Specific Activity Of Nitrate Reductase Research Articles

Inhibition of NO Biosynthetic Activities during Rehydration of Ramalina farinacea Lichen Thalli Provokes Increases in Lipid Peroxidation.

Lichens are poikilohydrous symbiotic associations between a fungus, photosynthetic partners, and bacteria. They are tolerant to repeated desiccation/rehydration cycles and adapted to anhydrobiosis. Nitric oxide (NO) is a keystone for stress tolerance of lichens; during lichen rehydration, NO limits free radicals and lipid peroxidation but no data on the mechanisms of its synthesis exist. The aim of this work is to characterize the synthesis of NO in the lichen Ramalina farinacea using inhibitors of nitrate reductase (NR) and nitric oxide synthase (NOS), tungstate, and NG-nitro-L-arginine methyl ester (L-NAME), respectively. Tungstate suppressed the NO level in the lichen and caused an increase in malondialdehyde during rehydration in the hyphae of cortex and in phycobionts, suggesting that a plant-like NR is involved in the NO production. Specific activity of NR in R. farinacea was 91 μU/mg protein, a level comparable to those in the bryophyte Physcomitrella patens and Arabidopsis thaliana. L-NAME treatment did not suppress the NO level in the lichens. On the other hand, NADPH-diaphorase activity cytochemistry showed a possible presence of a NOS-like activity in the microalgae where it is associated with cytoplasmatic vesicles. These data provide initial evidence that NO synthesis in R. farinacea involves NR.

Nitrogen uptake efficiency of finger millet grown in hydroponic culture

ABSTRACTThe optimum nitrogen requirement along with nitrogen uptake efficiency of finger millet (Eleusine coracana) for obtaining plants of a high phenotypic quality was studied in this research using hydroponic culture. 20 days old seedlings were transferred in nutrient solution containing all the essential macro and micro nutrients. Plants were subjected to three treatments of nitrate viz. 0.05 mM, 0.1 mM and 0.5 mM by dissolving it in the nutrient solution. Plants grown in nutrient solution devoid of nitrate were treated as control. Plants grown in 0.5 mM nitrate conc. attained more height compared to other low nitrogen treatments (0.1 mM and 0.05 mM). Similarly plants of 0.5 mM nitrate treatments possessed more number of lateral roots, surface area of leaves, dry weight of plants and chlorophyll content compared to other low nitrogen treatments. Specific activity of nitrate reductase in plants treated with 0.5 mM concentration was found nearly 3 times higher than that of control plants; similarly, specific activity of nitrite reductase in 0.5 mM treated plants was also high compared to other low nitrogen treatments followed by control. The results of the present investigation therefore indicate that nitrogen uptake efficiency as revealed by the activities of nitrate reductase and nitrite reductase was high in 0.5 mM nitrate treatment. It also shows that 0.5 mM nitrate is optimum nitrogen concentration for the growth of finger millet in hydroponic condition.

Green synthesis of silver nanoparticles using Rhodobacter Sphaeroides

The use of microorganisms in the synthesis of nanoparticles emerges as an eco-friendly and exciting approach. In this study, silver nanoparticles were successfully synthesized from AgNO3 by reduction of aqueous Ag+ ions with the cell filtrate of Rhodobacter sphaeroides. Nanoparticles were characterized by means of UV–vis absorption spectroscopy, X-Ray Diffraction (XRD), transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HRTEM). Crystalline nature of the nanoparticles in the fcc structure are confirmed by the peaks in the XRD pattern corresponding to (111), (200), (220) and (311) planes, bright circular spots in the selected are a electron diffraction (SAED) and clear lattice fringes in the high-resolution TEM image. Also, the size of silver nanoparticles was controlled by the specific activity of nitrate reductase in the cell filtrate.

Isolation and characterization of nitrate reductase from the halophilic sulfur-oxidizing bacterium Thioalkalivibrio nitratireducens

A novel nitrate reductase (NR) was isolated from cell extract of the haloalkaliphilic bacterium Thioalkalivibrio nitratireducens strain ALEN 2 and characterized. This enzyme is a classical nitrate reductase containing molybdopterin cofactor in the active site and at least one iron-sulfur cluster per subunit. Mass spectrometric analysis showed high homology of NR with the catalytic subunit NarG of the membrane nitrate reductase from the moderately halophilic bacterium Halomonas halodenitrificans. In solution, NR exists as a monomer with a molecular weight of 130-140 kDa and as a homotetramer of about 600 kDa. The specific nitrate reductase activity of NR is 12 micromol/min per mg protein, the maximal values being observed within the neutral range of pH. Like other membrane nitrate reductases, NR reduces chlorate and is inhibited by azide and cyanide. It exhibits a higher thermal stability than most mesophilic enzymes.

Role of individual nap gene cluster products in NapC-independent nitrate respiration of Wolinella succinogenes

Bacterial nap gene clusters, encoding periplasmic nitrate reductase (NapA), are complex and diverse, and the composition of the electron transport chain donating electrons to NapA is poorly characterized in most organisms. Exceptionally, Wolinella succinogenes transfers electrons from formate via the menaquinone pool to NapA independently of a membrane-bound c-type cytochrome of the NapC family. The role of individual ORFs of the W. succinogenes napAGHBFLD gene cluster is assessed here by characterizing in-frame gene inactivation mutants. The ability of the mutants to grow by nitrate respiration was tested and their NapA content and specific nitrate reductase activity were determined. The napB and napD gene products proved to be essential for nitrate respiration, with NapD being required for the production of mature NapA. Inactivation of either subunit of the putative membrane-bound menaquinol dehydrogenase complex NapGH almost abolished growth by nitrate respiration. Substitution of the twin-arginine sequence of NapG had the same effect as absence of NapG. Phenotypes of mutants lacking either NapF or NapL suggest that both proteins function in NapA assembly and/or export. The data substantiate the current model of the composition of the NapC-independent electron transport chain as well as of NapA maturation, and indicate the presence of an alternative electron transport pathway to NapA.

Mutagenesis study on the role of a lysine residue highly conserved in formate dehydrogenases and periplasmic nitrate reductases

Lysine 85 (K85) in the primary structure of the catalytic subunit of the periplasmic nitrate reductase (NAP-A) of Ralstonia eutropha H16 is highly conserved in periplasmic nitrate reductases and in the structurally related catalytic subunit of the formate dehydrogenases of various bacterial species. It is located between an [4Fe–4S] center and one of the molybdopterin-guanine dinucleotides mediating the through bonds electron flow to convert the specific substrate of the respective enzymes. To examine the role of K85, the structure of NAP-A of R. eutropha strain H16 was modeled on the basis of the crystal structure from the Desulfovibrio desulfuricans enzyme (Dias et al. Structure Fold Des. 7(1) (1999) 65) and K85 was replaced by site-directed mutagenesis, yielding K85R and K85M, respectively. The specific nitrate reductase activity was determined in periplasmic extracts. The mutant enzyme carrying K85R showed 23% of the wild-type activity, whereas the replacement by a polar, uncharged residue (K85M) resulted in complete loss of the catalytic activity. The reduced nitrate reductase activity of K85R was not due to different quantities of the expressed gene product, as controlled immunologically by NAP-specific antibodies. The results indicate that K85 is optimized for the electron transport flux to reduce nitrate to nitrite in NAP-A, and that the positive charge alone cannot meet further structural requirement for efficient electron flow.

Increased Arginine Biosynthesis during Phosphorus Deficiency

The accumulation of arginine in leaves of four citrus rootstock cultivars during P deficiency has been demonstrated to be due to increased de novo synthesis rather than decreased catabolism or increased protein degradation (E Rabe, CJ Lovatt, 1984, Plant Physiol 76: 747-752). In this report, we provide evidence (a) that the increased activity of the arginine biosynthetic pathway observed for citrus rootstocks grown under P-deficient conditions for 7 months is due to an increase in the concentration of ammonia in leaves of P-deficient plants and (b) that ammonia accumulation and removal through arginine systhesis are early responses to phosphorus deficiency for both a woody perennial, rough lemon (Citrus limon), and an herbaceous annual, summer squash (Cucurbita pepo). Transferring 5-day-old squash plants to a phosphorus-deficient nutrient solution for only 10 days resulted in a 2-fold increase in the concentration of nitrate in the youngest fully expanded leaves (YFE). Concomitantly, the specific activity of nitrate reductase doubled and the ammonia content of P-deficient YFE leaves increased to a concentration significantly greater that of leaves from healthy control plants (P < 0.05). Consistent with increased availability of ammonia, the incorporation of NaH(14)CO(3) into arginine plus urea doubled during phosphorus deficiency and arginine accumulated. Despite the accumulation of nitrate and ammonia in YFE leaves during phosphorus deficiency, the total nitrogen content of these leaves was less than that of the healthy control plants. Similar results were obtained for rough lemon. Nitrate content of the YFE leaves increased 1.5- and 3.0-fold in plants deprived of phosphorus for 6 and 12 weeks, respectively. Ammonia content of the leaves increased as P deficiency progressed to 1.4 +/- 0.08 mg (+/- se, n = 4) per gram dry weight, a level 1.8-fold greater than that of the P-sufficient control plants. During P deficiency de novo arginine biosynthesis in rough lemon increased 10-fold. Immersing the petiole of YFE leaves from P-sufficient squash and rough lemon plants in 50 millimolar NH(4) (+) for 3 hours resulted in the accumulation of ammonia in the leaves, and a 4-fold increase in the incorporation of NaH(14)CO(3) into arginine plus urea. Taken together, these results provide strong evidence that the accumulation of nitrate and ammonia in leaves is an early response of both woody and herbaceous plants to P deprivation. The data are consistent with the hypothesis that increased de novo arginine biosynthesis in leaves during P deficiency is in response to ammonia content of the leaves.

Manganese, copper and molybdenum nutrition of papaya

SummaryPapaya cv Honeydew, was grown in refined sand with complete nutrition and with deficiency levels of Mn, Cu and Mo. Deficiency symptoms of Mo and Cu were induced earlier than those of Mn. Depressions in growth and dry matter were less marked with Mn deficiency than with those of Mo and Cu. Mn, Cu or Mo deficiencies decreased the tissue concentrations of the deficient element and the specific activity of catalase, and increased the activities of peroxidase and ribonuclease. The specific activity of acid phosphatase was stimulated and that of cytochrome-c oxidase was depressed by deficiency of Mo or Cu. In addition to reduced soluble protein caused by deficiencies of Mn, Cu and Mo, the specific activity of nitrate reductase was depressed in the leaves of Mo-deficient plants.

Nitrate Metabolism in Alfalfa Root Nodules under Water Stress

Becana, M., Aparicio-Tejo, P. M. and Sanchez-Diaz, M. 1986. Nitrate metabolism in alfalfa root nodules under water stress.—J. exp. Bot. 37: 798-806. Three-month-old plants (vegetative stage) of alfalfa (Medicago sativa L. cv. Aragon) were supplied for one week with 10 dm3 (uniformly distributed) nutrient solutions containing 0 or 20 mol m 3 NO3 . One week after initiation of NO 3 treatment, the plants were subjected to drought by withholding water. Bacteroids and cytosol of nodules were obtained at different stages of stress, and used for enzyme assays and for determination of NH4, N02 and NO3 . Proteins of bacteroids were more stable than cytosolic proteins with respect to the detrimental effects of water stress and NO3. Protein contents of bacteroids and cytosol were inversely related to proteolytic activities against azocasein in both nodule fractions. Specific nitrate reductase activity (NRA) and nitrite reductase activity (NiRA) of bacteroids from NO J-treated plants were inhibited by c. 70% and 45%, respectively, as leaf water potential (ii/w) declined from —0-5 MPa (control) to —1-8 MPa. At still lower t//w, both activities began to increase: NRA was doubled, whereas NiRA only returned to its control level. Cytosolic NRA was strongly inhibited by drought, but the corresponding NiRA remained constant. Ammonia concentration in bacteroids and nodule cytosol kept basically constant, whereas NOJ accumulated in the cytosol at severe stress, due to the activation of bacteroid nitrate reductase. Results indicate that nitrate and nitrite reductases of the bacteroids and the nodule cytosol act in different form: assimilatory, the cytosolic enzymes; and dissimilatory, the enzymes of bacteroids at low 1/iw. The possibility that assimilation of NO3 also occurs in bacteroids at control or mild water stress conditions is suggested. Key words—Assimilatory and dissimilatory N03 reduction, bacteroids, Medicago sativa, nodule cytosol, water stress. Correspondence to: Departamento de Fisiologia Vegetal, Universidad de Navarra, Apartado 273,31080 Pamplona, Spain.

Nitrate and nitrite reduction by alfalfa root nodules: Accumulation of nitrite in Rhizobium melioti bacteroids and senescence of nodules

Addition of NO−3 rapidly induced senescence of root nodules in alfalfa (Medicago sativa L. cv. Aragon). Loss of nodule dry matter began at the lowest NO−3 concentration (10 mM) but degradation of bacteroid proteins was only detected when nodules were supplied with NO−3 concentrations above 20 mM.Bacteroids from Rhizobium meliloti contained high specific activities of nitrate reductase (NR) and nitrite reductase (NiR). Both enzymes were presumably substrate‐induced although substantial enzyme activities were present in the absence of NO−3 Typical specific activities for soluble NR and NiR of bacteroids under NO−3 free conditions were 1.2 and 1.4 μmol (mg protein)−1h−1, respectively. In the presence of NO−3, the specific activity of NR was considerably greater than that of NiR, thus causing NO−2 accumulation in bacteroids. Nitrite levels in the bacteroids were linearly correlated with specific activities of NR and NiR, indicating that NO−2 is formed by bacteroid NR and that this NO−2 in turn, induces bacteroid NiR. Accumulation of NO−2 within bacteroids also indicates that NO−2 inhibits nodule activity after feeding plants with NO−3

Construction in vitro of a cloned nar operon from Escherichia coli

To clone the nar operon of Escherichia coli without an effective selection procedure for the nar+ phenotype, a strategy utilizing nar::Tn5 mutants was employed. Partial segments of the nar operon containing Tn5 insertions were cloned into plasmid pBR322 by using the transposon resistance character for selection. A hybrid plasmid was constructed in vitro from two of these plasmids and isolated by a procedure that involved screening a population of transformed nar(Ts) mutant TS9A for expression of thermal stable nitrate reductase activity. A detailed restriction site map of the resulting plasmid, pSR95, corresponded closely to the composite restriction endonuclease map deduced for the nar region from maps of the cloned nar::Tn5 fragments. When transformed with pSR95, wild-type strain PK27 overproduced the alpha, beta, and gamma subunits of nitrate reductase, although nitrate reductase activity was only slightly increased. The alpha and beta subunits were overproduced about 5- to 10-fold and accumulated mostly as an inactive aggregate in the cytoplasm; the gamma subunit overproduction was detected as a threefold increase in the specific content of cytochrome b555 in the membrane fraction. Functional nitrate reductase and the cytochrome spectrum associated with functional nitrate reductase were restored in the nar::Tn5 mutant EE1 after transformation with pSR95. Although the specific activity of nitrate reductase in this case was less than that of the wild type, both the alpha and beta subunits appeared to be overproduced in an inactive form. In both strains PK27(pSR95) and EE1(pSR95), the formation of nitrate reductase activity and the accumulation of inactive subunits were repressed during aerobic growth. From these observations and the accumulation of inactive subunits were repressed during aerobic growth. From these observations and the demonstration that pSR95 contains a functional nor operon that encodes the alpha, beta, gamma subunits of nitrate reductase.

Isolation and characterisation of nitrate reductase mutants and regulation of nitrate reductase and nitrogenase in the cyanobacterium Nostoc muscorum

Chlorate resistant mutants of the cyanobacterium Nostoc muscorum isolated after N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) mutagenesis were found to be defective/blocked in nitrate reductase (NR). The parent strain possessed active NR in the presence of nitrogen as nitrate and only basal levels of activity in ammonia and N-free grown cultures. Addition of ammonia suppressed the NR activity in the parent strain whereas addition of L-methionine DL-sulphoximine (MSX) restored NR activity. A similar repression by ammonia, glutamine and derepression with MSX were also observed for nitrogenase synthesis. One class of mutants lacked NR activity (nar -) whereas the specific activity of NR was low in another class of mutants (nar def). Unlike the parent, the mutants synthesized nitrogenase and differentiated heterocysts in the presence of nitrate nitrogen. Uptake studies of nitrite and ammonia in mutants revealed that they possessed both nitrite reductase and glutamine synthetases (GS) at low levels, and the same level respectively in comparison with the parent.

Diverse effects of formate on the assimilatory metabolism of nitrate and nitrite inRhizobium

Two strains ofRhizobium, cowpeaRhizobium 32H1 andRhizobium japonicum CB 1809, showed a marked stimulation in growth on addition of formate to the minimal medium containing nitrate as the sole source of nitrogen. The amount of accumulated nitrite and specific nitrate reductase activity was much higher in cultures supplemented with formate than in the control medium. In contrast, growth, consumption of nitrite and specific nitrite reductase activity in minimal medium + nitrite was greatly reduced by the addition of formate. A chlorate resistant mutant (Chl-16) was isolated spontaneously which contained a nitrite reductase which was not inhibited by formate. The results suggest that formate serves as an electron donor for nitrate reductase and inhibits nitrite assimilation inRhizobium

Effect of ammonium on the regulation of nitrate assimilation in natural phytoplankton populations

The effect of ammonium additions (0.5 to 10 μg-at. N · 1 −1) on the nitrate reductase activity and on the nitrate uptake of natural marine phytoplankton populations was investigated. Specific nitrate reductase activity was significantly reduced at all ammonium concentrations after 24 h of the addition, but not after 6 h. Total nitrate reductase activity did not decrease even at the highest ammonium concentrations. From this it is concluded that the ammonium concentrations used during this study affected mostly the synthesis of new active enzyme. Nitrate uptake rates were also reduced by the addition of ammonium. In contrast to the nitrate reductase results, maximum inhibition was, however, observed after 6 h rather than after 24 h. The lag in the time of response between the inhibition after the ammonium addition suggests 1. (1) that NH 4 + affects nitrate assimilation in two steps, first nitrate uptake and second nitrate reduction; and 2. (2) that the mechanisms controlling these processes are to a certain extent independent of each other.

A comparison of the influence of iron on the growth and nitrate metabolism of Anabaena and Scenedesmus

AbstractA comparative study of growth and nitrate metabolism of Anabaena flos‐aquae (Lyng.) Bréb. and Scenedesmus bijugatus var. seriatus Chodat investigated possible mechanisms for the iron‐stimulated increases in growth specific for blue‐green algae in mixed algal communities. Algae were separately grown in an morganic medium with varying concentrations of iron and nitrate to determine the effects on each organism.Iron was found to be a limiting nutrient for cultures of both Anabaena and Scenedesmus as determined by chlorophyll a concentrations and cell enumeration. Both iron and nitrate stimulated the specific activity of nitrate reductase, nitrite reductase, and glutamine synthetase in Anabaena. Iron enrichment did not increase the activity of the enzymes in Scenedesmus, but inhibited the activity of nitrate reductase and glutamine synthetase. The stimulation of growth by iron in cells grown under iron limiting conditions was associated with increased nitrate metabolism in Anabaena but not in Scenedesmus.

Pathway for Nitrate Assimilation in Corn (Zea mays L.) Leaves

The localization of enzymes responsible for nitrate assimilation and the generation of NADH for nitrate reduction were studied in corn (Zea mays L.) leaf blades. The techniques used effectively separated mesophyll and bundle sheath cells as judged by microscopic observations, enzymic assays, chlorophyll a/b ratios and photochemical activities. Nitrate reductase, nitrite reductase, and the nitrate content of leaf blades were localized primarily in the mesophyll cells, although some nitrite reductase was found in the bundle sheath cells. Glutamine synthetase, NAD-malate dehydrogenase, NAD-glyceraldehyde-3-phosphate dehydrogenase, and NADP-glutamate dehydrogenase were found in both types of cells, however, more NADP-glutamate dehydrogenase was found in the bundle sheath cells than in the mesophyll cells. These data indicate that the mesophyll cells are the major site for nitrate assimilation in the leaf blade because they contained an ample supply of nitrate and the enzymes considered essential for the assimilation of nitrate into amino acids. Because the specific activity of nitrate reductase was severalfold lower than the other enzymes involved in nitrate assimilation, nitrate reduction is indicated as the rate-limiting step in situ. A sequence of reactions is proposed for nitrate assimilation in the mesophyll cells of corn leaves as related to the C-4 pathway of photosynthesis.

Nitrogen Assimilation and Protein Synthesis in Wheat Seedlings As Affected by Mineral Nutrition. I. Macronutrients

Deficiencies of each macronutrient (N, P, K, Ca. Mg, S, and Fe) decreased the specific activity of nitrate reductase from Triticum aestivum L. seedlings. Nitrate content was decreased by N, P, K, Ca, and Mg deficiencies and unaffected by S and Fe deficiencies. Glutamic acid dehydrogenase activity was decreased by N, P, and S deficiencies, unchanged by K deficiency, and increased by Ca, Mg, and Fe deficiencies. Glutamine synthetase activity closely paralleled nitrate reductase activity and was decreased by deficiencies of N, P, K, Ca, Mg, and S. Glutamic-oxaloacetic transaminase was not sensitive to macronutrient deficiencies. High (14)C-leucine incorporation into tissue sections of N-, P-, K-, Ca-, and S-deficient seedlings did not appear indicative of protein synthesis rates in intact seedlings. Nutritional deficiencies apparently depleted endogenous amino acid pools and caused less inhibition of exogenous (14)C-leucine incorporation into protein.

THE EFFECT OF NITROGEN STARVATION ON THE ACTIVITY OF NITRATE REDUCTASE AND OTHER ENZYMES IN CHLORELLA.

Chlorella vulgaris grown with nitrate as nitrogen source had high nitrate reductase activity. During nitrogen starvation the rate of nitrate assimilation increased but the specific activity of nitrate reductase in extracts decreased sharply; a similar decrease occurred after transfer to an ammonium medium. Organisms grown with ammonium as nitrogen source did not assimilate nitrate but acquired the ability to do so after nitrogen starvation; nitrate reductase activity in these organisms was initially very low but increased during nitrogen starvation and then decreased. After 18 hr of nitrogen starvation ammonium-grown and nitrate-grown organisms assimilated nitrate rapidly at a rate some ten times greater than the activity of nitrate reductase in cell-free extracts would allow. The specific activity of glutamic dehydrogenase was about 40% higher in extracts from ammonium-grown organisms than in extracts from nitrate-grown organisms and its activity increased by 40-100 % during nitrogen starvation. The activity of malic dehydrogenase was unaffected by 18 hr of nitrogen starvation. The activity of isocitrate lyase decreased by about one-quarter when acetate was present but decreased much more markedly when the organisms were nitrogen-starved in the presence of glucose.