this study, we report the intracellular synthesis of stable, mostly spherical silver nanoparticles (SNPs) by the haloarchaeal isolateHalococcus salifodinaeBK6. The isolate adapted to silver nitrate was found to exhibit growth kinetics similar to that of cells unexposed to silver nitrate. The nitrate reductase enzyme assay and the enzyme inhibitor studies showed the involvement of NADH dependent nitrate reductase in silver tolerance, reduction, and synthesis of SNPs. UV visible spectroscopy, XRD, TEM and EDAX were used for characterization of SNPs. The XRD exhibited characteristic Bragg peaks of face centered cubic silver with crystallite domain size of 26 nm and 12 nm for SNPs synthesized in NTYE and halophilic nitrate broth, respectively. TEM analysis exhibited an average particle size of 50.3 nm and 12 nm for SNPs synthesized in NTYE and halophilic nitrate broth (HNB), respectively. The as synthesized SNPs exhibited antimicrobial activity against both Gram positive and Gram negative organisms.

Preparation of stable cross-linked enzyme aggregates (CLEAs) of NADH-dependent nitrate reductase and its use for silver nanoparticle synthesis from silver nitrate

The NADH-dependent nitrate reductase from Fusarium oxysporum cell extract was directly immobilized as cross linked enzyme aggregates (CLEAs) and investigated for the synthesis of silver nanoparticles by a reduction of silver nitrate. The effects of precipitant type and cross-linking on activity recovery of enzyme in CLEAs were studied. After aggregation of enzyme with ammonium sulfate followed by cross-linking formed aggregates for 4h with 8mM glutaraldehyde, 93% activity recovery was achieved in CLEAs with enhanced thermal stability at 50°C and 40°C. Scanning electron microscopy analysis showed that immobilized NADH-dependent nitrate reductase was of spherical structure. CLEAs showed 90% catalytic yield even after 4cycles of repeated use in silver nanoparticle synthesis at pH7.2 and temperature 35°C.

Synthesis of silver nanoparticles using haloarchaeal isolate Halococcus salifodinae BK3

Numerous bacteria, fungi, yeasts and viruses have been exploited for biosynthesis of highly structured metal sulfide and metallic nanoparticles. Haloarchaea (salt-loving archaea) of the third domain of life Archaea, on the other hand have not yet been explored for nanoparticle synthesis. In this study, we report the intracellular synthesis of stable, mostly spherical silver nanoparticles (AgNPs) by the haloarchaeal isolate Halococcus salifodinae BK3. The culture on adaptation to silver nitrate exhibited growth kinetics similar to that of the control. NADH-dependent nitrate reductase was involved in silver tolerance, reduction, synthesis of AgNPs, and exhibited metal-dependent increase in enzyme activity. The AgNPs preparation was characterized using UV-visible spectroscopy, XRD, TEM and EDAX. The XRD analysis of the nanoparticles showed the characteristic Bragg peaks of face-centered cubic silver with crystallite domain size of 22 and 12 nm for AgNPs synthesized in NTYE and halophilic nitrate broth (HNB), respectively. The average particle size obtained from TEM analysis was 50.3 and 12 nm for AgNPs synthesized in NTYE and HNB, respectively. This is the first report on the synthesis of silver nanoparticles by haloarchaea.

Growth, photosynthesis, nitrogen partitioning and responses to CO2 enrichment in a barley mutant lacking NADH‐dependent nitrate reductase activity

Plant growth, photosynthesis and leaf constituents were examined in the wild-type (WT) and mutant nar1 of barley (Hordeum vulgare L. cv. Steptoe) that contains a defective structural gene encoding NADH-dependent nitrate reductase (NADH-NAR). In controlled environment experiments, total biomass, rates of photosynthesis, stomatal conductance, intercellular CO(2) concentrations and foliar non-structural carbohydrate levels were unchanged or differed slightly in the mutant compared with the WT. Both genotypes displayed accelerated plant growth rates when the CO(2) partial pressure was increased from 36 to 98 Pa. Total NADH-NAR activity was 90% lower in the mutant than in the WT, and this was further decreased by CO(2) enrichment in both genotypes. Inorganic nitrate was greater in the mutant than in the WT, whereas in situ nitrate assimilation by excised leaves was two-fold greater for the WT than for the mutant. Foliar ammonia was 50% lower in the mutant than in the WT under ambient CO(2). Ammonia levels in the WT were decreased by about one-half by CO(2) enrichment, whereas ammonia was unaffected by elevated CO(2) in mutant leaves. Total soluble amino acid concentrations in WT and mutant plants grown in the ambient CO(2) treatment were 30.1 and 28.4 micromol g(-1) FW, respectively, when measured at the onset of the light period. Seven of the twelve individual amino acids reported here increased during the first 12 h of light in the ambient CO(2) treatment, leading to a doubling of total soluble amino acids in the WT. The most striking effect of the mutation was to eliminate increases of glutamine, aspartate and alanine during the latter half of the photoperiod in the ambient CO(2) treatment. Growth in elevated CO(2) decreased levels of total soluble amino acids on a diurnal basis in the WT but not in mutant barley leaves. The above results indicated that a defect in NADH-NAR primarily affected nitrogenous leaf constituents in barley. Also, we did not observe synergistic effects of CO(2) enrichment and decreased foliar NADH-NAR activity on most N-containing compounds.

Modifications of the activity of nitrate reductase from cucumber roots

The regulatory properties of NADH-dependent nitrate reductase (NR) in desalted root extracts from hydroponically grown cucumber (Cucumis sativus L.) seedlings were examined. The lowest activity of NR was detected in extracts incubated with Mg2+ and ATP. An inhibitory effect of Mg-ATP was cancelled in the presence of staurosporine (the protein kinase inhibitor) and completely reversed after addition of ethylenediaminetetraacetate (EDTA) as well as AMP into reaction mixture. Reactivation of enzyme due to AMP presence, contrary to the chelator-dependent NR activation, was sensitive to microcystin LR (the protein phosphatase inhibitor). Above results indicated that the nitrate reductase in cucumber roots was regulated through reversible phosphorylation of enzyme protein. A drop in the activity of NR was also observed after incubation of enzyme at low pH. At low pH, the presence of ATP alone in the incubation medium was sufficient to inactivate NR, indicating that H+ can substitute the Mg2+ in formation of an inactive complex of enzyme. ATP-dependent inactivation of NR at low pH was prevented by staurosporine and reversed by AMP. However, AMP action was not altered by microcystin LR suggesting that in low pH the nucleotide induced reactivation of NR is not limited to the protein phosphorylation.

Nitric oxide generation during early germination of sorghum seeds

Germination of sorghum embryonic axes, defined as the development of a root 1–2 mm long, started after 24 h of imbibition. A distinctive EPR signal for the adduct MGD–Fe–NO was detected in the homogenates from axes isolated from seeds imbibed in the presence of 12 mM nitrate in a time-dependent manner. Also a close association between MGD–Fe–NO adduct content in the homogenates and nitrate supplementation to the incubation medium was observed. The activities of the NADH-dependent nitrate reductase (NR) and nitric oxide synthase were measured in axes, by the detection of NO by EPR under conditions of maximal supplementation of substrates. A significant increase in both enzymatic activities was determined between 24 and 30 h of imbibition. Total content in sorghum axes of β-carotene and α-tocopherol increased significantly as the germination occurred, however the total content of tocopherols was not affected over the studied period. The data reported here are the first observations employing EPR to assess for NR and NOS activities simultaneously in an active growing tissue. Moreover, since the increase in NO content preceded the initiation of phase II of development and the sharp increase in oxygen consumption, a potential role for NO as a signal molecule should be considered.

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.

Nitrate reductase activation state in barley roots in relation to the energy and carbohydrate status.

The NADH-dependent nitrate reductase (NR, EC 1.6.6.1) in roots of hydroponically grown barley seedlings was extracted, desalted and the activity measured in buffer containing either Mg2+ (10 mM) or EDTA (5 mM). The former gives the actual NR activity (NRact) equivalent to dephospho-NR, whereas the latter gives the maximum NR capacity of the dephospho-form (NRmax). Both values together permit an estimation of the NR-phosphorylation state. Changes in NRact and NRmax were followed in response to root aeration or to shoot illumination or shoot removal, and were correlated with sugar contents and adenylate levels. Ethanol formation was also measured in roots differing in NR activity in order to obtain information on the relation between anaerobic alcoholic fermentation and nitrate reduction. In aerated roots, NR was highly phosphorylated (about 80%) and largely inactive. It was partly dephosphorylated (activated) by anoxia or by cellular acidification (pH 4.8 plus propionic acid). Anaerobic activation (dephosphorylation) of NR was stronger at acidic external pH (5) than at slightly alkaline pH (8), although ATP levels decreased and AMP levels increased at pH 5 and at pH 8 to the same extent. Thus, rapid changes in the NR-phosphorylation state in response to anaerobiosis were not directly triggered by the adenylate pool, but rather by cytosolic pH. Under prolonged darkness (24 h) or after shoot removal. NRmax decreased slowly without a large change in the phosphorylation state. This decrease of NRmax was correlated with a large decrease in the sugar content, and was prevented by glucose feeding, which had only minor effects on the phosphorylation state. Cycloheximide also prevented the decrease in NRmax without affecting the phosphorylation state. In contrast, anaerobiosis or cellular acidification prevented the decrease of NRmax and at the same time decreased the NR-phosphorylation state. It is suggested that NR turnover in roots is controlled by several factors: NR synthesis appears to depend on sugar availability, which has little effect on the phosphorylation state; in addition, NR degradation appears to be strongly affected by the phosphorylation state in such a way that the inactive phospho-NR is a better substrate for NR degradation than the dephospho-form. The rate of anaerobic ethanol formation was not affected by NR activity, indicating that the purpose of NR activation under hypoxia or anoxia is not to decrease or prevent alcoholic fermentation.

Reduced Level of NADH-dependent Nitrate Reductase Activity in Rice Mutant M819 Due to Deletion of a Valine Residue in Heme Domain.

It was previously reported that the rice nitrate reductase (NR)-deficient mutant M819 (wild type: cv. Norin 8) Iacks the functional heme domain of NADH-NR polypeptide possibly due to a point mutation or small deletion. In this paper we were able to locate the mutation in the NR apoenzyme gene. Northern blot analysis using a rice NADH-NR genomic clone as a probe showed that the size of NR mRNA of M819 was the same as that of the wild type, suggesting that the mutation did not involve a large deletion or insertion in the NADH-NR coding sequence. The DNA fragment containing the coding region of NADH-NR heme domain was amplified by PCR from M 819 and Norin 8. The alignment of nucleotide sequences of the domain revealed that 3 nucleotides (GTC) encoding one valine residue (Val-561 or Val-562) were deleted in M819. Thus, it was demonstrated that there was a point mutation in the coding region of NADH-NR heme dornain in the mutant M819.

Possible interaction between peroxidase and NAD(P)H-dependent nitrate reductase activities of plasma membranes of corn roots

The relationship between the plasma membrane-bound NAD(P)H-nitrate reductase (NR) and a plasma membrane (PM)-bound peroxidase was investigated using highly purified PM vesicles isolated from corn roots. The PM-bound NR activity was strongly enhanced by MnCl 2 and SHAM, which stimulated peroxidase activity. Since both activities, the NAD(P)H-dependent NR and the peroxidase compete for NAD(P)H as electron donor, we propose a model in which a product of peroxidation is able to offer electrons to the nitrate reductase in a more reactive form with respect to NAD(P)H. Out hypothesis was confirmed by experiments in which the effects of inhibitors of peroxidative reactions, catalase, superoxide dismutase, and ascorbate on the PM-bound NR were studied. Results indicate that the putative electron donor for nitrate reduction could be a radicalic species, possibly NAD . . Furthermore, since cytochrome c decreased the activity of the plasma membrane-bound NAD(P)H-dependent NR, cytochrome b 557 might be the site of the enzyme accepting electrons from NAD . . Out results indicate that the PM environment of the NR may be involved in the extent of the membrane-associated nitrate reduction and that redox enzymes at the PM, the NAD(P)H-NR and a peroxidase-like NADH-oxidase, can interact.

Evidence for two different nitrate-reducing activities at the plasma membrane in roots of Zea mays L.

Plasma-membrane (PM) vesicles isolated from 6-d-old corn roots by sucrose gradient centrifugation or two-phase partitioning showed an NADH-dependent nitrate reductase (NR) activity averaging at 40 nmol per milligram PM protein per hour. This membrane-associated NR activity could not be removed from two-phase partitioned PM vesicles by salt washing, osmotic shock treatment, sonication, or freeze-thawing to reverse vesicle sidedness. Therefore, it could not be attributed to contamination of membrane vesicles by the soluble, cytosolic NR. Plasma-membrane vesicles reduced NO~ in the presence of the electron donors NADH or NADPH at an activity ratio of 2.2. The NADH- and NADPH-dependent NR activities of outside-out oriented PM vesicles differed in their sensitivity toward the detergent Brij 58,leading to a latency of 65% or 29% using NADH or NADPH as electron donor, respectively. The activities of NO 3 reduction in the presence of saturating concentrations of NADH and NADPH were additive. Furthermore,both activities were characterized by a different pH dependence with a pH optimum of 7.5 for the NADH-dependent activity and of 6.8 for the NADPH-dependent activity. The membrane-associated NAD(P)H-dependent NR activities responded to different nitrogen nutrition of plants in a manner different from the soluble forms of the enzyme. The data confirm the existence of a corn PM NR and suggest that there may be two different NO₃-reducing enzymes located at the PM of corn roots.

Tanzania's vanishing rain forests — assessment of nature conservation values, biodiversity and importance for water catchment: J. E. Bjorndalen, Agriculture, Ecosystems & Environment, 40(1–4), 1992, pp 313–334

Nitrogen is an essential nutrient for plant growth and development, and plays vital roles in crop yield. Assimilation of nitrogen is thus fine-tuned in response to heterogeneous environments. However, the regulatory mechanism underlying this essential process remains largely unknown. Here, we report that a zinc-finger transcription factor, drought and salt tolerance (DST), controls nitrate assimilation in rice by regulating the expression of OsNR1.2. We found that loss of function of DST results in a significant decrease of nitrogen use efficiency (NUE) in the presence of nitrate. Further study revealed that DST is required for full nitrate reductase activity in rice and directly regulates the expression of OsNR1.2, a gene showing sequence similarity to nitrate reductase. Reverse genetics and biochemistry studies revealed that OsNR1.2 encodes an NADH-dependent nitrate reductase that is required for high NUE of rice. Interestingly, the DST-OsNR1.2 regulatory module is involved in the suppression of nitrate assimilation under drought stress, which contributes to drought tolerance. Considering the negative role of DST in stomata closure, as revealed previously, the positive role of DST in nitrogen assimilation suggests a mechanism coupling nitrogen metabolism and stomata movement. The discovery of this coupling mechanism will aid the engineering of drought-tolerant crops with high NUE in the future.

Rubredoxin as an intermediary electron carrier for nitrate reduction by NAD(P)H in Clostridium perfringens.

The NAD(P)H-dependent nitrate reductase system in Clostridium perfringens was reconstituted with rubredoxin (Rd), nitrate reductase (NaR), and an unadsorbed fraction, on a DEAE-cellulose column, of the extract (designated as fraction A), under nitrogen gas. Ferredoxin in place of Rd was not effective as an electron carrier in this reconstituted system. NAD(P)H-dependent nitrate reducing activity was also obtained by replacing fraction A with ferredoxin-NADP+ reductase from spinach. We propose the following scheme for the electron transfer in this NAD(P)H dependent nitrate reduction system. NAD(P)H----NAD(P)H-Rd reductase----Rd----NaR----NO3-.

New Artificial Electron Donors for in Vitro Assay of Nitrate Reductase Isolated from Cultured Tobacco Cells and Other Organisms

The capacity of bromphenol blue and its analogs to act as electron donors for measurement of in vitro nitrate reductase activity from tobacco cells (Nicotiana tabacum var Techné SP 25 strain) was determined. Competitive inhibition was demonstrated to occur between NADH, the natural electron donor, and bromphenol blue, the artificial electron donor, suggesting that both donors bind to a similar active site on the enzyme. NADH-dependent or bromphenol blue-dependent nitrate reductase activity was carried out by a similar molecular weight protein exhibiting similar antigenic sites. Following ammonium sulfate precipitation, sucrose density gradient and two chromatographic steps, nitrate reductase activity from tobacco cells was purified near homogeneity using bromphenol blue as an electron donor in the absence of measurable NADH-dependent activity. The enzyme is composed of two identical subunits of 83 kilodaltons < Momega < 94 kilodaltons.

Binding of higher plant NADH-dependent nitrate reductase to different triazine dyes

Affinity partitioning in an aqueous two-phase system consisting of dextran, polyethylene glycol and triazine dye-substituted polyethylene glycol was applied to investigate the affinity of NADH-dependent nitrate reductases isolated from tobacco, barley and cucumber to six triazine dyes. The alteration of the partition coefficient of nitrate reductases in the presence and absence of dye-polyethylene glycol conjugates provided qualitative data for the affinity of the enzymes to the triazine dyes. Cibacron Blue F3G-A, widely used as a ligand for purification of nitrate reductase by affinity chromatography had a low affinity for higher plant nitrate reductases compared to other triazine dyes. The strength of interaction between dye and enzyme was not dependent on the enzyme source but on the dye used. The influence of NADH on the binding of triazine dyes to nitrate reductase was also investigated.