Inhibition of the nitrate reductase complex from spinach by oxylamines

The nitrate reductase complex from spinach (Spinacia oleracea) was found to be inhibited by oxylamine compounds such as aminooxyacetate, hydroxylamine and O-methoxylamine. These compounds appear to interact with reduced cytochrome b₅₅₇ during catalysis of the enzyme. However, if the enzyme is maintained in a reduced state by NADH in the absence of nitrate, an additional component involved in FMNH₂-nitrate reductase is also affected by them. The binding of the oxylamines with the enzyme is non-covalent in nature as the inhibition can be reversed by treatment with 2-oxoglutarate.     

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.     

Effect of sodium chloride on the nitrate reductase of Suaeda maritima var. macrocarpa

The nitrate reductase (NR) activity extracted from Suaeda maritima is reduced by half in the presence of 0.1 M sodium chloride. This effect of sodi     

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

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

Alleviation of nitrate inhibition of soybean nodulation by high inoculum does not involve bacterial nitrate metabolism

Inoculation of soybean (Glycine max. cv. Bragg) plants with high level inoculum partially alleviated the nitrate inhibition of nodule formation (3 to 4 fold), but not nodule growth. This alleviation did not require the bacterial nitrate reductase asBradyrhizobium japonicum mutant strains 110CR1 and 110CR2 (both lacking assimilatory nitrate reductase activity) gave the same results as the wild type parent 311b110. The study was carried out in the glasshouse, thereby confirming preliminary field data by Herridgeet al. (1984) using a wild type bacterial inoculant.     

NADH nitrate reductase and related NADH cytochrome c reductase species in barley

Nitrate and nitrate-less barley (Hordeum vulgare cv Golden Promise) shoot extracts were examined by Sephadex G200 gel filtration and sucrose density gradient analysis and the MWs of NR and CR species present were determined from their Stokes radii and sedimentation coefficients by the method of Siegel and Monty. Nitrate-less plant extracts possessed a CR species of MW 27 800 whilst nitrate-plant extracts possessed CR species of MW 203 000, 61 000, 40 000 and 27 800. The MW 203 000 CR species was associated with NADH-NR, FMNH-NR and MV°-NR activities and represents the NR complex. The MW 40 000 and 61 000 CR species were shown to be derived from the NR complex. We suggest that the MW 40 000 and 61 000 CR species represent either subunits of the NR complex or domains cleaved from the intact NR complex by endogenous proteinases.     

Effect of glutamine on the induction of nitrate reductase

Nitrate reductase (NR. EC 1.6.6.1/2) is a substrate inducible enzyme that could be repressed by its end product glutamine or amino acids. To test this hypothesis, 6-day-old maize seedlings ( Zea mays cv. W64A × W182E) were grown hydroponically in a 1/10 strength Hougland's salt solution modified to contain no nitrogen. Previous experiments had established that after a 24-h induction with NO₃⁻ (5 mM KNO₃⁻) the level of NR activity and protein had reached a constant level. In the present experiments when glutamine (5 mM) was included together with NO₃⁻, there was a significant reduction in NR activity (34% of the control values). NR protein and NR mRNA accumulation in the root. In the shoot, on the other hand, glutamine additions had little or no effect on the levels of either NR activity (81% of control) or NR protein. Inhibition of glutamine synthetase by methionine sulfoximine (MSX) resulted in reduced levels of glutamine in both root and shoot tissues. Contrary in our prediction, however, it had no effect on NR activity and mRNA content in roots. In the shoot, on the other hand, there was a marked reduction of NR activity (34% of the control value) and NR protein, but no apparent effect on NR mRNA. When detached shoots were treated with MSX and other inhibitors of glutamine synthetase (tabtoxinine-β-lactam or phosphinothricin) the induction of NR activity by NO₃⁻ was also inhibited. Glutamine additions 15 or 50 mM to detached shoots had essentially no effect on the induction of NR activity (90% of control). These results demonstrate that the influence of glutamine and MSX on the induction of NR in maize root and shoot tissues, respectively, is very different.     

Extraction of nitrate reductase from leaves of ericaceae

The inability to obtain active nitrate reductase (E.C. 1.6.6.2) preparations from leaf extracts of Leucothoe and Rhododendron (Ericaceae) was due to a galloyl ester-like compound, similar in nature to tannic acid.     

Stability of nitrate reductase and source of its reductant in Sorghum seedlings

Pre-incubation of nitrate reductase from Sorghum seedlings with NADH increased enzyme activity by 25%. Ferricyanide had no effect. NADH protected the enzyme from inactivation during storage. Malonate inhibited in vivo nitrate reduction in Sorghum leaves by 95%. The inhibitory effect of malonate was reversed by fumarate. Sodium fluoride in the presence of phosphate also inhibited in vivo nitrate reduction by 60%. It is suggested that NADH generated via the citric acid cycle is utilized for nitrate reduction in Sorghum seedlings.     

Properties of nitrate reductase from seedling leaves of selected barley genotypes

Crude extracts from leaves of 6-day barley seedlings of parental genotypes (cv. Aramir and primitive line R567) and selected doubled haploid (DH) lines were not found to have significant differences in the NADH:NR activity, while considerable differences between these genotypes were shown by the NAD(P)H:NR activity. The cv. Aramir and DH lines did not differ by nitrate accumulation in the leaves. However, the primitive line R567, as compared to the remaining genotypes, was characterized by an appreciably lower ability to accumulate nitrates. In partially purified leaf extracts, significant differences in total NADH:NR activity and in distal activity dependent on methyl viologen (MV:NR) were found between the parental genotypes and selected DH lines. The studied genotypes differed also in dehydrogenase NR activity, i.e. cytochrome c reductase activity in crude extracts. In the studied genotypes, the NADH:NR activity in partially purified leaf extracts did not substantially differ by Km values for nitrates. Calculated Vmax values for NADH:NR in these genotypes were similar to total NR activity in partially purified extracts. Significant differences between the parental genotypes and selected DH lines were found in the thermal NADH:NR stability in crude and partially purified leaf extracts. From the performed studies it follows that different NR stability was one of the reasons of revealed differences in total activity and in partial NR activities in the leaf extracts between the studied genotypes of spring barley. Besides, it is suggested that varied NR gene expression in the leaves of these barley genotypes could also influence NR activity.     

The activity and stability of wheat nitrate reductase in vitro

The activity and decay characteristics of nitrate reductase from wheat (Triticum aestivum) were studied in crude, partially-purified and highly-purified preparations. The decay of nitrate reductase activity in crude extracts was due to spontaneous dissociation of the enzyme and to the effects of two decay factors, one present in the 0–30% and the other in the 50–70% saturated (NH₄)₂SO₄ fraction of a crude extract. Low rates of factor-mediated NR decay in vitro were associated with high levels of NR activity in vivo.     

A method for the determination of nitrate reductase

A procedure for the assay of nitrate reductase based on Szekely's diaminodiphenylsulphone method of nitrate determination (Szekely, E. (1967) Talanta 14, 941–950) is described. The method is simple and sensitive, allowing determination of 1 μg of nitrate in a volume of 1 ml or less. It is particularly suited to the determination of nitrate reductase.     

Kinetics of substrate inhibition of periplasmic nitrate reductase

Periplasmic nitrate reductase catalyzes the reduction of nitrate into nitrite using a mononuclear molybdenum cofactor that has nearly the same structure in all enzymes of the DMSO reductase family. In previous electrochemical investigations, we found that the enzyme exists in several inactive states, some of which may have been previously isolated and mistaken for catalytic intermediates. In particular, the enzyme slowly and reversibly inactivates when exposed to high concentrations of nitrate. Here, we study the kinetics of substrate inhibition and its dependence on electrode potential and substrate concentration to learn about the properties of the active and inactive forms of the enzyme. We conclude that the substrate-inhibited enzyme never significantly accumulates in the EPR-active Mo(+ V) state. This conclusion is relevant to spectroscopic investigations where attempts are made to trap a Mo(+ V) catalytic intermediate using high concentrations of nitrate.     

Regulation of the assimilation of nitrate in Chlamydomonas reinhardii

In Chlamydomonas, The assimilation of ammonia proceeds through the glutamine synthetaseglutamate synthase pathway. The primary target in the regula     

Effect of ammonium and nitrate on ferric chelate reductase and nitrate reductase in Vaccinium species

BACKGROUND AND AIMS: Most Vaccinium species have strict soil requirements for optimal growth, requiring low pH, high iron availability and nitrogen primarily in the ammonium form. These soils are limited and are often located near wetlands. Vaccinium arboreum is a wild species adapted to a wide range of soils, including high pH, low iron, and nitrate-containing soils. This broader soil adaptation in V. arboreum may be related to increased efficiency of iron or nitrate uptake compared with the cultivated Vaccinium species. METHODS: Nitrate, ammonium and iron uptake, and nitrate reductase (NR) and ferric chelate reductase (FCR) activities were compared in two Vaccinium species grown hydroponically in either nitrate or ammonia, with or without iron. The species studied were the wild V. arboreum and the cultivated V. corymbosum interspecific hybrid, which exhibits the strict soil requirements of most Vaccinium species. RESULTS: Ammonium uptake was significantly greater than nitrate uptake in both species, while nitrate uptake was greater in the wild species, V. arboreum, compared with the cultivated species, V. corymbosum. The increased nitrate uptake in V. arboreum was correlated with increased root NR activity compared with V. corymbosum. The lower nitrate uptake in V. corymbosum was reflected in decreased plant dry weight in this species compared with V. arboreum. Root FCR activity increased significantly in V. corymbosum grown under iron-deficient conditions, compared with the same species grown under iron-sufficient conditions or with V. arboreum grown under either iron condition. CONCLUSIONS: V. arboreum appears to be more efficient in acquiring nitrate compared with V. corymbosum, possibly due to increased NR activity and this may partially explain the wider soil adaptation of V. arboreum.     

Age-dependent conversion of nitrate reductase to cytochrome c reductase species in barley leaf extracts

The stability of nitrate reductase (NR) in extracts from 4-, 5- and 6-day-old primary leaves of barley was examined. The half-time of loss of NR activity was found to be 358, 107 and 70 min, respectively. Bovine serum albumin (BSA) and phenylmethylsulphonylfluoride (PMSF) stabilized NR in extracts from 5- and 6-day-old primary leaves, but BSA was much more effective. The increased instability of NR with age correlated with increased conversion of the MW 203 000 NR complex to smaller NADH cytochrome c reductase (CR) species of MW 163 000, 61 000 and 40 000. The MW 163 000 CR species also possessed NR activity. BSA prevented and PMSF retarded the conversion of NR to the smaller CR species. The increased instability of NR in extracts from older tissue may be due to increased conversion of NR to smaller CR species. The ability of PMSF and BSA to stabilize NR and inhibit conversion of NR to the smaller CR species indicates that these phenomena are probably due to proteolytic degradation of NR. This suggestion is supported by the observation that trypsin cleaved NR to 3–4 S CR species and that cleavage was retarded by the presence of BSA. Endogenous proteinase attack at specific sites between domains of the barley NR complex may generate the CR species seen in barley extracts. The MW 40 000 CR species probably carries at least the FAD domain.     

Inactivation of nitrate reductase from wheat and rice leaves

In fresh leaves, the inactivation of nitrate reductase was rapid at high temperatures as compared to low temperatures. In leaves subjected to freeze-thaw treatment, the loss of enzyme activity was extremely rapid particularly at high temperatures. Pre-incubation with NADH not only protected the enzyme against inactivation, but also stimulated its activity. In dialysed extracts of rice leaves, NADH alone offered some protection while nitrate alone did not protect the enzyme from inactivation. Addition of both NADH and nitrate during pre-incubation enhanced the enzyme activity considerably. It is suggested that stimulation of nitrate reduction by NADH and nitrate may be of physiological significance to the plant, in the sense that in the presence of sufficient supplies of reluctant and nitrate, the process of nitrate assimilation would be accelerated.     

In vitro stability of nitrate reductase from barley leaves

Barley seedling nitrate reductase was stabilized in vitro without the use of extraneous protein by optimizing the buffer components. The extraction buffer (NRT 8.5) consists of 0.25 M Tris-HCl, pH 8.5, 3 mM DTT, 5 μM FAD, 1 μ M sodium molybdate and 1 mM EDTA. This buffer stabilizes the extracted nitrate reductase at O° and 30°, whereas the addition of extraneous protein to standard extraction buffers stabilizes the enzyme only at 0°.     

Substrates regulate the phosphorylation status of nitrate reductase

The effect of substrates on the phosphorylation status of nitrate reductase (NR; EC 1.6.6.1) was studied. The enzyme was obtained from the first leaf of 7-day-old oat (Avena sativa L. cv. Suregrain) plants, grown in the light. When desalted crude extracts were incubated with ATP, NR was strongly phosphorylated, as evidenced by the inhibition of the enzyme's activity in the presence of Mg²⁺. NR sensitivity to Mg²⁺ remained unchanged when 10 mM nitrate was added to crude extracts after ATP. Addition of nitrate before or simultaneously with ATP slightly decreased Mg²⁺ inhibition of NR, which was strongly diminished in the presence of 10 mM NO₃^?+ 100 µM NADH. Incubation with NADH alone did not affect the enzyme's susceptibility to Mg²⁺ inhibition. When ammonium sulfate was added to crude extracts, NR was recovered in a 0-40% saturation fraction (F1). After incubation of F1 with ATP, the sensitivity of the enzyme to Mg²⁺ inhibition remained low, but it strongly increased after mixing F1 with a 45-60% saturation fraction (F2) suggesting that also in oats an additional factor (inactivating protein, IP), which probably binds to phospho-NR, would be required to keep the phosphorylated enzyme inactive in a +Mg²⁺ medium. Addition of 10 mM NO₃^?+ 100 µM NADH together with desalted F2 did not prevent Mg²⁺ inhibition suggesting that NO₃^? did not interfere with IP binding to phospho-NR. Again, incubation of F1 with both substrates during in vitro phosphorylation kept the enzyme active after adding F2, even in the presence of Mg²⁺, After in vitro phosphorylation, NR in crude extract was hardly reactivated when incubated alone or in the presence of 10 mM NO₃^? at 30°C. On the other hand, a strong and very rapid reactivation was found when the extract was incubated with both nitrate and NADH. Microcystine, an inhibitor of types 1 and 2A phosphoprotein phosphatases, inhibited the reactivation of phospho-NR induced by the substrates. The results presented here show that the substrates could prevent NR phosphorylation and induce the enzyme's dephosphorylation, but they were effective only after their binding to the NR protein. Thereby, they seemed to affect the NR protein itself and not the phosphatase- or the kinase-proteins. It has been reported that nitrate binding to the enzyme's active site induces conformational changes in the NR protein. We propose that this conformational change would prevent NR phosphorylation, by converting the enzyme into a form in which the site recognized by the protein kinase is no longer accessible, and, simultaneously, stimulate NR dephophorylation by allowing the specific phosphatases to recognize NR.