Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.7.1.1 (nitrate reductase)
3,728 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Partially purified soluble rat liver guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] was activated by superoxide dismutase (superoxide: superoxide oxidoreductase, EC 1.15.1.1). This activation was prevented with KCN or glutathione, inhibitors of superoxide dismutase. Guanylate cyclase preparations formed superoxide ion. Activation by superoxide dismutase was further enhanced by the addition of nitrate reductase. Although guanylate cyclase activity was much greater with Mn2+ than with Mg2+ as sole cation cofactor, activation with superoxide dismutase was not observed when Mn2+ was included in incubations. Catalase also decreased the activation induced with superoxide dismutase. Thus, activation required the formation of both superoxide ion and H2O2 in incubations. Activation of guanylate cyclase could not be achieved by the addition of H2O2 alone. Scavengers of hydroxyl radicals prevented the activation. It is proposed that superoxide ion and hydrogen peroxide can lead to the formation of hydroxyl radicals that activate guanylate cyclase. This mechanism of activation can explain numerous observations of altered guanylate cyclase activity and cyclic GMP accumulation in tissues with oxidizing and reducing agents. This mechanism will also permit physiological regulation of guanylate cyclase and cyclic GMP formation when there is altered redox or free radical formation in tissues in response to hormones, other agents, and processes.
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PMID:Activation of guanylate cyclase by superoxide dismutase and hydroxyl radical: a physiological regulator of guanosine 3',5'-monophosphate formation. 2 77

Spinach (Spinacia oleracea L.) leaf nitrate reductase (NADH:NR;NADH:nitrate oxidoreductase, EC 1.6.6.1) activity was found to rapidly change during light/dark transitions. The most rapid and dramatic changes were found in a form of NR which was sensitive to inhibition by millimolar concentrations of magnesium. This form of NR predominated in leaves in the dark, but was almost completely absent from leaves incubated in the light for only 30 min. When the leaves were returned to darkness, the NR rapidly became sensitive to Mg2+ inhibition. Modulation of the overall reaction involving NADH as electron donor was also found when reduced methyl viologen was the donor (MV:NR), indicating that electron transfer had been blocked, at least in part, at or near the terminal molybdenum cofactor site. Changes in activity appear to be the result of a covalent modification that affects sensitivity of NR to inhibition by magnesium, and our results suggest that protein phosphorylation may be involved. NR was phosphorylated in vivo after feeding excised leaves [32P]Pi. The NR subunit was labeled exclusively on seryl residues in both light and dark. Tryptic peptide mapping indicated three major 32P-labeled phosphopeptide (Pp) fragments. Labeling of two of the P-peptides (designated Pp1 and 3) was generally correlated with NR activity assayed in the presence of Mg2+. In vivo, partial dephosphorylation of these sites (and activation of NR assayed with Mg2+) occurred in response to light or feeding mannose in darkness. The light effect was blocked completely by feeding okadaic acid via the transpiration stream, indicating the involvement of type 1 and/or type 2A protein phosphatases in vivo. While more detailed analysis is required to establish a causal link between the phosphorylation status of NR and sensitivity to Mg2+ inhibition, the current results are highly suggestive of one. Thus, in addition to the molecular genetic mechanisms regulating this key enzyme of nitrate assimilation, NR activity may be controlled in leaves by phosphorylation/dephosphorylation of the enzyme protein resulting from metabolic changes taking place during light/dark transitions.
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PMID:Reversible light/dark modulation of spinach leaf nitrate reductase activity involves protein phosphorylation. 160 45

The toxicity of chromium and tin on growth, uptake of NO3- and NH4+, nitrate reductase and glutamine synthetase activity of Anabaena doliolum, and its interaction with bivalent cations, viz. Ca2+, Mg2+, Mn2+, Ni2+, Co2+, and Zn2+, has been studied. Some interacting cations, viz. Ca, Mg, and Mn, substantially antagonized the toxic effects of chromium and tin with reference to growth and nutrient (NO3- and NH4+) uptake in the hierarchical sequence Ca greater than Mg greater than Mn, whereas the sequence of hierarchy was Mn greater than Mg greater than Ca for nitrate reductase and glutamine synthetase activity of A. doliolum. A synergistically inhibitory pattern of interaction was noted for all the parameters, viz. growth, uptake of NO3- and NH4+, nitrate reductase and glutamine synthetase activity of A. doliolum, when Ni, Co, and Zn were used in combination with test metals in the growth medium. These bivalent cations followed the synergistic inhibition sequence Ni greater than Co greater than Zn and potentiated the toxicity of test metals in the N2-fixing cyanobacterium under study.
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PMID:Impact of chromium and tin on a nitrogen-fixing cyanobacterium Anabaena doliolum: interaction with bivalent cations. 256 5

The soluble subcellular fraction of a chlB mutant contains an inactive precursor form of the molybdoenzyme nitrate reductase, which can be activated by the addition to the soluble fraction of protein FA, which is thought to be the active product of the chlB locus. Dialysis or desalting of the chlB soluble fraction leads to the loss of nitrate reductase activation, indicating that some low-molecular-weight material is required for the activation. The protein FA-dependent activation of nitrate reductase can be restored to the desalted chlB soluble fraction by the addition of a clarified extract obtained after heating the chlB soluble fraction at 100 degrees C for 8 min. The heat-stable substance present in this preparation has a molecular weight of approximately 1,000. This substance is distinct from the active molybdenum cofactor since its activity is unimpaired in heat-treated extracts prepared from the organism grown in the presence of tungstate, which leads to loss of cofactor activity. Mutations at the chlA or chlE locus, which are required for molybdenum cofactor biosynthesis, similarly do not affect the activity of the heat-treated extract in the in vitro activation process. Moreover, the active material can be separated from the molybdenum cofactor activity by gel filtration. None of the other known pleiotropic chlorate resistance loci (chlD, chlG) are required for the expression of its activity. Magnesium ATP appears to have a role in the formation of the active substance. We conclude that a low-molecular-weight substance, distinct from the active molybdenum cofactor, is required to bestow activity on the molybdoenzyme nitrate reductase during its biosynthesis.
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PMID:Involvement of a low-molecular-weight substance in in vitro activation of the molybdoenzyme respiratory nitrate reductase from a chlB mutant of Escherichia coli. 330 48

1. The b-type haem centres of the three (alpha, beta and gamma) subunit nitrate reductase from Paracoccus denitrificans have been analysed by redox potentiometry. Two components were identified with mid-point potentials +95 mV and +210 mV. 2. Washing, in the absence of Mg2+ ions, of cytoplasmic membrane vesicles from P. denitrificans promoted selective release of nitrate reductase activity. The released enzyme was purified by chromatography and shown to contain alpha and beta, but not gamma polypeptides. A haem spectrum was absent, consistent with the lack of the gamma subunit. The alpha and beta polypeptides of the water-soluble nitrate reductase had molecular masses that were identical to those of the detergent-purified enzyme and also of the nitrate reductase in cytoplasmic membranes. This observation, together with the failure of protease inhibitors to prevent release from the membrane, indicates that the release is not related to limited proteolysis of the alpha and/or beta polypeptides. The relative molecular mass of the water-soluble alpha beta enzyme was estimated to be approximately 200,000. 3. The water-soluble nitrate reductase was released from intact inverted cytoplasmic membrane vesicles as judged by loss of NADH-NO3- reductase activity and retention by the vesicles after washing of uncoupler-sensitive NADH-oxidase activity. These observations show that alpha and beta polypeptides, and therefore the active site for nitrate reduction, are located on the cytoplasmic side of the membrane. 4. Attempts to reverse the nitrate reductase activity of the enzyme, using nitrate as reductant plus ferricyanide or chlorate as tested oxidants, were unsuccessful. The implications for the mechanism of the enzyme are discussed.
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PMID:Respiratory nitrate reductase from Paracoccus denitrificans. Evidence for two b-type haems in the gamma subunit and properties of a water-soluble active enzyme containing alpha and beta subunits. 337 62

The NADH-dependent nitrate reductase (NR, EC 1.6.6.1) in roots of hydroponically grown barley seedlings was extracted, desalted and the activity measured in buffer containing either Mg2+ (10 mM) or EDTA (5 mM). The former gives the actual NR activity (NRact) equivalent to dephospho-NR, whereas the latter gives the maximum NR capacity of the dephospho-form (NRmax). Both values together permit an estimation of the NR-phosphorylation state. Changes in NRact and NRmax were followed in response to root aeration or to shoot illumination or shoot removal, and were correlated with sugar contents and adenylate levels. Ethanol formation was also measured in roots differing in NR activity in order to obtain information on the relation between anaerobic alcoholic fermentation and nitrate reduction. In aerated roots, NR was highly phosphorylated (about 80%) and largely inactive. It was partly dephosphorylated (activated) by anoxia or by cellular acidification (pH 4.8 plus propionic acid). Anaerobic activation (dephosphorylation) of NR was stronger at acidic external pH (5) than at slightly alkaline pH (8), although ATP levels decreased and AMP levels increased at pH 5 and at pH 8 to the same extent. Thus, rapid changes in the NR-phosphorylation state in response to anaerobiosis were not directly triggered by the adenylate pool, but rather by cytosolic pH. Under prolonged darkness (24 h) or after shoot removal. NRmax decreased slowly without a large change in the phosphorylation state. This decrease of NRmax was correlated with a large decrease in the sugar content, and was prevented by glucose feeding, which had only minor effects on the phosphorylation state. Cycloheximide also prevented the decrease in NRmax without affecting the phosphorylation state. In contrast, anaerobiosis or cellular acidification prevented the decrease of NRmax and at the same time decreased the NR-phosphorylation state. It is suggested that NR turnover in roots is controlled by several factors: NR synthesis appears to depend on sugar availability, which has little effect on the phosphorylation state; in addition, NR degradation appears to be strongly affected by the phosphorylation state in such a way that the inactive phospho-NR is a better substrate for NR degradation than the dephospho-form. The rate of anaerobic ethanol formation was not affected by NR activity, indicating that the purpose of NR activation under hypoxia or anoxia is not to decrease or prevent alcoholic fermentation.
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PMID:Nitrate reductase activation state in barley roots in relation to the energy and carbohydrate status. 915 51

In this report we address two questions regarding the regulation of phosphorylated nitrate reductase (pNR; EC 1.6.6.1) by 14-3-3 proteins. The first concerns the requirement for millimolar concentrations of a divalent cation in order to form the inactive pNR:14-3-3 complex at pH 7.5. The second concerns the reduced requirement for divalent cations at pH 6.5. In answering these questions we highlight a possible general mechanism involved in the regulation of 14-3-3 binding to target proteins. We show that divalent cations (e.g. Ca2+, Mg2+ and Mn2+) bind directly to 14-3-3s, and as a result cause a conformational change, manifested as an increase in surface hydrophobicity. A similar change is also obtained by decreasing the pH from pH 7.5 to pH 6.5, in the absence of divalent cations, and we propose that protonation of amino acid residues brings about a similar effect to metal ion binding. A possible regulatory mechanism, where the 14-3-3 protein has to be "primed" prior to binding a target protein, is discussed.
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PMID:Biological significance of divalent metal ion binding to 14-3-3 proteins in relationship to nitrate reductase inactivation. 987 66

The proteins commonly referred to as 14-3-3s have recently come to prominence in the study of protein:protein interactions, having been shown to act as allosteric or steric regulators and possibly scaffolds. The binding of 14-3-3 proteins to the regulatory phosphorylation site of nitrate reductase (NR) was studied in real-time by surface plasmon resonance, using primarily an immobilized synthetic phosphopeptide based on spinach NR-Ser543. Both plant and yeast 14-3-3 proteins were shown to bind the immobilized peptide ligand in a Mg2+-stimulated manner. Stimulation resulted from a reduction in KD and an increase in steady-state binding level (Req). As shown previously for plant 14-3-3s, fluorescent probes also indicated that yeast BMH2 interacted directly with cations, which bind and affect surface hydrophobicity. Binding of 14-3-3s to the phosphopeptide ligand occurred in the absence of divalent cations when the pH was reduced below neutral, and the basis for enhanced binding was a reduction in K(D). At pH 7.5 (+Mg2+), AMP inhibited binding of plant 14-3-3s to the NR based peptide ligand. The binding of AMP to 14-3-3s was directly demonstrated by equilibrium dialysis (plant), and from the observation that recombinant plant 14-3-3s have a low, but detectable, AMP phosphatase activity.
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PMID:Modulation of 14-3-3 protein interactions with target polypeptides by physical and metabolic effectors. 1084 67

To assess the role of 14-3-3 proteins in the magnesium-dependent inhibition of nitrate reductase (NR) we tested the effect of magnesium on NR binding to 14-3-3s by coimmunoprecipitation and gel filtration. The stability of the 14-3-3 complex of NR was, unlike its activity, unaffected by magnesium. We therefore conclude that binding to 14-3-3s per se does not inhibit NR. Magnesium inhibited 14-3-3-bound NR much more strongly than 14-3-3-free NR. 14-3-3s possibly reinforce NR inhibition by magnesium.
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PMID:Binding to 14-3-3 proteins is not sufficient to inhibit nitrate reductase in spinach leaves. 1103 32

Binding of 14-3-3 proteins to nitrate reductase phosphorylated on Ser543 (phospho-NR) inhibits activity and is responsible for the inactivation of nitrate reduction that occurs in darkened leaves. The 14-3-3-dependent inactivation of phospho-NR is known to require millimolar concentrations of a divalent cation such as Mg2+ at pH 7.5. We now report that micromolar concentrations of the polyamines, spermidine(4+) and spermine(3+), can substitute for divalent cations in modulating 14-3-3 action. Effectiveness of the polyamines decreased with a decrease of polycation charge: spermine(4+) > spermidine(3+) >>> cadavarine(2+) approximately putrescine(2+) approximately agmatine(2+) approximately N1-acetylspermidine(2+), indicating that two primary and at least one secondary amine group were required. C-terminal truncations of GF14 omega, which encodes the Arabidopsis 14-3-3 isoform omega, indicated that loop 8 (residues 208-219) is the likely cation-binding site. Directed mutagenesis of loop 8, which contains the EF hand-like region identified in earlier studies, was performed to test the role of specific amino acid residues in cation binding. The E208A mutant resulted in a largely divalent cation-independent inhibition of phospho-NR activity, whereas the D219A mutant was fully Mg(2+)-dependent but had decreased affinity for the cation. Mutations and C-terminal truncations that affected the Mg(2+) dependence of phospho-NR inactivation had similar effects on polyamine dependence. The results implicate loop 8 as the site of divalent cation and polyamine binding, and suggest that activation of 14-3-3s occurs, at least in part, by neutralization of negative charges associated with acidic residues in the loop. We propose that binding of polyamines to 14-3-3s could be involved in their regulation of plant growth and development.
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PMID:Divalent cations and polyamines bind to loop 8 of 14-3-3 proteins, modulating their interaction with phosphorylated nitrate reductase. 1185 16


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