NADH-nitrate Reductase Research Articles

Nitrite Reductase 1 Is a Target of Nitric Oxide-Mediated Post-Translational Modifications and Controls Nitrogen Flux and Growth in Arabidopsis.

Plant growth is the result of the coordinated photosynthesis-mediated assimilation of oxidized forms of C, N and S. Nitrate is the predominant N source in soils and its reductive assimilation requires the successive activities of soluble cytosolic NADH-nitrate reductases (NR) and plastid stroma ferredoxin-nitrite reductases (NiR) allowing the conversion of nitrate to nitrite and then to ammonium. However, nitrite, instead of being reduced to ammonium in plastids, can be reduced to nitric oxide (NO) in mitochondria, through a process that is relevant under hypoxic conditions, or in the cytoplasm, through a side-reaction catalyzed by NRs. We use a loss-of-function approach, based on CRISPR/Cas9-mediated genetic edition, and gain-of-function, using transgenic overexpressing HA-tagged Arabidopsis NiR1 to characterize the role of this enzyme in controlling plant growth, and to propose that the NO-related post-translational modifications, by S-nitrosylation of key C residues, might inactivate NiR1 under stress conditions. NiR1 seems to be a key target in regulating nitrogen assimilation and NO homeostasis, being relevant to the control of both plant growth and performance under stress conditions. Because most higher plants including crops have a single NiR, the modulation of its function might represent a relevant target for agrobiotechnological purposes.

The occurence of a nitrate reductase inactivating factor in extracts of Spirodela polyrrhiza

NADH-nitrate reductase (NR) had a low activity immediately after extraction from 14 day-old cultures of <em>Spirodela polyrrhiza</em> with the basic extraction solution (50 mM K-phosphate buffer, pH 7.5, 5 mM cysteine and 3 mM EDTA) whereas nitrite reductase (NiR) activity in the same extract was very high. Increasing the EDTA or cysteine concentration or replacing cysteine by dithiothreitol had no effect on the level of NR activity. The addition of 3% bovine serum albumine (BSA) and 0.4 mM phenylmethyl sulfonyfluoride (PMSF) or 3% BSA and 1.5% polyvinylpolypyrrolidone (PVPP) to the extraction solution greatly increased the NR activity. The enzyme activity increased 40 times in the presence of 1.5% PVPP with 3% BSA and 0.4 mM PMSF. The highest NR activity (over 100-fold) was found in the presence of 3% caseine. The probable nature of the factor inactivating nitrate reductase in <em>S. polyrrhiza</em> extracts is discussed.

Denitrification by the FungusFusarium oxysporumInvolves NADH-Nitrate Reductase

Fusarium oxysporum JCM11502 expresses a denitrifying (nitrate (NO(3)(-))-respiring) mechanism and can thrive under oxygen (O(2)) limitation. The fungus reduces NO(3)(-) to nitrite at the initial step of denitrification. In this study, we cloned the gene coding NADH-NO(3)(-) reductase (NADH-Nar) (niaD) from F. oxysporum JCM11502. The niaD gene complemented the defective NO(3)(-) assimilation by mutant strain M10, indicating that the fungus reduced NO(3)(-) through NADH-Nar activity and assimilated it like other fungi. We found that the transcription of niaD and the production of NADH-Nar activity were enhanced under O(2)-limited denitrifying conditions relative to aerobic conditions. Strain M10 produced less NADH-Nar activity and less denitrified product than the wild-type strain. Introducing niaD into the mutant also restored these defects, indicating that niaD is involved in denitrification. These results indicate that the fungus denitrified NO(3)(-) through NADH-Nar activity in addition to the ubiquinol-Nar mechanism.

Induction of Class-1 Non-symbiotic Hemoglobin Genes by Nitrate, Nitrite and Nitric Oxide in Cultured Rice Cells

Non-symbiotic hemoglobins (ns-Hbs) are found in all plants, although their physiological function remains to be determined. The present study was undertaken to explore the mode of induction of ns-Hb genes by metabolites of nitrate assimilation using cultured rice (Oryza sativa L.) cells. Two class-1 ns-Hb genes, ORYsa GLB1a and ORYsa GLB1b, were strongly induced by nitrate, nitrite and nitric oxide (NO) donors, S-nitroso-N-acetylpenicillamine and sodium nitroprusside. The rapid and transient accumulation of ORYsa GLB1a and ORYsa GLB1b transcripts in response to nitrate, nitrite and NO donors was similar to that of nia1, which encodes NADH-nitrate reductase (NR), although repression by glutamine and asparagines was significant only for nia1. In the mutants defective in NR mRNA expression, nitrate, nitrite and NO donors failed to induce not only nia1 but also ORYsa GLB1a and ORYsa GLB1b transcripts, indicating that the induction of ns-Hb genes is closely associated with that of the NR gene. Although the kinetics of induction by nitrate, nitrite and NO donors are similar for the two ns-Hb genes, an inhibitor study demonstrated that de novo synthesis of the protein in cytoplasm is essential for inducing ORYsa GLB1b. In contrast, ORYsa GLB1a, like nia1, can be induced in the primary response to these signals without de novo protein synthesis. The role of nitrate, nitrite and NO in the induction of ns-Hb gene expression in rice cells and the possible cellar function of ns-Hbs were discussed in relation to nitrate reduction pathways.

Influence of plant age and growth conditions on nitrate assimilation in roots of Lotus japonicus plants.

In roots of Lotus japonicus (Regel) Larsen cv. Gifu, the level of NADH-nitrate reductase (NR) activity and protein, as well as ferredoxin-nitrite reductase activity and nitrate accumulation, were higher in roots of young plants, and decreased in mature plants grown in seed trays. When plants were grown in larger pots, the decrease in NR activity and nitrate accumulation took place at a later stage of growth, suggesting that the cessation of nitrate assimilation in mature plants could be related to both ageing and a lower availability of space for roots. Low NR activity was detected in leaves, whereas nitrate accumulation in this tissue could reach relatively high levels. NR activity and protein, and nitrate accumulation, also decreased in leaves of mature plants, indicating that the diminution in nitrate accumulation and NR in roots from mature plants is not related to a shift of the nitrate assimilation process from roots to leaves. Measurement of the extent of total and active NR according to the phosphorylation/dephosphorylation inactivating mechanism described for spinach leaf NR, indicates that this mechanism is operative on NR from L. japonicus roots, being responsible for a great proportion of inactive NR protein. The amount of inactive NR protein in roots due to the aforementioned phosphorylation mechanism also increased in mature plants.

Rhodococcus sp. RB1 grows in the presence of high nitrate and nitrite concentrations and assimilates nitrate in moderately saline environments.

Rhodococcus sp. RB1 was able to thrive in media with up to 0.9 M NaCl or KCl and in the presence of high concentrations of nitrate (up to 0.9 M) and nitrite (up to 60 mM), but only under oxic conditions. An adaptation period was not required for salt tolerance, but a rapid extrusion of K+ and intake of Na+ was observed after addition of 0.5 M NaCl. Nitrate assimilation was limited by the carbon supply, but nitrite was not accumulated in the culture medium, even at nitrate concentrations as high as 0.8 M, thus suggesting that nitrite reduction does not limit nitrate assimilation. The presence of NaCl or KCl did not affect nitrate or nitrite uptake, which were completely inhibited by ammonium or glutamine. Rhodococcus sp. RB1 nitrate reductase had an apparent molecular mass of 142 kDa and used NADH and reduced bromophenol blue or viologens as electron donors, independently of the presence of salt. The enzyme was associated with an NADH-diaphorase activity and was induced by nitrate and repressed by ammonium or glutamine, thus showing typical biochemical and regulatory properties of bacterial assimilatory NADH-nitrate reductases. The enzyme was active in vitro in the presence of 3 M NaCl or KCI, but the maximal activity was observed at 0.5 M salt. Addition of 2 M NaCl increased the optimal temperature of the enzyme from 12 to 32 degrees C, but the optimal pH (10.3) was unaffected.

Physiological adaptations of glutamate dehydrogenase isozyme activities and other nitrogen-assimilating enzymes in the macroalga Bryopsis maxima

The activities of nitrogen-assimilating enzymes in the marine macroalga, Bryopsis maxima Okamura ex Segawa (Chlorophyta) were studied. Changes in these enzymatic activities were observed during cultivation in a laboratory setting, and after the addition of chemicals. The following results were obtained. (1) The chloroplastic NADP-glutamate dehydrogenase (NADP-GDH; EC 1.4.1.4) increased by 49%, while the cytosolic/mitochondrial NADP-GDH decreased to 23% of its original level, showing organelle-specificity. Chloroplastic and cytosolic glutamine synthetases (GS; EC 6.3.1.2) showed 46% increases in 3 days, followed by decreases to their original levels. Chloroplastic NADPH-glutamate synthase (NADPH-GOGAT; EC 1.4.1.13) showed an increase. Cytosolic NADH-nitrate reductase (NADH-NR; EC 1.6.6.1) decreased to about half its original level in 7 days. (2) A compensatory enhancement of NADP-GDH activity was not observed when the GS activity was completely inhibited by methionine sulfoximine. (3) The chloroplastic NADP-GDH decreased to 82% while cytosolic/mitochondrial NADP-GDH increased to 250% with ATP treatment. These results suggest that the activities of nitrogen-assimilating enzymes are regulated independently in this alga.

Increased glutamine synthetase activity and changes in amino acid pools in leaves treated with 5-aminoimidazole-4-carboxiamide ribonucleoside (AICAR).

Feeding 5-aminoimidazole-4-carboxiamide ribonucleoside (AICAR) through the petiole of detached young barley leaves rapidly increased activities of NADH-nitrate reductase (NR) and glutamine synthetase (GS) in leaf extracts and at least partly prevented the usual slow decrease of these enzyme activities during prolonged illumination. Further, AICAR caused drastic changes in amino acid levels: glutamine and serine levels were increased whereas glutamate and glycine were decreased, probably indicating a higher GS activity and more rapid conversion of glycine into serine. The latter may be responsible for the higher ammonium contents found in AICAR treated leaves. We tentatively suggest that GS (located in the chloroplast) and glycine decarboxylase (located in the mitochondria) are regulated in a manner similar to NR. This is discussed in the light of recent reports that 14-3-3 isoforms exist in chloroplasts and that GS binds to 14-3-3s in vitro.

Nitrate reductase in Zea mays L. under salinity

ABSTRACTYoung maize seedlings (Zea mays L. cv.Giza 2) were exposed to moderate salinity in hydroponic culture. NADH‐nitrate reductase (NR) activity (E.C. 1·6.6·1), NR activation state, NR‐mRNA‐steady state levels and major solute contents in leaves and roots were investigated. With increasing external salt concentration, Na+, Cl−, sugars, amino acids and quarternary ammonium compounds accumulated in leaves and roots, with concentrations in leaves exceeding those in roots. The nitrate content of leaves decreased, but increased in roots. The diurnal pattern of NR activity and of NR‐mRNA was also changed under salinity, but the NR activation state was not affected. In the first light phase, maximum NR activity increased rapidly in leaves of control plants, but was much slower in leaves from salinized plants. Thus, integrated over the whole day, the NR activity of salt‐stressed plants was lower than in control plants. NR transcript levels of control plants were low in the early night phase, started to increase in the second night phase, followed by a distinct peak at 2 h into the light period. This large ‘early morning peak’ of NR‐mRNA was hardly affected by salinity, whereas the initial slow increase of m‐RNA levels in the early night phase was almost absent in salinized plants. This is considered as one reason for the low NR activity of salinized plants in the first half of the day. It is also suggested that nitrate is a major signal affecting NR expression and activity under salinity. Sugars and amino acids appeared less important.

Ferricyanide restores nitrate reductase activity in leaf extracts of water stressed wheat seedlings

Summary Nitrate reductase (EC 1.6.6.1) activity declined rapidly in leaves of wheat seedlings following polyethylene glycol-induced water stress. The decrease in NADH-nitrate reductase was accompanied by a concomitant loss of FMNH 2 − and reduced benzyl viologen-linked nitrate reductase activities. Incubation of dialysed leaf extracts with potassium ferricyanide restored the enzyme activity almost to the same level as that in non-stressed plants. The decline in nitrate reductase, at least during the initial stages of water stress, hence seems to be primarily due to redox-mediated inactivation rather than diminished synthesis of the enzyme.

Responses of NADH and NADPH nitrate reductase activities and nitrate uptake to chlorate supply in maize seedlings

Summary Experiments were conducted to examine the responses of NADH and NADPH specific nitrate reductase (NR) activities from various organs of maize seedlings to chlorate, with a view to understand the mechanism of chlorate toxicity and to ellucidate the significance of NAD(P)H: NR under toxic conditions. Supply of 1, 5, 10 or 20 mmol/L KCIO 3 under inducing conditions (10 mmol/L nitrate) to 4-5-day-old seedlings grown without nitrogen for 24 h in light (67 W m −2 ) at 25°C inhibited NADH:NR activity in the shoots and scutellum, and to some extent in the roots also. On the other hand, NADPH: NR activity was increased substantially in the scutellum and decreased (above 1 mmol/L chlorate) in roots by chlorate supply. Total protein content of the shoot or scutellum was inhibited by 20 mmol/L chlorate and was almost unaffected at lower concentrations in these organs; it increased slightly at all concentrations of chlorate in the roots. Nitrate acquisition by the seedlings was inhibited to an almost similar magnitude at each concentration of chlorate used. In another experiment involving short term (3h) supply of 5 mmol/L chlorate to the seedlings under inducing (+nitrate) or non-inducing (−nitrate) conditions, NADH:NR activity was usually inhibited and NADPH:NR activity was increased in scutellum or unaffected in roots. During in vitro incubation of enzyme preparations for 1 h at 4°C with 5 mmol/L chlorate, 10 mmol/L nitrate or chlorate + nitrate, NADH:NR activity increased in shoots and to some extent in roots also, but had variable effects in scutellum. Root NADPH:NR activity also increased by incubation with either of these ions, while in the scutellum it was drastically reduced. The experiments demonstrate an organ specific and isoform specific response of NR to chlorate and also that chlorate was acting directly on enzyme molecules under in vitro conditions.

Nitrate reductase from rice seedlings: Partial purification, characterization and the effects of in situ and in vitro NaCl salinity

Summary The effect of NaCl salinity in situ on NADH-nitrate reductase (EC 1.6.6.1) activity was studied in rice seedlings grown in sand cultures. NaCl salinity levels of 7 dS m−1 and 14 dS m−1 caused significant inhibition in NR activity in 10- and 20-day-grown seedlings. The enzyme was partially purified from leaves of 15-day-grown non-salinized seedlings using ammonium sulphate (25–45 percent) precipitation and affinity chromatography on a Blue-Sepharose CL-6B column. The purification achieved was 303-fold with 53 percent yield and the purified enzyme showed a specific activity of 3.6 μmoles NO2− min−1 mg−1 protein. NR activity was inhibited in vitro with an increase in NaCl in the assay medium. With 1000 mmol/L NaCl in the assay medium almost 85 percent NR activity was lost. The purified enzyme showed optimum activity at pH 7.5 and temperature optima of 30°C for 30 min. Preincubation of NR with 0.4 mmol/L NADH caused a significant increase in its activity. Apparent Km values of the enzyme for nitrate and NADH were 0.286 mmol/L and 4 mmol/L, respectively. The enzyme NR thus appears to be fairly sensitive to in situ as well as in vitro effects of NaCl.

Spectroscopic and Kinetic Characterization of the Recombinant Cytochrome c Reductase Fragment of Nitrate Reductase: IDENTIFICATION OF THE RATE-LIMITING CATALYTIC STEP

The recombinant NADH-cytochrome c reductase fragment of spinach NADH-nitrate reductase (EC 1.6.6.1), consisting of the contiguous heme-containing cytochrome b domain and flavin-containing NADH-cytochrome b reductase fragment, has been characterized spectroscopically and kinetically. Reductive titration with sodium dithionite indicates heme reduction takes place prior to flavin reduction, which correlates well with the reduction potentials for enzyme-bound heme (15 mV) and FAD (-280 mV). Reductive titration with NADH also indicates that the reduced enzyme forms a charge-transfer complex with NAD+. The circular dichroism spectrum of the oxidized fragment is primarily due to the flavin, whereas the ferrous heme dominates the circular dichroism spectrum of reduced enzyme. Three kinetic phases are observed in the course of the reaction of the enzyme with NADH, each with a distinct spectral signature. The fast phase represents flavin reduction, concomitant with the formation of a charge-transfer complex between reduced flavin and NAD+, and exhibits hyperbolic dependence on NADH concentration with a Kd of 3 microM and a limiting rate constant of 560 s-1. Electron transfer from reduced flavin to heme with a rate constant of 12 s-1 is the intermediate phase, which is rate-limited by breakdown of the charge-transfer complex between NAD+ and reduced flavin. The slow phase is dismutation of a pair of molecules of two-electron reduced enzyme (generated at the end of the second phase of the reaction) to give one molecule each of one- and three- electron reduced enzyme, with a second order rate constant of 2 x 10(6) M-1 s-1. In the presence of excess NADH, this dismutation reaction is followed by the rapid reaction of the one-electron reduced enzyme with a second equivalent of NADH to generate fully reduced enzyme. On the basis of this work, it appears that dissociation of NAD+ from the reduced flavin site rate limits electron transfer to the cytochrome and likely represents the overall rate-limiting step of catalysis.

The Impact of Ozone on the15N Incorporation and Nitrogen Assimilation of Wheat and Maize

Abstract Young wheat (C3) and maize (C4) plants were exposed to near-ambient concentrations of ozone in open-top chambers in order to investigate the possible effects of ozone on nitrogen metabolism. Nitrogen was supplied to the plants by adding (15)N-labelled tracer substances via the soil substrate. Enzyme activities (NADH nitrate reductase, nitrite reductase, glutamine synthetase and NADH glutamate dehydrogenase) and the incorporation of (15)N were determined. The findings show that nitrogen metabolism was affected by O(3), however, there were distinct differences between the two species. In plants treated with O(3), NADH nitrate reductase activity in maize leaves was reduced, while NR activity in wheat leaves only slightly declined. Only minor changes were observed with respect to the activities of nitrite reductase, glutamine synthetase and NADH glutamate dehydrogenase. Feeding experiments using (15)NO(3) (-) showed that the incorporation of nitrate nitrogen in wheat plants exposed to ozone remains virtually unchanged, whereas in maize plants reduced incorporation rates were observed for nitrate nitrogen. The incorporation of ammonium nitrogen was distinctly increased in wheat and maize by the impact of ozone. When investigating pigment contents, reduced levels of chlorophyll a and b and carotenoids were observed, whereas the pigment content of wheat leaves remained unchanged. These results indicate that young maize plants are more susceptible than wheat plants to short-term ozone exposure.

Physiology and interaction of nitrate and nitrite reduction in Staphylococcus carnosus.

Staphylococcus carnosus reduces nitrate to ammonia in two steps. (i) Nitrate was taken up and reduced to nitrite, and nitrite was subsequently excreted. (ii) After depletion of nitrate, the accumulated nitrite was imported and reduced to ammonia, which again accumulated in the medium. The localization, energy gain, and induction of the nitrate and nitrite reductases in S. carnosus were characterized. Nitrate reductase seems to be a membrane-bound enzyme involved in respiratory energy conservation, whereas nitrite reductase seems to be a cytosolic enzyme involved in NADH reoxidation. Syntheses of both enzymes are inhibited by oxygen and induced to greater or lesser degrees by nitrate or nitrite, respectively. In whole cells, nitrite reduction is inhibited by nitrate and also by high concentrations of nitrite (> or = 10 mM). Nitrite did not influence nitrate reduction. Two possible mechanisms for the inhibition of nitrite reduction by nitrate that are not mutually exclusive are discussed. (i) Competition for NADH nitrate reductase is expected to oxidize the bulk of the NADH because of its higher specific activity. (ii) The high rate of nitrate reduction could lead to an internal accumulation of nitrite, possibly the result of a less efficient nitrite reduction or export. So far, we have no evidence for the presence of other dissimilatory or assimilatory nitrate or nitrite reductases in S. carnosus.

Variation of nitrate reductase genes in selected grass species.

In order to study the variation of nitrate reductase (NR) genes among grass species, gene number, intron size and number, and the heme-hinge fragment sequence of 25 grass species were compared. Genomic DNA cut with six restriction enzymes and hybridized with the barley NAD(P)H and NADH NR gene probes revealed a single NAD(P)H NR gene copy and two or more NADH NR gene copies per haploid genome in most of the species examined. Major exceptions were Hordeum vulgare, H. vulgare ssp. spontaneum, and Avena strigosa, which appeared to have a single NADH NR gene copy. The NADH NR gene intron number and lengths were examined by polymerase chain reaction amplification. Introns I and III appeared to be absent in at least one of the NADH NR genes in the grass species, while intron II varied from 0.8 to 2.4 kilobases in length. The NADH NR gene heme-hinge regions were amplified and sequenced. The estimated average overall nucleotide substitution rate in the sequenced region was 7.8 x 10(-10) substitutions/site per year. The synonymous substitution rate was 2.11 x 10(-9) substitutions/synonymous site per year and the nonsynonymous substitution rate was 4.10 x 10(-10) substitutions/nonsynonymous site per year. Phylogenetic analyses showed that all of the wild Hordeum species examined clustered in a group separate from H. vulgare and H. vulgare ssp. spontaneum.

Possible interaction between peroxidase and NAD(P)H-dependent nitrate reductase activities of plasma membranes of corn roots

The relationship between the plasma membrane-bound NAD(P)H-nitrate reductase (NR) and a plasma membrane (PM)-bound peroxidase was investigated using highly purified PM vesicles isolated from corn roots. The PM-bound NR activity was strongly enhanced by MnCl 2 and SHAM, which stimulated peroxidase activity. Since both activities, the NAD(P)H-dependent NR and the peroxidase compete for NAD(P)H as electron donor, we propose a model in which a product of peroxidation is able to offer electrons to the nitrate reductase in a more reactive form with respect to NAD(P)H. Out hypothesis was confirmed by experiments in which the effects of inhibitors of peroxidative reactions, catalase, superoxide dismutase, and ascorbate on the PM-bound NR were studied. Results indicate that the putative electron donor for nitrate reduction could be a radicalic species, possibly NAD . . Furthermore, since cytochrome c decreased the activity of the plasma membrane-bound NAD(P)H-dependent NR, cytochrome b 557 might be the site of the enzyme accepting electrons from NAD . . Out results indicate that the PM environment of the NR may be involved in the extent of the membrane-associated nitrate reduction and that redox enzymes at the PM, the NAD(P)H-NR and a peroxidase-like NADH-oxidase, can interact.

An NADH nitrate reductase gene copy appears to have been deleted in barley

Cultivated barley,Hordeum vulgare L., has a single NADH nitrate reductase (NR) gene while diploid wheat,Triticum monococcum, and cultivated hexaploid wheat,Triticum aestivum L., have two NADH NR genes. To determine whether the NADH NR gene was duplicated since the divergence ofTriticum fromHordeum or was deleted from barley, theT. Monococcum NADH NR gene heme-hinge regions were sequenced and compared with the barley NADH NR gene sequence. Sequence identity and phylogenetic analyses showed that one of theT. Monococcum NADH NR genes is more-closely related to the barley NADH NR gene than to the otherT. Monococcum NADH NR gene. The heme-hinge region of all three NR genes appeared to have evolved at a constant rate. These results suggest that the NADH NR gene duplicated before the divergence ofTriticum andHordeum and that a deletion resulted in the loss of one NADH NR gene from cultivated barley.

Modulation of nitrate reductase in vivo and in vitro: Effects of phosphoprotein phosphatase inhibitors, free Mg2+ and 5?-AMP

Nitrate reductase in spinach (Spinacia oleracea L.) leaves was rapidly inactivated in the dark and reactivated by light, whereas in pea (Pisum sativum L.), roots, hyperoxic conditions caused inactivation, and anoxia caused reactivation. Reactivation in vivo, both in leaves and roots, was prohibited by high concentrations (10–30 μM) of the serine/threonine-protein phosphatase inhibitors okadaic acid or calyculin, consistent with the notion that protein dephosphorylation catalyzed by type-1 or type-2A phosphatases was the mechanism for the reactivation of NADH-nitrate reductase (NR). Following inactivation of leaf NR in vivo, spontaneous reactivation in vitro (in desalted extracts) was slow, but was drastically accelerated by removal of Mg2+ with excess ethylenediaminetetraacetic acid (EDTA), or by desalting in a buffer devoid of Mg2+. Subsequent addition of either Mg2+, Mn2+ or Ca2+ inhibited the activation of NR in vitro. Reactivation of NR (at pH 7.5) in vitro in the presence of Mg2+ was also accelerated by millimolar concentrations of AMP or other nucleoside monophosphates. The EDTA-mediated reactivation in desalted crude extracts was completely prevented by protein-phosphatase inhibitors whereas the AMP-mediated reaction was largely unaffected by these toxins. The Mg2+-response profile of the AMP-accelerated reactivation suggested that okadaic acid, calyculin and microcystin-LR were rather ineffective inhibitors in the presence of divalent cations. However, with partially purified enzyme preparations (5–15% polyethyleneglycol fraction) the AMPmediated reactivation was also inhibited (65–80%) by microcystin-LR. Thus, the dephosphorylation (activation) of NR in vitro is inhibited by divalent cations, and protein phosphatases of the PP1 or PP2A type are involved in both the EDTA and AMP-stimulated reactions. Evidence was also obtained that divalent cations may regulate NR-protein phosphatase activity in vivo. When spinach leaf slices were incubated in Mg2+ -and Ca2+-free buffer solutions in the dark, extracted NR was inactive. After addition of the Ca2+ /Mg2+-ionophore A 23187 plus EDTA to the leaf slices, NR was activated in the dark. It was again inactivated upon addition of divalent cations (Mg2+ or Ca2+). It is tentatively suggested that Mg2+ fulfills several roles in the regulatory system of NR: it is required for active NR-protein kinase, it inactivates the protein phosphatase and is, at the same time, necessary to keep phospho-NR in the inactive state. The EDTA- and AMP-mediated reactivation of NR in vitro had different pH optima, suggesting that two different protein phosphatases may be involved. At pH 6.5, the activation of NR was relatively slow and the addition or removal of Mg2+ had no effect. However, 5′-AMP was a potent activator of the reaction with an apparent Km of 0.5 mM. There was also considerable specificity for 5′AMP relative to 3′- or 2′-AMP or other nucleoside monophoposphates. We conclude that, depending upon conditions, the signals triggering NR modulation in vivo could be either metabolic (e.g. 5′-AMP) or physical (e.g. cytosolic [Mg2+]) in nature.

Magnesium and Calcium Inhibition of Squash Leaf NADH Nitrate Reductase

Magnesium and Calcium Inhibition of Squash Leaf NADH Nitrate Reductase