Correlations between the Electronic Properties of Shewanella oneidensis Cytochrome c Nitrite Reductase (ccNiR) and Its Structure: Effects of Heme Oxidation State and Active Site Ligation.

The electrochemical properties of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR), a homodimer that contains five hemes per protomer, were investigated by UV-visible and electron paramagnetic resonance (EPR) spectropotentiometries. Global analysis of the UV-vis spectropotentiometric results yielded highly reproducible values for the heme midpoint potentials. These midpoint potential values were then assigned to specific hemes in each protomer (as defined in previous X-ray diffraction studies) by comparing the EPR and UV-vis spectropotentiometric results, taking advantage of the high sensitivity of EPR spectra to the structural microenvironment of paramagnetic centers. Addition of the strong-field ligand cyanide led to a 70 mV positive shift of the active site's midpoint potential, as the cyanide bound to the initially five-coordinate high-spin heme and triggered a high-spin to low-spin transition. With cyanide present, three of the remaining hemes gave rise to distinctive and readily assignable EPR spectral changes upon reduction, while a fourth was EPR-silent. At high applied potentials, interpretation of the EPR spectra in the absence of cyanide was complicated by a magnetic interaction that appears to involve three of five hemes in each protomer. At lower applied potentials, the spectra recorded in the presence and absence of cyanide were similar, which aided global assignment of the signals. The midpoint potential of the EPR-silent heme could be assigned by default, but the assignment was also confirmed by UV-vis spectropotentiometric analysis of the H268M mutant of ccNiR, in which one of the EPR-silent heme's histidine axial ligands was replaced with a methionine.

Lead-induced nitric oxide generation plays a critical role in lead uptake by Pogonatherum crinitum root cells.

The effects of lead (Pb) on endogenous nitric oxide (NO) generation, the role of NO in Pb uptake and the origin of Pb-induced NO production in Pogonatherum crinitum root cells were evaluated. Pb treatment induced rapid NO generation, showing that Pb exposure triggered endogenous NO signaling of the cells. Pre-treatment of the cells with the NO-specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline -1-oxyl-3-oxide (cPTIO) not only abolished the Pb-triggered NO burst but also reduced Pb contents of the cells. Moreover, Pb exposure enhanced nitrate reductase (NR) activity of the cells. The NR inhibitors tungstate and glutamine not only suppressed the Pb-enhanced NR activities but also reduced the Pb-triggered NO generation. Pre-treatment of the cells with tungstate and glutamine suppressed Pb accumulation and the suppression could be restored by application of exogenous NO via its donors sodium nitroprusside (SNP) and S-nitrosoglutathione (GSNO). Together, our results indicated that Pb exposure enhanced NR activity and triggered the NO burst of P. crinitum root cells. Furthermore, the data demonstrated that NR was responsible for the Pb-triggered NO burst and that NR-mediated NO generation played a critical role in Pb uptake by P. crinitum root cells. Thus, our results suggest a potential strategy for controlling Pb uptake by plants by targeting NR as a source of Pb-triggered NO production.

Calcium is involved in nitric oxide- and auxin-induced lateral root formation in rice.

In the present study, the role of nitric oxide (NO) in the regulation of lateral root (LR) formation in rice was examined. Application of sodium nitroprusside (SNP; a NO donor) and indole-3-butyric acid (IBA; a naturally occurring auxin) to rice seedlings induced LR formation. The effect is specific for NO because the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3- oxide (cPTIO) blocked the action of SNP and IBA. Endogenous NO was detected by the specific fluorescence probe 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate. SNP- and IBA-induced NO fluorescence was specifically suppressed by cPTIO. Nitrate reductase (NR) inhibitor sodium tungstate completely inhibited IBA-induced LR formation and NO fluorescence. However, nitric oxide synthase inhibitor N (G)-nitro-L: -arginine methyl ester hydrochloride slightly reduced IBA-induced LR formation and NO generation. It appears that NO generation that occurs in response to IBA might primarily involve NR activity. Moreover, NO production caused by SNP and IBA was localized in root area corresponding to LR emergence. The effects of Ca(2+) chelators, Ca(2+)-channel inhibitors, and calmodulin antagonists on LR formation induced by SNP and IBA were also examined. All these inhibitors were effective in reducing the action of SNP and IBA. However, Ca(2+) chelators and Ca(2+)-channel inhibitors had no effect on SNP- and IBA-induced NO generation. It is concluded that cytosolic levels of Ca(2+) may regulate SNP and IBA action through calmodulin-dependent mechanism.

Nitrate reduction associated with respiration in Sinorhizobium meliloti 2011 is performed by a membrane-bound molybdoenzyme.

The purification and biochemical characterization of the respiratory membrane-bound nitrate reductase from Sinorhizobium meliloti 2011 (Sm NR) is reported together with the optimal conditions for cell growth and enzyme production. The best biomass yield was obtained under aerobic conditions in a fed-batch system using Luria-Bertani medium with glucose as carbon source. The highest level of Sm NR production was achieved using microaerobic conditions with the medium supplemented with both nitrate and nitrite. Sm NR is a mononuclear Mo-protein belonging to the DMSO reductase family isolated as a heterodimeric enzyme containing two subunits of 118 and 45 kDa. Protein characterization by mass spectrometry showed homology with respiratory nitrate reductases. UV-Vis spectra of as-isolated and dithionite reduced Sm NR showed characteristic absorption bands of iron-sulfur and heme centers. Kinetic studies indicate that Sm NR follows a Michaelis-Menten mechanism (K (m) = 97 ± 11 µM, V = 9.4 ± 0.5 µM min(-1), and k (cat) = 12.1 ± 0.6 s(-1)) and is inhibited by azide, chlorate, and cyanide with mixed inhibition patterns. Physiological and kinetic studies indicate that molybdenum is essential for NR activity and that replacement of this metal for tungsten inhibits the enzyme. Although no narGHI gene cluster has been annotated in the genome of rhizobia, the biochemical characterization indicates that Sm NR is a Mo-containing NR enzyme with molecular organization similar to NarGHI.

pHg/pSILBAγ vector system for efficient gene silencing in homobasidiomycetes: optimization of ihpRNA - triggering in the mycorrhizal fungus Laccaria bicolor.

pSILBAγ silencing vector was constructed for efficient RNA silencing triggering in the model mycorrhizal fungus Laccaria bicolor. This cloning vector carries the Agaricus bisporus gpdII promoter, two multiple cloning sites separated by a L. bicolor nitrate reductase intron and the Aspergillus nidulans trpC terminator. pSILBAγ allows an easy oriented two-step PCR cloning of hairpin sequences to be expressed in basidiomycetes. With one further cloning step into pHg, a pCAMBIA1300-based binary vector carrying a hygromycin resistance cassette, the pHg/pSILBAγ plasmid is used for Agrobacterium-mediated transformation. The pHg/pSILBAγ system results in predominantly single integrations of RNA silencing triggering T-DNAs in the fungal genome and the integration sites of the transgenes can be resolved by plasmid rescue. pSILBAγ construct and two other pSILBA plasmid variants (pSILBA and pSILBAα) were evaluated for their capacity to silence Laccaria nitrate reductase gene. While all pSILBA variants tested resulted in up to 65-76% of transformants with reduced growth on nitrate, pSILBAγ produced the highest number (65%) of strongly affected fungal strains. The strongly silenced phenotype was shown to correlate with T-DNA integration in transcriptionally active genomic sites. pHg/pSILBAγ was shown to produce T-DNAs with minimum CpG methylation in transgene promoter regions which assures the maximum silencing trigger production in Laccaria. Methylation of the target endogene was only slight in RNA silencing triggered with constructs carrying an intronic spacer hairpin sequence. The silencing capacity of the pHg/pSILBAγ was further tested with Laccaria inositol-1,4,5-triphosphate 5-phosphatase gene. Besides its use in silencing triggering, the herein described plasmid system can also be used for transgene expression in Laccaria. pHg/pSILBAγ silencing system is optimized for L. bicolor but it should be highly useful also for other homobasidiomycetes, group of fungi currently lacking molecular tools for RNA silencing.

A simple whole cell based high throughput screening protocol using Mycobacterium bovis BCG for inhibitors against dormant and active tubercle bacilli.

This study aimed at developing a whole cell based high throughput screening protocol to identify inhibitors against both active and dormant tubercle bacilli. A respiratory type of nitrate reductase (NarGHJI), which was induced during dormancy, could reflect the viability of dormant bacilli of Mycobacterium bovis BCG in microplate adopted model of in vitro dormancy. Correlation between reduction in viability and nitrate reductase activity was seen clearly when dormant stage inhibitor metronidazole and itaconic anhydride were applied in this in vitro microplate model. Active replicating stage could also be monitored in the same assay by measuring the A(620) of the culture. MIC values of 0.08, 0.075, 0.3 and 3.0 microg/ml, determined through monitoring A(620) in this assay for rifampin, isoniazid, streptomycin and ethambutol respectively, were well in agreement with previously reported by BACTEC and Bio-Siv assays. S/N ratio and Z' factor for the assay were 8.5 and 0.81 respectively which indicated the robustness of the protocol. Altogether the assay provides an easy, inexpensive, rapid, robust and high content screening tool to search novel antitubercular molecules against both active and dormant bacilli.

Inhibiting Escherichia coli cytochrome c nitrite reductase: voltammetry reveals an enzyme equipped for action despite the chemical challenges it may face in vivo.

Escherichia coli cytochrome c nitrite reductase is one of a large family of homologous enzymes that are particularly prevalent in pathogenic enterobacteria. The enzymes are periplasmic and in vivo may find themselves challenged by molecules that could enhance or compromise their performance. In the present study, we describe protein film voltammetry in which the activity of E. coli cytochrome c nitrite reductase is challenged by the presence of a number of small molecules. These results are discussed in light of the environment(s) that the enzyme may face before and after colonization of a human host.

Energy coupling to nitrate uptake into the denitrifying cells of Paracoccus denitrificans.

This study deals with the effects of the agents that dissipate the individual components of the proton motive force (short-chain fatty acids, nigericin, and valinomycin) upon the methyl viologen-coupled nitrate reductase activity in intact cells. Substitution of butyrate or acetate for chloride in Tris-buffered assay media resulted in a marked inhibition at pH 7. In a Tris--chloride buffer of neutral pH, the reaction was almost fully inhibitable by nigericin. Alkalinisation increased the IC(50) value for nigericin and decreased the maximal inhibition attained. Both types of inhibitions could be reversed by the permeabilisation of cells or by the addition of nitrite, and that caused by nigericin disappeared at high extracellular concentrations of potassium. These data indicate that nitrate transport step relies heavily on the pH gradient at neutral pH. Since the affinity of cells for nitrate was strongly diminished by imposing an inside-positive potassium (or lithium) diffusion potential at alkaline external pH, a potential dependent step may be of significance in the transporter cycle under these conditions. Experiments with sodium-depleted media provided no hints for Na(+) as a possible H(+) substitute.

Resolving complexity in the interactions of redox enzymes and their inhibitors: contrasting mechanisms for the inhibition of a cytochrome c nitrite reductase revealed by protein film voltammetry.

Cytochrome c nitrite reductase is a dimeric decaheme-containing enzyme that catalyzes the reduction of nitrite to ammonium. The contrasting effects of two inhibitors on the activity of this enzyme have been revealed, and defined, by protein film voltammetry (PFV). Azide inhibition is rapid and reversible. Variation of the catalytic current magnitude describes mixed inhibition in which azide binds to the Michaelis complex (approximately 40 mM) with a lower affinity than to the enzyme alone (approximately 15 mM) and leads to complete inhibition of enzyme activity. The position of the catalytic wave reports tighter binding of azide when the active site is oxidized (approximately 39 microM) than when it is reduced. By contrast, binding and release of cyanide are sluggish. The higher affinity of cyanide for reduced versus oxidized forms of nitrite reductase is immediately revealed, as is the presence of two sites for cyanide binding and inhibition of the enzyme. Formation of the monocyano complex by reduction of the enzyme followed by a "rapid" scan to high potentials captures the activity-potential profile of this enzyme form and shows it to be distinct from that of the uninhibited enzyme. The biscyano complex is inactive. These studies demonstrate the complexity that can be associated with inhibitor binding to redox enzymes and illustrate how PFV readily captures and deconvolves this complexity through its impact on the catalytic properties of the enzyme.

Redox-triggered events in cytochrome c nitrite reductase.

Escherichia coli cytochrome c nitrite reductase is a homodimeric enzyme whose 10 heme centres range in reduction potential from ca. -30 to -320 mV. Protein film voltammetry (PFV) was performed to assess how the reactivity of the enzyme towards a number of small molecules was influenced by heme oxidation state. The experimental approach provided a high-resolution description of activity across the electrochemical potential domain by virtue of the fact that the enzyme sample was under the precise potential control of an electrode at all times. The current potential profiles displayed by nitrite reductase revealed that heme oxidation state has a profound, and often unanticipated, effect on the interactions with substrate molecules, nitrite and hydroxylamine, as well as the inhibitor, cyanide. Thus, PFV provides a powerful route to define redox-triggered events in this complex multi-centred redox enzyme.

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.

Effects of site-directed mutations on heme reduction in Escherichia coli nitrate reductase A by menaquinol: a stopped-flow study.

We have studied the effects of site-directed mutations in Escherichia coli nitrate reductase A (NarGHI) on heme reduction by a menaquinol analogue (menadiol) using the stopped-flow method. For NarGHI(H66Y) and NarGHI(H187Y), both lacking heme b(L) but having heme b(H), the heme reduction by menadiol is abolished. For NarGHI(H56R) and NarGHI(H205Y), both without heme b(H) but with heme b(L), a smaller and slower heme reduction compared to that of the wild-type enzyme is observed. These results indicate that electrons from menadiol oxidation are transferred initially to heme b(L). A transient species, likely to be associated with a semiquinone radical anion, was generated not only on reduction of the wild-type enzyme as observed previously (1) but also on reduction of NarGHI(H56R) and NarGHI(H205Y). The inhibitors 2-n-heptyl-4-hydroxyquinoline-N-oxide and stigmatellin both have significant effects on the reduction kinetics of NarGHI(H56R) and NarGHI(H205Y). We have also investigated the reoxidation of menadiol-reduced heme by nitrate in the mutants. Compared to the wild type, no significant heme reoxidation is observed for NarGHI(H56R) and NarGHI(H205Y). This result indicates that a single mutation removing heme b(H) blocks the electron-transfer pathway from the subunit NarI to the catalytic dimer NarGH.

The role of nitrate reductase in the regulation of the nitrate assimilation pathway in the yeast Hansenula polymorpha.

The role of nitrate reductase (NR) in the regulation of the nitrate assimilation pathway was evaluated in the yeast Hansenula polymorpha. Posttranscriptional regulation of NR in response to reduced nitrogen sources and the effect of a heterologous NR on the transcriptional regulation of nitrate-assimilatory gene expression was examined. The strain bearing YNR1 (nitrate reductase gene) under the control of the methanol-induced MOX (methanol oxidase) promoter showed that NR is active in the presence of reduced nitrogen sources. In cells incubated with glutamine plus nitrate, rapamycin abolished nitrogen catabolite repression, NR activity being very similar to that in cells induced by nitrate alone. This reveals the involvement of the Tor-signalling pathway in the transcriptional regulation of H. polymorpha nitrate assimilation genes. To assess the role of NR in nitrate-assimilatory gene expression, different strains lacking YNR1, or both YNR1 and YNT1 (high-affinity nitrate transporter) genes, or expressing the tobacco NR under the YNR1 promoter, were used. Tobacco NR abolished the constitutive nitrate-assimilatory gene induction shown by an NR gene disruptant strain. Moreover, in strains lacking the high-affinity nitrate transporter and NR this deregulation disappeared. These facts discard the role of NR protein in the transcriptional induction of the nitrate-assimilatory genes and point out the involvement of the high-affinity nitrate transporter as a part of the nitrate-signalling pathway.

Temporal changes in the toxicity of pentachlorophenol to Chlorella pyrenidosa algae.

The toxicity of pentachlorophenol (PCP) on Chlorella pyrenidosa algae was investigated with specific attention given to possible variation of toxic effects with time. A concentration-effect relationship was observed in which there was significant inhibition of PCP on cell density and chlorophyll A content. The inhibition rate of PCP on cell density was dependent on exposure time. The IC50 values after exposure times of 2, 4 and 6 days for cell growth were 4.18 +/- 0.49, 3.49 +/- 0.40 and 3.30 +/- 0.26 mg/L, respectively. There was also inhibition of chlorophyll A production, which appeared to increase marginally with exposure time for a given concentration of PCP. The corresponding IC50 values on day 2, 4 and 6 were 2.30 +/- 0.12, 2.63 +/- 0.38 and 3.30 +/- 0.34 mg/L, respectively. The effect of PCP on nitrate reductase (NR), was first stimulation followed by an inhibition phase. It is postulated that the observed temporal changes in the activity of nitrate reductase (NR) may occur through the addition or loss of phosphorus in the NR protein.

Denitrification of nitrate by the fungus Cylindrocarpon tonkinense.

The denitrifying fungus Cylindrocarpon tonkinense was thought to be able to denitrify only nitrite (NO2-) but not nitrate (NO3-) to form nitrous oxide (N2O). Here we found, however, that C. tonkinense can denitrify NO3- under certain conditions. Presence of ammonium (NH3+) in addition to NO3- and the use of a fermentable sugar as an electron donor were key conditions for inducing the denitrifying activity. Such induction accompanied a remarkable increase in the intracellular level of the enzyme activities related to NO3- metabolism. These activities contained assimilatory type NADPH (or NADH)-dependent NO3- reductase (aNar), dissimilatory nitrite reductase (dNir), and nitric oxide reductase (P450nor), but did not contain ubiquinol-dependent, dissimilatory NO3- reductase (dNar). The denitrification was inhibited by tungstate, an inhibitor of Nar. These results demonstrated occurrence of a novel type of denitrification in C. tonkinense, in which assimilatory type Nar is possibly involved.

Transient kinetic studies of heme reduction in Escherichia coli nitrate reductase A (NarGHI) by menaquinol.

We have studied the transient kinetics of quinol-dependent heme reduction in Escherichia coli nitrate reductase A (NarGHI) by the menaquinol analogue menadiol using the stopped-flow method. Four kinetic phases are observed in the reduction of the hemes. A transient species, likely to be associated with a semiquinone radical anion, is observed with kinetics that correlates with one of the phases. The decay of the transient species and the formation of the second reduction phase of the hemes can be fitted to a double-exponential equation giving similar rate constants, k(1) = 9.24 +/- 0.9 s(-1) and k(2) = 0.22 +/- 0.02 s(-1) for the decay of the transient species, and k(1) = 9.23 +/- 0.9 s(-1) and k(2) = 0.22 +/- 0.02 s(-1) for the formation of the reduction phase. The quinol-binding-site inhibitors 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) and stigmatellin have significant and different inhibitory effects on the reduction kinetics. The kinetics of heme reduction in NarI expressed in the absence of the NarGH catalytic dimer (NarI(DeltaGH) exhibits only two kinetic phases, and the decay of the transient species also correlates kinetically with the second reduction phase of the hemes. We have also studied nitrate-dependent heme reoxidation following quinol-dependent heme reduction using a sequential stopped-flow method. HOQNO elicits a much stronger inhibitory effect than stigmatellin on the reoxidation of the hemes. On the basis of our results, we propose schemes for the mechanism of NarGHI reduction by menaquinol and reoxidation by nitrate.

Cloning of a nitrate reductase inactivator (NRI) cDNA from Spinacia oleracea L. and expression of mRNA and protein of NRI in cultured spinach cells.

The spinach ( Spinacia oleracea L. (cv. Hoyo) nitrate reductase inactivator (NRI) is a novel protein that irreversibly inactivates NR. Using degenerate primers based on an N-terminal amino acid sequence of NRI purified from spinach leaves and a cDNA library, we isolated a full-length NRI cDNA from spinach that contains an open reading frame encoding 479 amino acid residues. This protein shares 67.4% and 51.1-68.3% amino acid sequence similarities with a nucleotide pyrophosphatase (EC 3.6.1.9) from rice and three types of the nucleotide pyrophosphatase-like protein from Arabidopsis thaliana, respectively. Immunoblot analysis revealed that NRI was constitutively expressed in suspension-cultured spinach cells; however, its expression level is quite low in 1-day-subcultured cells. Moreover, northern blot analysis indicated that this expression was regulated at the mRNA level. These results suggest that NRI functions in mature cells.

Regulation of the alternative oxidase Aox1 gene in Chlamydomonas reinhardtii. Role of the nitrogen source on the expression of a reporter gene under the control of the Aox1 promoter.

In higher plants, various developmental and environmental conditions enhance expression of the alternative oxidase (AOX), whereas its induction in fungi is mainly dependent on cytochrome pathway restriction and triggering by reactive oxygen species. The AOX of the unicellular green alga Chlamydomonas reinhardtii is encoded by two different genes, the Aox1 gene being much more transcribed than Aox2. To analyze the transcriptional regulation of Aox1, we have fused its 1.4-kb promoter region to the promoterless arylsulfatase (Ars) reporter gene and measured ARS enzyme activities in transformants carrying the chimeric construct. We show that the Aox1 promoter is generally unresponsive to a number of known AOX inducers, including stress agents, respiratory inhibitors, and metabolites, possibly because the AOX activity is constitutively high in the alga. In contrast, the Aox1 expression is strongly dependent on the nitrogen source, being down-regulated by ammonium and stimulated by nitrate. Inactivation of nitrate reductase leads to a further increase of expression. The stimulation by nitrate also occurs at the AOX protein and respiratory levels. A deletion analysis of the Aox1 promoter region demonstrates that a short upstream segment (-253 to +59 with respect to the transcription start site) is sufficient to ensure gene expression and regulation, but that distal elements are required for full gene expression. The observed pattern of AOX regulation points to the possible interaction between chloroplast and mitochondria in relation to a potential increase of photogenerated ATP when nitrate is used as a nitrogen source.

Purification of a plant nucleotide pyrophosphatase as a protein that interferes with nitrate reductase and glutamine synthetase assays.

An activity that inhibited both glutamine synthetase (GS) and nitrate reductase (NR) was highly purified from cauliflower (Brassica oleracea var. botrytis) extracts. The final preparation contained an acyl-CoA oxidase and a second protein of the plant nucleotide pyrophosphatase family. This preparation hydrolysed NADH, ATP and FAD to generate AMP and was inhibited by fluoride, Cu2+, Zn2+ and Ni2+. The purified fraction had no effect on the activity of NR when reduced methylviologen was used as electron donor instead of NADH; and inhibited the oxidation of NADH by both spinach NR and an Escherichia coli extract in a time-dependent manner. The apparent inhibition of GS and NR and the ability of ATP and AMP to relieve the inhibition of NR can therefore be explained by hydrolysis of nucleotide substrates by the nucleotide pyrophosphatase. We have no evidence that the nucleotide pyrophosphatase is a specific physiological regulator of NR and GS, but suggest that nucleotide pyrophosphatase activity may underlie some confusion in the literature about the effects of nucleotides and protein factors on NR and GS in vitro.