Effect of nitrogen source on the levels of nitrate reductase in the yeast Hansenula anomala.

Levels of nitrate reductase (NR) protein in Hansenula anomala and Hansenula wingei were determined using specific antiserum raised against the enzyme from H. anomala. Extracts from nitrate-grown cells contained NR protein, while in those from cells grown on ammonium, glutamine or peptone, no cross-reacting material could be observed. Enzyme activity correlated with the levels of cross-reacting material. When nitrate was used as nitrogen source, NR was always present, even in cultures with ammonium, glutamine or peptone, although in these cases both the levels of activity and protein were lower. NR activity was consistently two to four times higher in cells grown in glucose than in cells grown in ethanol. Nitrate was required for NR induction, and deprivation of nitrate from nitrate-grown cells resulted in a rapid loss of NR activity.     

Cytometric quantification of nitrate reductase by immunolabeling in the marine diatom Skeletonema costatum

BACKGROUND: The uptake of nitrate by phytoplankton is a central issue in biological oceanography due to its importance to primary production and vertical flux of biogenic carbon. Nitrate reductase catalyzes the first step of nitrate assimilation, the reduction of NO(3) to NO(2). A cytometric protocol to detect and quantify relative changes in nitrate reductase (NR) protein content of the marine centric diatom Skeletonema costatum is presented. METHODS: Immunolabeling of NR protein was achieved with polyclonal antibodies raised against S.costatum NR. Antisera specific to a NR protein subunit and to a NR polypeptide sequence were compared, and cytometric results of NR protein abundance were related to Western analyses. Changes in cellular NR abundance and activity were followed during an upwelling simulation experiment in which S. costatum was exposed to a shift from ammonia to nitrate as major nitrogen source. RESULTS: NR protein could be detected in NO(3)-grown cells and at extremely low levels hardly discernible by Western Blot densiometry in NH(4)-grown cells. The protocol allowed observation of early stages of NR induction during an upwelling simulation. NR abundance increased after the nutrient shift to reach a new physiological "steady-state" 96 hrs later. NR activity exhibited diel variation with maxima at mid-day. NR abundance as estimated by both flow cytometry and Western analysis exhibited a hyperbolic relationship to NR activity. This pattern suggests post-translational activation of NR protein. CONCLUSIONS: The presented protocol allows the differentiation of NH(4)- versus NO(3)-grown algae as well as the monitoring of early stages in the induction of nitrate assimilatory capacities.     

Rapid and slow modulation of nitrate reductase activity in the red macroalga <Emphasis Type="Italic">Gracilaria chilensis</Emphasis> (Gracilariales,Rhodophyta): influence of different nitrogen sources

Nitrate is one of the most important stimuli in nitrate reductase (NR) induction, while ammonium is usually an inhibitor. We evaluated the influence of nitrate, ammonium or urea as nitrogen sources on NR activity of the agarophyte Gracilaria chilensis. The addition of nitrate rapidly (2 min) induced NR activity, suggesting a fast post-translational regulation. In contrast, nitrate addition to starved algae stimulated rapid nitrate uptake without a concomitant induction of NR activity. These results show that in the absence of nitrate, NR activity is negatively affected, while the nitrate uptake system is active and ready to operate as soon as nitrate is available in the external medium, indicating that nitrate uptake and assimilation are differentially regulated. The addition of ammonium or urea as nitrogen sources stimulated NR activity after 24 h, different from that observed for other algae. However, a decrease in NR activity was observed after the third day under ammonium or urea. During the dark phase, G. chilensis NR activity was low when compared to the light phase. A light pulse of 15 min during the dark phase induced NR activity 1.5-fold suggesting also fast post-translational regulation. Nitrate reductase regulation by phosphorylation and dephosphorylation, and by protein synthesis and degradation, were evaluated using inhibitors. The results obtained for G. chilensis show a post-translational regulation as a rapid response mechanism by phosphorylation and dephosphorylation, and a slower mechanism by regulation of RNA synthesis coupled to de novo NR protein synthesis.     

The roles of nitrate and ammonium in the regulation of the development of nitrate reductase in Chlamydomonas reinhardii

The regulation of the development of nitrate reductase (NR) activity in Chlamydomonas reinhardii has been compared in a wild-type strain and in a mutant (nit-A) which possesses a modified nitrate reductase enzyme that is non-functional in vivo. The modified enzyme cannot use NAD(P)H as an electron donor for nitrate reduction and it differs from wild-type enzyme in that NR activity is not inactivated in vitro by incubation with NAD(P)H and small quantities of cyanide; it is inactivated when reduced benzyl viologen or flavin mononucleotide is present. After short periods of nitrogen starvation mutant organisms contain much higher levels of terminal-NR activity than do similarly treated wild-type ones. Despite the inability of the mutant to utilize nitrate, no nitrate or nitrite was found in nitrogen-starved cultures; it is therefore concluded that the appearance of NR activity is not a consequence of nitrification. After prolonged nitrogen starvation (22 h) the NR level in the mutant is low. It increases rapidly if nitrate is then added and this increase in activity does not occur in the presence of ammonium, tungstate or cycloheximide. Disappearance of preformed NR activity is stimulated by addition of tungstate and even more by addition of ammonium. The results are interpreted as evidence for a continuous turnover of NR in cells of the mutant with ammonium both stimulating NR breakdown and stopping NR synthesis. Nitrate protects the enzyme from breakdown. Reversible inactivation of NR activity is thought to play an insignificant rôle in the mutant.Abbreviations NR nitrate reductase - BV benzyl viologen     

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.     

Regulation of Maize Leaf Nitrate Reductase Activity Involves Both Gene Expression and Protein Phosphorylation

Nitrate reductase (NR; EC 1.6.6.1) activity increased at the beginning of the photoperiod in mature green maize (Zea mays L.) leaves as a result of increased enzyme protein level and protein dephosphorylation. In vitro experiments suggested that phosphorylation of maize leaf NR affected sensitivity to Mg2+ inhibition, as shown previously in spinach. When excised leaves were fed 32P-labeled inorganic phosphate, NR was phosphorylated on seryl residues in both the light and dark. Tryptic peptide mapping of NR labeled in vivo indicated three major 32P-phosphopeptide fragments, and labeling of all three was reduced when leaves were illuminated. Maize leaf NR mRNA levels that were low at the end of the dark period peaked within 2 h in the light and decreased thereafter, and NR activity generally remained high. It appears that light signals, rather than an endogenous rhythm, account primarily for diurnal variations in NR mRNA levels. Overall, regulation of NR activity in mature maize leaves in response to light signals appears to involve control of gene expression, enzyme protein synthesis, and reversible protein phosphorylation.     

Immunological approach to the regulation of nitrate reductase in Monoraphidium braunii

The effects of different culture conditions on nitrate reductase activity and nitrate reductase protein from Monoraphidium braunii have been studied, using two different immunological techniques, rocket immunoelectrophoresis and an enzyme-linked immunosorbent assay, to determine nitrate reductase protein. The nitrogen sources ammonium and glutamine repressed nitrate reductase synthesis, while nitrite, alanine, and glutamate acted as derepressors. There was a four- to eightfold increase of nitrate reductase activity and a twofold increase of nitrate reductase protein under conditions of nitrogen starvation versus growth on nitrate. Nitrate reductase synthesis was repressed in darkness. However, when Monoraphidium was grown under heterotrophic conditions with glucose as the carbon and energy source, the synthesis of nitrate reductase was maintained. With ammonium or darkness, changes in nitrate reductase activity correlated fairly well with changes in nitrate reductase protein, indicating that in both cases loss of activity was due to repression and not to inactivation of the enzyme. Experiments using methionine sulfoximine, to inhibit ammonium assimilation, showed that ammonium per se and not a product of its metabolism was the corepressor of the enzyme. The appearance of nitrate reductase activity after transferring the cells to induction media was prevented by cycloheximide and by 6-methylpurine, although in this latter case the effect was observed only in cells preincubated with the inhibitor for 1 h before the induction period.     

Regulation of molybdenum cofactor species in the green alga Chlamydomonas reinhardtii

Molybdenum cofactor (MoCo) of molybdoenzymes is constitutively produced in cells of the green alga Chlamydomonas reinhardtii grown in ammonium media, under which conditions certain molybdoenzymes are not synthesized. In soluble form, MoCo was found to be present in several forms: (i) as a low Mr free species; (ii) bound to a MoCo-carrier protein of about 50 kDa that could release MoCo to directly reconstitute in vitro nitrate reductase activity in the nit-1 mutant of Neurospora crassa, but not to Thiol-Sepharose which, in contrast, bonded free MoCo; and (iii) bound to other proteins, putatively constitutive molybdoenzymes, which only released MoCo after a denaturing treatment. The amount of total MoCo (free, carrier-bound and heat releasable forms) was dependent on the growth phase of cell cultures. Constitutive levels of total MoCo in ammonium-grown cells markedly increased when cells were transferred to media lacking ammonium (nitrate, urea or nitrogen-free media). This increase did not require de novo protein synthesis and was stimulated by light. Levels of both total MoCo and free plus carrier-bound MoCo seemed to be unrelated to either nitrate reductase synthesis or functioning of nit-1 and nit-2 genes responsible for nitrate reductase structure and regulation, respectively. Results suggest that MoCo is continuously synthesized in C. reinhardtii and that its levels are regulated by ammonium in a way independent of nitrate reductase synthesis.     

Inhibition of tobacco nitrite reductase activity by expression of antisense RNA

A tobacco nitrite reductase (NiR) cDNA and its corresponding gene were isolated from cDNA and genomic libraries. An NiR antisense mRNA was expressed in transgenic tobacco under the control of a double 35S promoter. Transformants were obtained on a medium containing ammonium as the sole source of nitrogen. One plant growing normally on ammonium but displaying drastically reduced development and chlorotic leaves when grown on nitrate as the sole source of nitrogen was studied further. This plant accumulated nitrite fivefold over wild-type level and showed reduced amounts of ammonium (11% wild-type level), glutamine (19%), and total protein (8%). NiR mRNA and activity were below detectable levels. Under these conditions, nitrate reductase (NR) activity and mRNA were overexpressed, suggesting that N-metabolites resulting from nitrate reduction are responsible for the repression of the expression of the NR gene, independently from the presence or absence of a functional NR protein.     

Synthesis and Degradation of Nitrate Reductase during the Cell Cycle of Chlorella Sorokiniana

Studies on the diurnal variations of nitrate reductase (NR) activity during the life cycle of synchronized Chlorella sorokiniana cells grown with a 7:5 light-dark cycle showed that the NADH:NR activity, as well as the NR partial activities NADH:cytochrome c reductase and reduced methyl viologen:NR, closely paralleled the appearance and disappearance of NR protein as shown by sodium dodecyl sulfate gel electrophoresis and immunoblots. Results of pulse-labeling experiments with [³⁵S]methionine further confirmed that diurnal variations of the enzyme activities can be entirely accounted for by the concomitant synthesis and degradation of the NR protein.     

Synthesis and degradation of barley nitrate reductase

Nitrate and light are known to modulate barley (Hordeum vulgare L.) nitrate reductase activity. The objective of this investigation was to determine whether barley nitrate reductase is regulated by enzyme synthesis and degradation or by an activation-inactivation mechanism. Barley seedling nitrate reductase protein (cross-reacting material) was determined by rocket immunoelectrophoresis and a qualitative immunochemical technique (western blot) during the induction and decay of nitrate reductase activity. Nitrate reductase cross-reacting material was not detected in root or shoot extracts from seedlings grown without nitrate. Low levels of nitrate reductase activity and cross-reacting material were observed in leaf extracts from plants grown on nitrate in the dark. Upon nitrate induction or transfer of nitrate-grown etiolated plants to the light, increases in nitrate reductase activity were positively correlated with increases in immunological cross-reactivity. Root and shoot nitrate reductase activity and cross-reacting material decreased when nitrate-induced seedlings were transferred to a nitrate-free nutrient solution or from light to darkness. These results indicate that barley nitrate reductase levels are regulated by de novo synthesis and protein degradation.     

NITROGEN METABOLISM OF AQUATIC ORGANISMS. II. THE ASSIMILATION OF NITRATE,NITRITE, AND AMMONIA BY BIDDULPHIA AURITA1

Biddulphia aurita, a centric diatom, can grow on either nitrate, nitrite, or ammonia as its sole nitrogen, source. Cells remove ammonium nitrogen from the medium 2.3–2.4 times faster than either nitrate or nitrite nitrogen and, when grown for 24 hr in the ammonium medium, contain higher levels of non-protein nitrogen than cells grown in the nitrate or nitrite medium for the same period of time. The nitrogenous compounds in the nonprotein nitrogen fraction from cells grown in the nitrate, nitrite, or ammonium medium contain the same level of soluble-free amino nitrogen, combined amino nitrogen, and ammonium nitrogen. The high level of soluble nonprotein nitrogen in the medium of the cells grown in the ammonium medium is due to soluble amide nitrogen which represents 18% of the total soluble nitrogen present in these cells, whereas it represents only 2% in cells from the nitrite medium, and its level is negligible in cells from the nitrate medium. Cells grown in the nitrate medium have both nitrate- and nitrite-reductase activity. Cells grown in the nitrite medium have only nitrite-reductase activity in significant levels, while cells grown in the ammonium medium lack both enzymes.     

Nitrate reductase and glutamate dehydrogenase activities in Skeletonema costatum as measures of nitrogen assimilation rates

Nitrate reductase (NADH-NR) and glutamate dehydrogenase (NADPH-GDH)activities were measured in Skeletonema costatum (Grev.) Clevein ammonium and nitrate limited continuous cultures before andafter additions of nitrate and/or ammonium. Comparisons of enzymicactivity with nitrogen uptake and assimilation rates, externaland internal nitrate concentrations, and external ammonium concentrationswere made in order to assess the roles of NR and GDH in nitrogenassimilation and to determine their suitability as measuresof nitrogen assimilation rates. NR activity appeared to be inducedby internal rather than external nitrate concentrations. Ammoniumin the medium reduced NR activity under some environmental conditions,but not others. However, ammonium acted indirectly, perhapsby causing the accumulation of an internal pool of an intermediateof ammonium assimilation. NR activity was found to approximatenitrate assimilation rates during growth limited by the nitratesupply and undeT some conditions in the presence of high nitrateand ammonium concentrations in the medium. Under other environmentalconditions, NR activity did not agree with nitrate assimilationrates; a second nitrate reducing mechanism may operate whenthese conditions prevail. GDH activities were consistently low,representing less than 5% of the ammonium uptake and assimilationrates. Consequently, it is proposed that ODH is not the primaryammonium assimilating enzyme under most environmental conditionsand cannot be used as a measure of ammonium assimilation. ¹ Contribution number 1095 from the Department of Oceanography,University of Washington     

Regulation of aldehyde oxidase and nitrate reductase in roots of barley (Hordeum vulgare L.) by nitrogen source and salinity

The molybdenum cofactor (MoCo) is a component of aldehyde oxidase (AO EC 1.2.3.1), xanthine dehydrogenase (XDH EC 1.2.1.37) and nitrate reductase (NR, EC 1.6.6.1). The activity of AO, which catalyses the last step of the synthesis of abscisic acid (ABA), was studied in leaves and roots of barley (Hordeum vulgare L.) plants grown on nitrate or ammonia with or without salinity. The activity of AO in roots was enhanced in plants grown with ammonium while nitrate-grown plants exhibited only traces. Root AO in barley was enhanced by salinity in the presence of nitrate or ammonia in the nutrient medium while leaf AO was not significantly affected by the nitrogen source or salinity of the medium.Salinity and ammonium decreased NR activity in roots while increasing the overall MoCo content of the tissue. The highest level of AO in barley roots was observed in plants grown with ammonium and NaCl, treatments that had only a marginal effect on leaf AO. ABA concentration in leaves of plants increased with salinity and ammonium.Keywords: ABA, aldehyde oxidase, ammonium, nitrate, salinity.