NR Enzyme Research Articles

Hydrochloric acid-modified biochar enhances nitrogen retention and microbial diversity in mollisols

The annual decline in the quality of China's mollisols and associated nitrogen loss necessitates effective interventions. This study investigates the effects of hydrochloric acid-modified biochar (BC-H700), derived from corn straw, on nitrogen retention and microbial diversity in mollisols through pot experiments. Through 100 days of continuous study found the application of 6 % BC-H700 significantly improved soil properties, increasing pH by 18.60 %, organic matter by 1.6 %, ammonium nitrogen by 26.96 %, and nitrate nitrogen by 23.04 %. By inquiring impact of BC-H700 on microbial enzyme activities in 60-day revealing BC-H700 also enhanced the activities of key nitrogen-transforming enzymes (AMO, NOR, NOS) and functional genes (nirK, norB, nosZ) while reducing narG gene abundance and NR enzyme activity. These changes indicate that BC-H700 promotes nitrification, inhibits denitrification, enhances nitrogen retention, and reduces N2O emissions. Furthermore, BC-H700 improved microbial diversity, increasing the abundance of beneficial bacteria such as Actinobacteria, Proteobacteria, Nitrospirae, and Gemmatimonadetes. The main environmental factors affecting soil bacterial community structure were identified as organic matter, NO3-, NO2-, AMO, NOR, and NOS. Overall, BC-H700 facilitates nitrogen transformation and retention through complex interactions with soil properties and microbes. This study contributes to understanding biochar's role in soil nitrogen management and offers practical insights for improving mollisol health.

Can Foliar-Applied Omeprazole Improve the Yield, Assimilation, Recovery and Nitrogen Use Efficiency in Bean Plants?

The low efficiency of nitrogen (N) fertilizers is a frequent problem in agriculture that impacts the environment. Omeprazole (OMP) has been reported to promote N uptake and assimilation in tomato, basil, and corn. However, information about the effect of omeprazole on N assimilation, recovery, and N use efficiency parameters for bean plants is limited. Therefore, the objective of the present study was to determine the effect of foliar applications of OMP at 0, 1, 10, and 100 µM on nitrogen assimilation, growth, yield, nitrogen use efficiency parameters, and recovery percentage in green bean plants. Green bean plants cv. Strike grown in pots were used. Biomass, yield, nitrate reductase activity, photosynthetic pigments concentration, soluble amino acids and protein concentrations, total nitrogen concentration, nitrogen use efficiency parameters, and nitrogen recovery were analyzed. The results obtained indicate that the application of OMP at 1 µM increased yield and biomass, promoted N assimilation through higher NR enzyme activity, higher amino acid concentration, higher N use efficiency coefficient, and allowed a more efficient nitrogen recovery percentage.

Gene expression of a nitrogen tolerance gene ZmNR1 under the influence of different levels of nitrogen in maize

A field experiment was carried out in the College of Agricultural Engineering Sciences - University of Baghdad, during the fall season of 2021 to find out which cultivated cultivars of maize are efficient under nitrogen fertilization. The experiment was applied according to an RCBD (split-plot design with three replications). The cultivars of the experiment (Baghdad, 5018, Sarah) supply three levels of nitrogen fertilizer, which are N1 (100 kg.N/ha), N2 (200 kg.N/ha) and N3 (300 kg.N/ha). The statistical analysis results showed the superiority of the Sarah genotype, which gave the highest value of SOD and CAT enzymes, reaching 11.59 units mg-1 and 10.76 units mg-1 . Protein sequentially, while cultivar5018 outperformed as it gave the highest value of POD enzyme, which was 5.43 units mg-1.protein, and there were no significant differences between genotypes in NR enzyme. The increase in nitrogen fertilizer caused an increase in the values of oxidation and reduction enzymes, as the nitrogen level N3 gave the highest value for SOD, POD, CAT and nitrate reduction enzymes NR, reaching 11.59 mg-1 and 6.94 mg-1 units. Protein and 16.40 mg-1 units. Protein and 6.30 mg-1 units. Protein sequentially. The results of the molecular analysis using the Real-Time PCR technique showed the expression of the ZmNR1 gene. The analysis showed that the cultivated genotypes contained the gene in varying proportions as the gene expression increased in the compositions to which the nitrogen fertilizer was added. The value ranged from (0.16) to (49.46) times (a copy of the gene), where the highest expression of the gene was (49.46) for the Sarah cultivar when The nitrogen level N2 also gave the same gene expression ZmNR1 (15.01) folds. The cultivars of maize varied among them in their tolerance to excess or deficiency of nitrogen and in their ability to express the ZmNR1 gene, one of the most important nitrogen-carrying genes for maize crops. Keywords: maize, gene expression, nitrate reductase, antioxidant enzymes

INTRODUCING SEVERAL WHEAT GENOTYPES AND TESTING THEM FOR PLANTING UNDER WESTERN PARTS OF IRAQ CONDITIONS

A field experiment was carried out in the western parts of Iraq aimed to screen 21 wheat genotypes introduced to arid and semi-arid areas by the Ministry of Science and technology beside 3 registered genotypes (IRAQ, Diar, and Mahmodiah) for comparison. A randomized completely block design was used to achieve this experiment with three replications for each genotype. The results of this study showed that recently introduced genotypes and planted for the second season sequentially in the western parts of Iraq of which promising genotypes to be nominated for other plant breeding programs beside they can be registered after planting them for other seasons to ensure their stability in terms of growth and production under such conditions. The seeds of genotypes varied in their content of Mo and N and this indicates their variation in growth and production as well as metabolism processes in the plant. Also, this was confirmed the variation in the activity of NR enzyme which in turn depends on Mo and assimilates N in the plant. It is worth mentioning that the activity of this enzyme was superior in genotype 29 which also was superior in yield (ton ha-1). Genotype 31 was superior by giving the highest average of yield over most introduced genotypes as well as registered ones. It can be recommended to use genotypes with high yields to be used in plant breeding programs.

Effect of LED Spectrum on the Quality and Nitrogen Metabolism of Lettuce Under Recycled Hydroponics.

Light quality optimization is an efficient method for improving the growth and quality of lettuce in plant factories. In this study, lettuce seedlings were illuminated under different light-emitting diode (LED) lights, namely, red-blue (RB), red-blue-green (RBG), red-blue-purple (RBP), and red-blue-far-red (RBF) LED lights, to investigate the effect of light quality on growth, quality, and nitrogen metabolism. The combination of 75% red and 25% blue light was set as the basic light source, and 20% of green, purple and far-red light were added to basic light source, respectively. All the treatments were set to 200 μmol m–2 s–1. Results showed that the fresh weight and dry weight of aboveground lettuce under RBG, RBP, and RBF treatments were significantly lower than those under the RB treatment because of the decrease in the effective photon flux density for chlorophyll absorption. The vitamin C content of the lettuce leaves was increased by about 23% with the addition of purple light. For nitrate reduction, the addition of green light significantly increased the nitrite content of the lettuce leaves. It also promoted the reduction from nitrite to ammonium through the activation of the nitrite reductase (NiR) expression and enzyme activity. The nitrate and ammonium content decreased with the addition of purple light because of the inhibited NR and NiR expression and enzyme activity. For nitrogen assimilation, individual (e.g., Asp, Glu, and Leu) and total amino acids were induced to increase by adding green, purple, and far-red light. The addition of light was hypothesized to have inhibited protein biosynthesis, thereby causing the accumulation of amino acids. Correlation analysis showed that the relative expression levels between HY5 and NR/NiR presented a significantly negative correlation. Transcription factor HY5 might mediate the regulation of light quality on nitrogen metabolism by inhibiting NR and NiR expressions. It might also exert a negative effect on nitrate reduction. Further studies via genome editing techniques on the identification of HY5 functions for nitrate assimilation will be valuable. Nevertheless, the results of this work enrich the understanding of the effect of light quality on nitrate metabolism at the level of gene expression and enzyme activity.

Atmospheric ammonia concentration modulates soil enzyme and microbial activity in an oak forest affecting soil microbial biomass

The present work was carried out to assess the effect of atmospheric ammonia (NH3) on soil physicochemical properties, soil enzymatic activities (β-glucosidase -β-GLU-, nitrate reductase –NR-, urease –UR-, protease –PRO-, acid phosphatase –PHO-, dehydrogenase –DHA-), soil microbial biomass and soil respiration. The study was conducted along a NH3 gradient in a Q. pubescens Milld. forest in the vicinity of two livestock farms. Because of NHy (NHy: NH3 and NH4+) deposition, N saturation was detected up to 330 m from the farms. This excess of N led to a decrease in soil C:N and an increase in soil nitrification processes, which resulted in an accumulation of the heavy N isotope (15N) in the soil. N saturation was also reflected in the activity of NR enzyme, which was inhibited. On the other hand, while UR enzyme was inhibited close to the farms possibly due to the high amount of N-NH4+ resulting from the hydrolysis of NH3, PRO activity was stimulated by the presence of organic nitrogen compounds and the need of soil organisms to meet the C demand. In addition, the activity of PHO and β-GLU enzymes was regulated by the relative amount of C and P that organisms need. Regarding biological variables, enhanced NH3 reduced soil microbial biomass and biomass respiratory efficiency. Finally, soil enzyme activities and soil microbial biomass have proved to be good biological indicators of soil quality.

Effect of Pgrs and Nutrients on Growth, Nitrate Reductase Activity, Soluble Protein, Proline Content and Yield in Bhendi Hybrid Under Salinity

An experiment was conducted to study the effect of PGRs { gibberellic acid (10 ppm), brassinolide (0.5 ppm), salicylic acid (100 ppm), ascorbic acid (100 ppm), benzyl amino purine (5 ppm)} and nutrients { K2SO4 (0.5%) + FeSO4 (0.5%) + Borax (0.3%) mixture and 19:19:19 (1%) mixture} on growth, NR enzyme activity, proline, soluble protein content and yield of bhendi hybrid (COBh H 1) under saline condition. The treatments were given at 25 and 45 DAS as foliar spray. The results showed that, nitrate reductase activity, soluble protein and plant growth were reduced under saline condition where as proline content was increased compared to absolute control. Foliar application of PGRs and nutrients enhanced the NR activity, proline, soluble protein and plant growth under saline condition. Among the PGRs and nutrients, foliar application of salicylic acid (100 ppm) and brassinolide (0.5 ppm) showed the better performance to mitigate the effect of salinity.

Controlled biosynthesis of silver nanoparticles using nitrate reductase enzyme induction of filamentous fungus and their antibacterial evaluation

The controlled synthesis of silver nanoparticles (AgNPs) using cell-free filtrate of Fusarium oxysporum fungus was investigated. The effect of fungal incubation period on nanoparticle formation and nitrate reductase enzyme activity was studied using UV–visible spectroscopy and Harley assay, respectively. The highest AgNP formation was observed in the cell-free filtrate of biomass harvested at the early stationary phase where the NR enzyme activity is the maximum. Mixing of the cell-free filtrates of fungal cultures obtained at 23, 28, and 33 °C with silver nitrate solution confirms the higher productivity of AgNP biosynthesis using the cell-free filtrate of fungus incubated at 28 °C. The effect of some factors such as carbon and nitrate sources and light in fungal incubation period on nitrate reductase induction and AgNP formation was also evaluated. In conclusion, increasing nitrate and carbon sources and presence of light induced NR enzyme and produced AgNPs with smaller size, higher monodispersity, and productivity. Results revealed that the presence of ammonium prevents the NR enzyme secretion and causes to the lower productivity of AgNPs.

Biogenic synthesis of Iron oxide nanoparticles via optimization of nitrate reductase Enzyme using statistical experimental design

Statistically designed experiments were proceeded to evaluate nutritional and environmental parameters that affect nitrate reductase enzyme (NR) activity in Achromobacter sp. KT735046. Plackett-Burman design was performed for screening and identifying efficiently the significance of 15 culture conditions influencing NR activity. FeCl3·6H2O, Na2MoO4·2H2O and CuSO4.5H2O were the most significant variables positively influencing, whereas pH was the most significant negative contributors. The optimal levels of significant factors were further predicted from five level factorial designs, Central Composite Design (CCD). The optimum parameter values were CuSO4.5H2O (60.375) mg/l, FeCl3·6H2O (240 mg /L), pH (6.135) and Na2MoO4·2H2O (150 mg /L). The biogenic Iron oxide nanoparticles "IONPs" were characterized; the dark brown IONPs exhibit maximum absorption from 400 to 464 nm, XRD reveals crystallite rhombohedral hematite, EDX confirms presence of 62% of iron, TEM illustrates formation of tiny IONPs, 1.4 and 2.8 nm in size, ξ potential and PDI recorded -42.9 mV and 0.268 respectively exhibiting high stability and monodispersed with no agglomeration by the help of biomolecules. This study conclude that Achromobacter sp. KT735046 consider being biofactory for synthesis nanoparticles by the help of NR enzyme and it may be the first time to optimize Achromobacter sp. KT735046 for IONPs biosynthesis using statistical designs.Â

Reduction of nitrates in Cucumis sativus L. seedlings II. Influence of tungsten and vanadium on nitrate reductase and adenosine triphosphatase activities

ATPases isolated from the roots of cucumber seedlings activated by Mg<sup>+2</sup> ions in experiments in vitro, were fairly distinctly inhibited by Ca<sup>-2</sup> ions, very slightly inhibited by fluorides and molybdenum ions while NO<sub>3</sub><sup>-</sup> anions had no effect on the level of ATPase activity studied. Introduction into the nutrient of 10<sup>-4</sup> M Na<sub>2</sub>WO<sub>4</sub> or 10<sup>-3</sup> M Na VO<sub>3</sub> (inhibitors of nitrate reductase NR) distinctly inhibited activity of the ATPase under study especially of fractions IIa and III, and inhibited NR activity and lowered uptake of NO<sub>3</sub><sup>-</sup>. WO<sub>4</sub><sup>-2</sup> and VO<sub>3</sub><sup></sup> inhibited to the same extent absorption and reduction of NO<sub>3</sub><sup>-</sup> in the initial phase of NR induction, whereas at a later stage both inhibitors checked reduction to a greater degree than uptake of NO<sub>3</sub><sup>-</sup>. The results indicate the possibility of certain ATPase participation in assimilating nitrates, and suggest that in the initial stage of biosynthesis of the NR enzyme system, activity of the enzyme is distinctly dependent upon NO<sub>3</sub><sup>-</sup> transport and the level of NR activity limited by the amount of nitrate taken up. At a later an additional mechanism of NO<sub>3</sub><sup>-</sup> transport probably functions, not connected with simultaneous reduction of nitrates. On the basis of results the Butz and Jackson (1977) hypothesis concerning a model for the absorption and reduction of NO<sub>3</sub><sup>-</sup> by plant tissues is discussed.

Application of exogenous substances reduces tobacco-specific nitrosamines content by regulating biosynthesis of nicotine and nitrite in burley tobacco

Tobacco-specific nitrosamines (TSNAs) are carcinogenic chemicals found in tobacco plants. The increasing health consciousness of individuals had led to an increased interest in research on reducing TSNAs content. The aim of this study was to use a pot experiment in which exogenous substances were applied to burley tobacco to dissect the mechanism of TSNAs production. The results indicated that spraying the exogenous substances IAA, NAA, SA and combination thereof on burley tobacco after topping decreased TSNAs content by 2.69–29.4 % in upper leaves and 0.23–39.3 % in middle leaves without affecting total sugar, total nitrogen, potassium and chlorine contents. The application of exogenous substances could down-regulate expression of the NR gene and the activity of the NR enzyme, resulting in less accumulation of the TSNAs precursor nitrite. The exogenous substances significantly reduced nicotine accumulation, which was consistent with low enzyme activities and the down-regulated expressions of genes involved in nicotine biosynthesis, especially significant in the case of quinolinate phosphoribosyltransferase. These results suggested that the application of exogenous substances on burley tobacco after topping could reduce TSNAs content which may be attributed to the regulation of exogenous substances on nitrite and nicotine. This also implies one potential improvement to agronomic practices aimed at controlling the accumulation of TSNAs in burley tobacco.

Effect of Sewage Irrigation on Nitrate Accumulation and Nitrate Reductase Activity in Leafy Vegetables

The effect of sewage water irrigation along with the N-fertilizers on NO3 accumulation and on the in vivo NR assay in three leafy vegetable crops were studied. It was found that the NO3 content in the leaves from the experimental sites were around two fold than the control and the NR enzyme activity was comparatively more in the control samples. The in vivo NRA with NaCl treatment increased at low level of NaCl where as with high level of NaCl, the enzyme activity decreased considerably. This is suggestive of the fact that NO3 accumulation may be due to antagonistic effect of chloride ions.

Nitrate reduction associated with respiration in Sinorhizobium meliloti 2011 is performed by a membrane-bound molybdoenzyme

The purification and biochemical characterization of the respiratory membrane-bound nitrate reductase from Sinorhizobium meliloti 2011 (Sm NR) is reported together with the optimal conditions for cell growth and enzyme production. The best biomass yield was obtained under aerobic conditions in a fed-batch system using Luria-Bertani medium with glucose as carbon source. The highest level of Sm NR production was achieved using microaerobic conditions with the medium supplemented with both nitrate and nitrite. Sm NR is a mononuclear Mo-protein belonging to the DMSO reductase family isolated as a heterodimeric enzyme containing two subunits of 118 and 45 kDa. Protein characterization by mass spectrometry showed homology with respiratory nitrate reductases. UV-Vis spectra of as-isolated and dithionite reduced Sm NR showed characteristic absorption bands of iron-sulfur and heme centers. Kinetic studies indicate that Sm NR follows a Michaelis-Menten mechanism (K (m) = 97 ± 11 μM, V = 9.4 ± 0.5 μM min(-1), and k (cat) = 12.1 ± 0.6 s(-1)) and is inhibited by azide, chlorate, and cyanide with mixed inhibition patterns. Physiological and kinetic studies indicate that molybdenum is essential for NR activity and that replacement of this metal for tungsten inhibits the enzyme. Although no narGHI gene cluster has been annotated in the genome of rhizobia, the biochemical characterization indicates that Sm NR is a Mo-containing NR enzyme with molecular organization similar to NarGHI.

Control of nitrate reductase by circadian and diurnal rhythms in tomato.

Nitrate reductase (NR, EC 1.6.6.1) is a key regulatory enzyme in the assimilation of nitrate into amino acids in plant leaves. NR activity is intricately controlled by multifarious regulatory mechanisms acting at different levels ranging from transcription to protein degradation. It is among the few enzymes known to have a robust circadian rhythm of enzyme activity in many plant species. Although many aspects of NR regulation have been studied in depth, how these different types of control interact in a plant to deliver integrated control of activity in leaves over the course of the day has not been systematically investigated. This work documents that NR in young tomato (Lycopersicon esculentum Mill.) leaves has an endogenous rhythm in mRNA and protein level, which in nearly all circumstances are in phase with the rhythm in NR enzyme activity. Our data show that the diurnal control of NR activity in tomato leaves rests primarily with circadian regulation of Nia gene expression. The accompanying oscillations in protein level in tomato are made possible by a short half-life of NR protein that is approx. 6 h under normal conditions and approx. 2.5 h when plants are darkened during mid-day. NR post-transcriptional regulation via phosphorylation and subsequent 14-3-3 protein binding has a physiologically vital but secondary regulatory role in tomato of rapidly deactivating NR in response to changes in light intensity that cannot be anticipated by circadian timing. The post-translational reactivation of phosphorylated NR appears to have its primary physiological role in tomato leaves in reversing the down regulation of NR following transient shading events. Although there is a significant steady-state pool of apparently inactive NR throughout the diurnal, our data indicate that tomato leaves are unable to draw on this reserve to compensate for NR protein that is degraded during shading.

Inhibition of Wheat Nitrate Reductase Activity by Zinc

The effect of Zn2- on nitrate reductase (NR, EC 1.6.6.1) activity was studied in bota wheat (Triticum aestivum cv. Oasis) leaves and in the NR enzyme partially purified from wheat leaves. Leaf segments were floated on 0 to 5 mM ZnSO4 solutions (pH 6.0) for 24 h under continuous light. Zn2- at 250 μM decreased NR activity and increased membrane permeability. However, parameters of cellular oxidative damage were scarcely affected by Zn2- treatments. Accordingly, the decrease of NR activity induced by Zn2- was not prevented by benzoate (a scavenger of oxygen radicals). The effect of Zn2- was dependent on leaf age: it decreased NR activity in mature but not in young leaves. Zn2 inhibited the partially purified NR. This inhibition was not reversed by either co- or post-incubation with cysteine, and the amount of -SH groups of the purified NR was not affected by Zn2+ indicating that Zn2- inhibition does not involve key -SH groups of the enzyme. However, o-phenantroline both prevented and reversed Zn2+-induced NR inhibition. We concluded that the effect of Zn2+ on NR activity in vivo is not associated with an increase in active oxygen generation and involves a direct and reversible inhibition of the enzyme.

Nitrate assimilation and biomass production in Sesamum indicuml. Seedlings in a lead enriched environment

Seed germination was delayed and seedling growth inhibited by 0.04 to 1.9 mM Pb+2 in Sesamum indicum L. var HT-I. In root, shoot and leaf Pb+2 accumulation increased with increasing Pb+2 concentration in the nutrient solution. In root and leaf tissues in vivo and in vitro nitrate reductase activity was inhibited significantly which was well correlated with the concentration of Pb+2 supplied and its accumulation in the plant parts. The inhibition of the NR enzyme activity could be, however, reversed by simultaneous treatment of Sesamum seedlings with K2HPO4, CaCl2 and KNO3 dissolved in nutrient solution. Total organic N and soluble protein of roots and shoots/leaves, on the other hand, increased with increasing concentration of Pb+2 while the same treatment caused a decrease in the N content of cotyledons. It appears therefore, that the increase in N and protein in the roots, shoots/leaves may be a result of increased translocation of N from the cotyledons to the roots and shoots/leaves during early seedling growth in a Pb+2 enriched environment.

Nitrate reductase and nitrate accumulation in relation to nitrate toxicity in Boronia megastigma

Moderate levels of N were toxic to the native Australian plant boronia (Boronia megastigma Nees). As NO‐3 is the major N form available for plants under cultivated conditions, NO‐3 reduction and accumulation patterns in boronia were examined following the supply of various levels of NO‐3 to understand the physiological basis of this toxicity. At a low level of supplied NO‐3 [15 mmol (plant)‐1], NO‐3 was reduced without any detectable accumulation and without nitrate reductase activity (NRA) reaching its maximum capacity. When higher NO‐3 levels [≥25 mmol (plant)‐1] were supplied, both NRA and NO‐3 accumulation increased further. However, NRA increased to a maximum of ca 500 nmol NO‐3 (g fresh weight)‐1 h‐1, both in the roots and leaves, irrespective of a 4‐fold difference in the levels of supplied NO‐3, whereas NO‐3 continued to accumulate in proportion to the level of supplied NO‐3. Chlorotic toxicity symptoms appeared on the leaves at an accumulation of ca 32 μmol NO‐3 (g fresh weight)‐1. High endogenous NO‐3 concentrations inhibited NRA. The low level of NRA in boronia was not limited by NO‐3 or electron donor availability. It is concluded that the low NR enzyme activity is a genetic adaptation to the low NO‐3 availability in the native soils of boronia. Thus, when NO‐3 supply is high, the plat cannot reduce it at high rates, leading to large and toxic accumulations of the ion in the leaf tissues.

The Conversion of Nitrite to Nitrogen Oxide(s) by the Constitutive NAD(P)H-Nitrate Reductase Enzyme from Soybean

A two-step purification protocol was used in an attempt to separate the constitutive NAD(P)H-nitrate reductase [NAD(P)H-NR, pH 6.5; EC 1.6.6.2] activity from the nitric oxide and nitrogen dioxide (NO((x))) evolution activity extracted from soybean (Glycine max [L.] Merr.) leaflets. Both of these activities were eluted with NADPH from Blue Sepharose columns loaded with extracts from either wild-type or LNR-5 and LNR-6 (lack constitutive NADH-NR [pH 6.5]) mutant soybean plants regardless of nutrient growth conditions. Fast protein liquid chromatography-anion exchange (Mono Q column) chromatography following Blue Sepharose affinity chromatography was also unable to separate the two activities. These data provide strong evidence that the constitutive NAD(P)H-NR (pH 6.5) in soybean is the enzyme responsible for NO((x)) formation. The Blue Sepharose-purified soybean enzyme has a pH optimum of 6.75, an apparent K(m) for nitrite of 0.49 millimolar, and an apparent K(m) for NADPH and NADH of 7.2 and 7.4 micromolar, respectively, for the NO((x)) evolution activity. In addition to NAD(P)H, reduced flavin mononucleotide (FMNH(2)) and reduced methyl viologen (MV) can serve as electron donors for NO((x)) evolution activity. The NADPH-, FMNH(2)-, and reduced MV-NO((x)) evolution activities were all inhibited by cyanide. The NADPH activity was also inhibited by p-hydroxymer-curibenzoate, whereas, the FMNH(2) and MV activities were relatively insensitive to inhibition. These data indicate that the terminal molybdenum-containing portion of the enzyme is involved in the reduction of nitrite to NO((x)). NADPH eluted both NR and NO((x)) evolution activities from Blue Sepharose columns loaded with extracts of either nitrate- or zero N-grown winged bean (Psophocarpus tetragonolobus [L.]), whereas NADH did not elute either type of activity. Winged bean appears to contain only one type of NR enzyme that is similar to the constitutive NAD(P)H-NR (pH 6.5) enzyme of soybean.

Immunological comparison of spinach and Chlorella nitrate reductase

Polyclonal antibodies raised against spinach ( Spinacea oleracea) and Chlorella vulgaris nitrate reductases, cross-react with the enzymes with limited recognition. Monoclonal antibodies, previously raised against spinach NR, which bound to Chlorella NR were detected by direct enzyme linked immunosorbent assay (ELISA) and immunoblotting but did not inhibit Chlorella NR enzyme activities. Two new monoclonal antibodies raised against spinach NR (designated AFRC MAC 231 and 232) have been obtained, which recognised both native and denatured spinach NR and Chlorella NR with high avidity without inhibiting their enzyme activities.