The Influence of Temperature on Nitrate Reductase Activity of Agrostis palustris and Cynodon dactylon

The influence of temperature was studied in relation to nitrate reductase activity of creeping bentgrass (Agrostis palustris Huds. cv. ‘Toronto’) a cool season grass and bermudagrass (Cynodon dactylon L. cv. ‘Tifgreen’) a warm season grass. Maximum nitrate reductase activity of both species occurred at 20°C. The nitrate reductase level in bentgrass leaves was reduced when grown at 35°C while bermudagrass leaves were relatively unaffected. The activity per se of the bentgrass enzyme preparation was inhibited rather than synthesis of the enzyme.     

Characterization of the Nitrate-Nitrite Transporter of the Major Facilitator Superfamily (the nrtP Gene Product) from the Cyanobacterium Nostoc punctiforme Strain ATCC 29133

The products of the NpR1527 and NpR1526 genes of the filamentous, diazotrophic, fresh-water cyanobacterium Nostoc punctiforme strain ATCC 29133 were identified as a nitrate transporter (NRT) and nitrate reductase (NR) respectively, by complementation of nitrate assimilation mutants of the cyanobacterium Synechococcus elongatus strain PCC 7942. While other fresh-water cyanobacteria, including S. elongatus, have an ATP-binding cassette (ABC)-type NRT, the NRT of N. punctiforme belongs to the major facilitator superfamily, being orthologous to the one found in marine cyanobacteria (NrtP). Unlike the ABC-type NRT, which transports both nitrate and nitrite with high affinity, Nostoc NrtP transported nitrate preferentially over nitrite. NrtP was distinct from ABC-type NRT also in its insensitivity to ammonium-promoted regulation at the post-translational level. The nitrate reductase of N. punctiforme was, on the other hand, inhibited upon addition of ammonium to medium, lending ammonium sensitivity to nitrate assimilation.     

Purification and Characterization of Nitrate Reductase from the Halotolerant Cyanobacterium <Emphasis Type="Italic">Aphanothece halophytica</Emphasis>

Summary Factors affecting the activity of nitrate reductase (E.C.1.7.7.2) from the halotolerant cyanobacterium Aphanothece halophytica were investigated. Cells grown in nitrate-containing medium exhibited higher nitrate reductase activity than cells grown in medium in which nitrate was replaced by glutamine. When ammonium was present in the medium instead of nitrate, the activity of nitrate reductase was virtually non-detectable, albeit with normal cell growth. The enzyme was localized mainly in the cytoplasm. The enzyme was purified 406-fold with a specific activity of 40.6 μmol/min/mg protein. SDS-PAGE revealed a subunit molecular mass of 58 kDa. Gel filtration experiments revealed a native molecular mass of 61 kDa. The K _m value for nitrate was 0.46 mM. Both methyl viologen and ferredoxin could serve as electron donor with K _m values of 4.3 mM and 5.2 μM, respectively. The enzyme was strongly inhibited by sulfhydryl-reactive agents and cyanide. Nitrite, the product of the enzyme reaction, showed little inhibition. Chlorate, the substrate analog, could moderately inhibit the enzyme activity. NaCl up to 200 mM stimulated the activity of the enzyme whereas enzyme inhibition was observed at ≥300 mM NaCl.     

Enhanced frequency of karyogamy in electrofusion of yeast protoplasts by means of preceding G1 arrest

Abstract The cellular glycogen pool and nitrate reductase activity were measured in the cyanobacterium Phormidium uncinatum after infection with cyanophage LPP-1, under both light and dark conditions. While dark incubation of the cyanobacterium reduced the glycogen level, the nitrate reductase (NR) activity remained almost unchanged. Furthermore, cyanophage multiplication enhances cellular glycogen level and NR activity in both illuminated and dark-incubated cyanobacterial cultures. Cyanophage-mediated increase in host nitrate assimilation appears to support the high protein demand for its reproductive cycle.     

Inactivation of Nitrate Reductase of the Yeast, Rhodotorula glutinis

Nitrate reductase (NR) from the yeast, Rhodotorula glutinis var. salinaria was composed of two enzymatic components, diaphorase and terminal nitrate reducing moieties. The enzyme used NADPH as electron donor and FAD as cofactor. The synthesis of nitrate reductase was promoted specifically by nitrate and repressed by ammonium and amino acids. Nitrate reductase from this yeast had an inactive as well as an active form. Inactive enzyme was reactivated by oxidation with ferricyanide in vitro. Hydroxylamine promoted the formation of inactive enzyme in vivo. Ammonium could neither promote the inactivation nor reduce the total level of nitrate reductase activity. Nitrate could protect nitrate reductase from inactivation caused by nitrogen starvation or hydroxylamine.     

Complex formation between ferredoxin and Synechococcus ferredoxin: nitrate oxidoreductase

The ferredoxin-dependent nitrate reductase from the cyanobacterium Synechococcus sp. PCC 7942 has been shown to form a high-affinity complex with ferredoxin at low ionic strength. This complex, detected by changes in both the absorbance and circular dichroism (CD) spectra, did not form at high ionic strength. When reduced ferredoxin served as the electron donor for the reduction of nitrate to nitrite, the activity of the enzyme declined markedly as the ionic strength increased. In contrast, the activity of the enzyme with reduced methyl viologen (a non-physiological electron donor) was independent of ionic strength. These results suggest that an electrostatically stabilized complex between Synechococcus nitrate reductase and ferredoxin plays an important role in the mechanism of nitrate reduction catalyzed by this enzyme. Treatment of Synechococcus nitrate reductase with either an arginine-modifying reagent or a lysine-modifying reagent inhibited the ferredoxin-dependent activity of the enzyme but did not affect the methyl viologen-dependent activity. Treatment with these reagents also resulted in a large decrease in the affinity of the enzyme for ferredoxin. Formation of a nitrate reductase complex with ferredoxin prior to treatment with either reagent protected the enzyme against loss of ferredoxin-dependent activity. These results suggest that lysine and arginine residues are present at the ferredoxin-binding site of Synechococcus nitrate reductase. Results of experiments using site-specific, charge reversal variants of the ferredoxin from the cyanobacterium Anabaena sp. PCC 7119 as an electron donor to nitrate reductase were consistent with a role for negatively charged residues on ferredoxin in the interaction with Synechococcus nitrate reductase.     

Effect of nitrate,ammonia and nitrogen starvation on the regulation of nitrate reductase in Cyanidium caldarium

Summary Cells of Cyanidium caldarium grown with ammonia or ammonium nitrate as nitrogen source do not contain appreciable nitrate reductase activity. The alga develops the capacity to synthesize the enzyme when it is transferred from the ammonium medium to a nitrogen-free medium. Nitrate is not needed as an inducer and no enhancement in the rate of enzyme synthesis is observed when it is present. By contrast, whereas the synthesis of the enzyme in nitrogen-free medium proceeds at an increasing rate, in the nitrate medium it attains a stationary level after a short time.Nitrate grown cells possess variable amount of inactive nitrate reductase (from 9 to 60%) whereas in nitrogen-free medium the enzyme occurs principally in a fully active form. Addition of ammonia inactivates reversibly the preexisting enzyme. The inactive enzyme is measurable in the crude extract after activation by heating.It is suggested that in Cyanidium the inactivating effect of ammonia, which is the end product of nitrate reduction, in association with the repression of enzyme controls the level of nitrate reductase activity.     

Induction of nitrate reductase and membrane cytochromes in wild type and chlorate-resistant Paracoccus denitrificans

An experimental system has been devised for induction of nitrate reductase in suspensions of wild type Paracoccus denitrificans incubated with limited aeration in the presence of azide, nitrate or nitrite. Azide promoted maximum synthesis of enzyme, accompanied by formation of excess b-type cytochrome; the level of enzyme attained with nitrate was less and c-type cytochrome predominated in the membrane. The nitrate reductase was solubilized with deoxycholate from membranes of azide-induced cells and was identified as a major polypeptide M _r =150,000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Mutants strains lacking nitrate reductase activity were isolated on the basis of resistance to chlorate and mutant M-1 was examined in detail. When incubated in the cell suspension system M-1 formed a membrane protein M _r =150,000 similar to that attributed to nitrate reductase in the wild type. Maximum formation of the protein by M-1 occurred without inducer and it was accompanied by synthesis of excess b-type cytochrome. The observations with wild type and M-1 indicate that nitrate reductase protein and b-type cytochrome are coregulated and that the active enzyme has a role in regulating its own synthesis.Non-standard Abbreviations SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - DOC sodlum deoxycholate     

Role of Protein Synthesis in Recovering Nitrate Reductase Activity in Wheat Leaves Exposed to Heat Stress

Heating intact leaves of 14–15-day-old seedlings of wheat (Triticum aestivumL.), cv. Albidum 29, for 10 min at 44–45°C brought about a decrease in nitrate reductase activity by 50–90% of the initial level. The complete recovery of the enzyme activity occurred one to two days after the plants were returned to normal temperature conditions. Darkening plants or adding cycloheximide to the nutrient medium did not interfere with the recovery of nitrate reductase activity. The plants grown in darkness or on a nitrate-free medium were devoid of nitrate reductase activity. The transfer of these plants to the light or the addition of nitrate resulted in the induction of enzyme activity. In the untreated plants, nitrate reductase activity attained the control level in 48 h; in the heated plants, this process was considerably retarded. After heating, the activity of the preexisting enzyme recovered at a higher rate than the ability for enzyme induction. This means that the reactivation of nitrate reductase occurred even when the induction of the enzyme was almost entirely suppressed. We conclude that after the short-term effect of high temperatures, the functional activity of nitrate reductase may recover without the de novosynthesis of the enzyme protein.     

The development of nitrate reductase in Chlorella and its repression by ammonium

Summary Chlorella vulgaris, grown with ammonium sulphate as nitrogen source, contains very little nitrate reductase activity in contrast to cells grown with potassium nitrate. When ammonium-grown cells are transferred to a nitrate medium, nitrate reductase activity increases rapidly and the increase is partially prevented by chloramphenicol and by p-fluorophenylalanine, suggesting that protein synthesis is involved. The increase in nitrate reductase activity is prevented by small quantities of ammonium; this inhibition is overcome, in part, by raising the concentration of nitrate. Although nitrate stimulates the development of nitrate reductase activity, its presence is not essential for the formation of the enzyme since this is formed when ammonium-grown cells are starved of nitrogen and when cells are grown with urea or glycine as nitrogen source. It is concluded that the formation of the enzyme is stimulated (induced) by nitrate and inhibited (repressed) by ammonium.     

Nitrate and Nitrite Reduction in Wheat Leaves as Affected by Different Types of Water Stress

The effect of different types of water stress on nitrate and nitrite reductases of wheat (Triticum vulgare L. cv. Mivhor) leaves was investigated. Water stress was applied either to leaf tissue by its incubation in mannitol or various salt solutions, or to intact plants by exposure of the root system to low temperatures or to salinity. Nitrite reductase was much less sensitive to water stress than nitrate reductase, and was not sensitive to salinity up to osmotic potentials of about — 13 bars. The decrease in nitrite reductase activity by water stress was attributed to a direct inhibition of the enzyme rather than to a repression of enzyme synthesis. This was based on the fast response of the enzyme after exposure of leaf tissue to reduced osmotic potential, on the lack of a continuous decrease in enzyme activity during a prolonged stress, and on the fact that light activation of reductase was unaffected by water stress. The inhibition of nitrate reductase under water stress was attributed to both a direct inhibition and a reduced rate in enzyme synthesis. This is concluded from the fact that a decrease in its activity was obtained already within 1 h after stress application and from the fact that light induction of the enzyme was inhibited by stress.     

Isolation and characterization of Anacystis nidulans R2 mutants affected in nitrate assimilation: Establishment of two new mutant types

Summary Eighteen mutant strains of the unicellular cyanobacterium Anacystis nidulans R2 that are unable to assimilate nitrate have been isolated after transposon Tn901 mutagenesis. Characterization of phenotypes and transformation tests have allowed the distinction of five different mutant types. The mutants exhibiting a nitrate reductase-less phenotype were identified as being affected in previously defined loci, as they could be transformed to the wild type by one of the plasmids pNR12, pNR63 or pNR193, which contain cloned genes of A. nidulans R2 involved in nitrate reduction. The mutations in strains FM2 and FM16 appear to affect two other genes involved in nitrate assimilation. Strain FM2 apparently bears a single mutation which results in both lack of nitrite reductase activity and loss of ammonium-promoted repression of nitrate reductase synthesis. FM16 has a low but significant level of nitrate reductase that is also freed from repression by ammonium, and an increased level of nitrite reductase activity. FM16 exhibited properties which indicate that this mutant strain might also be affected in the transport of nitrate into the cell.Abbreviations EDTA ethylenediamine-tetraacetic acid - MTA mixed alkyltrimethylammonium bromide - TES N-tris (hydroxymethyl)methyl-2-aminoethane sulfonic acid - Tricine N-[2-hydroxy-1,1-bis (hydroxymethyl)ethyl]-glycine - Tris Tris(hydroxymethyl)aminomethane     

Biochemical analysis of mutants defective in nitrate assimilation in Neurospora crassa: evidence for autogenous control by nitrate reductase

Summary A biochemical analysis of mutants altered for nitrate assimilation in Neurospora crassa is described. Mutant alleles at each of the nine nit (nitrate-nonutilizing) loci were assayed for nitrate reductase activity, for three partial activities of nitrate reductase, and for nitrite reductase activity. In each case, the enzyme deficiency was consistent with data obtained from growth tests and complementation tests in previous studies. The mutant strains at these nit loci were also examined for altered regulation of enzyme synthesis. Such exeriments revealed that mutations which affect the structural integrity of the native nitrate reductase molecule can result in constitutive synthesis of this enzyme protein and of nitrite reductase. These results provide very strong evidence that, as in Aspergillus nidulans, nitrate reductase autogenously regulates the pathway of nitrate assimilation. However, only mutants at the nit-2 locus affect the regulation of this pathway by nitrogen metabolite repression.     

Effect of N oxyanions on anaerobic induction of nitrate reductase in subcellular fractions of <Emphasis Type="Italic">Bradyrhizobium</Emphasis> sp. (Lupinus)

Anaerobic induction of nitrate reductase in subcellular fractions of Bradyrhizobium sp. strain USDA 3045 showed fivefold increase of the enzyme activity in spheroplasts, considered as the source of intact-membrane-bound nitrate reductase, within a 3 h time frame after nitrate addition. Such a dynamics was confirmed at the protein level, with antibodies specific to membrane-bound nitrate reductase. Nitrate reductase activity in the periplasm was one order of magnitude lower and significant only at initial 3 h of induction, within a narrow range of nitrate added. Nitrite induced the membrane-bound nitrate reductase at least 70% as effectively as nitrate, as judged from its activity pattern and Western blot analysis. The limited ability of Bradyrhizobium sp. to dissimilate ≥5 mM nitrate is not due to direct inhibition of respiratory nitrate reductase by accumulated nitrite. Moreover, a synergistic induction of membrane-bound nitrate reductase by nitrate and nitrite was indicated due to a twofold higher protein synthesis after simultaneous addition of these N oxyanions than when they were given separately.     

Effect of polyamines and guanidines on the growth,nitrogen assimilation and reserve mobilization in germinating radish seeds

Polyamines and guanidines enhanced the growth of radish seedlings grown in dark or light, irrespective of the supply of nitrogen. All the compounds inhibited ntirate reducatase and glutamine synthetase in the cotyledons of light-grown but not in dark-grown seeds. Nitrite reductase and glutamate dehydrogenase were not affected. Protease activity was enhanced by all the compounds in dark-as well as in light-grown seeds. Alanine aminotransferase activity was increased only in the light-grown seeds. The inhibition of nitrate reductase was not due to decreased nitrate uptake but was due to a decreased metabolic pool of nitrate and a decline in enzyme synthesis. The inhibition of glutamine synthetase and activation of alanine aminotransferase by the compounds was found only in the chloroplast fraction. The activation of protease was due to the release or activation of preexisting enzyme while that of alanine aminotransferase was dependent on the de novo protein synthesis which was abolished by cycloheximide.     

Phytochrome mediated induction of nitrate reductase activity in etiolated maize leaves

The effects of red and far-red light on the enhancement of in vitro nitrate reductase activity and on nitrate accumulation in etiolated excised maize leaves were examined. Illumination for 5 min with red light followed by a 4-h dark period caused a marked increase in nitrate reductase activity, whereas a 5-min illumination with far-red light had no effect on the enzyme activity. The effect of red light was completely reversed by a subsequent illumination with the same period of far-red light. Continuous far-red light also enhanced nitrate reductase activity. Both photoreversibility by red and far-red light and the operation of high intensity reaction under continuous far-red light indicated that the induction of nitrate reductase was mediated by phytochrome. Though nitrate accumulation was slightly enhanced by red and continuous far-red light treatments by 17% and 26% respectively, this is unlikely to account for the entire increase of nitrate reductase activity. The far-red light treatments given in water, to leaves preincubated in nitrate, enhanced nitrate reductase activity considerably over the dark control. The presence of a lag phase and inhibition of increase in enzyme activity under continuous far-red light-by tungstate and inhibitors of RNA synthesis and protein synthesis-rules out the possibility of activation of nitrate reductase and suggests de novo synthesis of the enzyme affected by phytochrome.     

Studies on nitrate reductase from Cyanidium caldarium

Summary A nitrate reductase from the thermophilic acidophilic alga, Cyanidium caldarium, was studied. The enzyme utilises the reduced forms of benzyl viologen and flavins as well as both NADPH₂ and NADH₂ as electron donors to reduce nitrate.Heat treatment has an activating effect on the benzyl viologen (FMNH₂, FADH₂) nitrate reductase. At 50°C the activation of the enzyme is complete in about 20 min of exposure, whereas at higher temperatures (until 75°C) it is virtually an instantaneous phenomenon. The observed increase in activity is very low in extracts from potassium nitrate grown cells, whereas it is 5 or more fold in extracts from ammonium sulphate supplied cells. The benzyl viologen nitrate reductase is stable at 60°C and is destroyed at 75°C after 3 min; the NADPH₂ nitrate reductase is destroyed at 60°C. The pH optimum for both activities was found in the range 7.8–8.2.Ammonium nitrate grown cells possess a very low level of nitrate reductase: when they are transferred to a nitrate medium a rapid synthesis of enzyme occurs. By contrast, when cells with fully induced activity are supplied with ammonia, a rapid loss of NADPH₂ and benzyl viologen nitrate reductase occurs; however, activity measured with heated extracts shows that the true level of benzyl viologen nitrate reductase is as high as before ammonium addition. It is suggested that the presence of ammonia causes a rapid inactivation but no degradation of the enzyme.Cycloheximide inhibits the formation of the enzyme; the drug is without effect on the loss of nitrate reductase activity induced by ammonium. The nitrate reductase is reactivated in vivo by the removal of the ammonium, in the absence as well as in the presence of cycloheximide.     

A nitrate reductase gene of the cyanobacterium Synechococcus PCC6301 inferred by heterologous hybridization,cloning and targeted mutagenesis

DNA probes from the narG gene of Escherichia coli, which encodes the large polypeptide of respiratory nitrate reductase, show cross-hybridization at low stringency to a single region of the genome of the cyanobacterium Synechococcus PCC6301. This segment of cyanobacterial DNA was cloned as the insert of plasmid pDN1 and characterized. RNA complementary to pDN1 was shown to be substantially more abundant in nitrate grown cells of Synechococcus PCC6301 than in ammonium grown cells, thus parallelling the nitrate induction and ammonium repression of nitrate reductase activity in cultures of this cyanobacterium. A mutant of Synechococcus PCC6301 deficient in nitrate reductase activity was obtained after a potentially mutagenic transformation treatment using pDN1 as a donor. This mutant was restored to the wild type phenotype following stable integrative transformation with pDN1 DNA. Taken together these data suggest that pDN1 might encode a polypeptide of nitrate reductase. pDN1 is distinct from three clones of genes involved in nitrate assimilation that were isolated previously from the related cyanobacterium Synechococcus PCC7942 (Kuhlemeier et al., 1984a, J.Bact. 159, 36–41, and 1984b, Gene 31, 109–116).     

Inhibition of nitrate reductase induction by canavanine in maize roots

The induction of nitrate reductase activity in maize root tips was inhibited by canavanine and the inhibition increased with increasing concentration of canavanine between 0·1 and 1 mM. Addition of canavanine to the induced enzyme had little effect on the disappearance of the enzyme when nitrate was removed, and it is likely that the canavanine reduces the activity of the nitrate reductase by inhibiting its synthesis rather than by accelerating its breakdown.