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Query: EC:1.7.1.2 (nitrate reductase)
 3,861 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Stoicheometries and rates of proton translocation associated with respiratory reduction of NO3- have been measured for spheroplasts of Escherichia coli grown anaerobically in the presence of NO3-. Observed stoicheiometries [leads to H+/NO3- ratio; P. Mitchell (1966) Chemiosmotic Coupling in Oxidative and Photosynthetic Phosphorylation, Glynn Research, Bodmin] were approx. 4 for L-malate oxidation and approx. 2 for succinate, D-lactate and glycerol oxidation. Measurements of the leads to H+/2e- ratio with formate as the reductant and oxygen or NO3- as the oxidant were complicated by pH changes associated with formate uptake and CO2 formation. Nevertheless, it was possible to conclude that the site of formate oxidation is on the inner aspect of the cytoplasmic membrane, that the leads to H+/O ratio for formate oxidation is approx. 4, and that the leads to H+/NO3- ratio is greater than 2. Measurements of the rate of NO3- penetration into osmotically sensitive spheroplasts demonstrated an electrogenic entry of NO3- anion. The permeability coefficient for nitrate entry at 30 degrees C was between 10(-9) and 10(-10) cm- s(-1). The calculated rate of nitrate entry at the concentration typically used for the assay of nitrate reductase (EC 1.7.99.4) activity was about 0.1% of that required to support the observed rate of nitrate reduction by reduced Benzyl Viologen. Measurements of the distribution of nitrate between the intracellular and extracellular spaces of a haem-less mutant, de-repressed for nitrate reductase but unable to reduce nitrate by the respiratory chain, showed that, irrespective of the presence or the absence of added glucose, nitrate was not concentrated intracellularly. Osmotic-swelling experiments showed that the rate of diffusion of azid anion across the cytoplasmic membrane is relatively low in comparison with the fast diffusion of hydrazoic acid. The inhibitory effect of azide on nitrate reductase was not altered by treatments that modify pH gradients across the cytoplasmic membrane. It is concluded that the nitrate-reducing azide-sensitive site of nitrate reductase is located on the outer aspect of the cytoplasmic membrane. The consequences of this location for mechanisms of proton translocation driven by nitrate reduction are discussed, and lead to the proposal that the nitrate reductase of the cytoplasmic membrane is vectorial, reducing nitrate on the outer aspect of the membrane with 2H+ and 2e- that have crossed from the inner aspect of the membrane.
Biochem J 1975 Dec
PMID:Proton translocation and the respiratory nitrate reductase of Escherichia coli. 0 96

Xanthine oxidase is stable and active in aqueous dimethyl sulphoxide solutions of up to at least 57% (w/w). Simple techniques are described for mixing the enzyme in this solvent at--82 degrees C, with its substrate, xanthine. When working at high pH values under such conditions, no reaction occurred, as judged by the absence of e.p.r. signals. On warming to--60 degrees C, for 10 min, however, the Very Rapid molybdenum(V) e.p.r. signal was obtained. This signal did not change on decreasing the pH, while maintaining the sample in liquid nitrate reductase, caused its molybdenum(V) e.p.r. signal to change from the high-pH to the low-pH form. These findings are not compatible with the conclusions of Edmondson, Ballou, Van Heuvelen, Palmer & Massey [J. Biol. Chem. (1973) 248, 6135-6144], that the Very Rapid signal is in prototropic equilibrium with the Rapid signal, and should be important in understanding the mechanism of action of the enzyme. They emphasize the unique nature of the intermediate represented by the Very Rapid e.p.r. signal. The possible value of the pK for loss of an exchangeable proton from the Rapid signal is discussed.
Biochem J 1978 Dec 01
PMID:pH-jump studies at subzero temperatures on an intermediate in the reaction of xanthine oxidase with xanthine. 3 66

A molybdenum cofactor (Mo-co) from xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2) can be isolated from the enzyme by a technique that has been used to isolate an iron-molybdenum cofactor (FeMo-co) from component I of nitrogenase. N-Methylformamide is used for the extraction of these molybdenum cofactors. Mo-co from xanthine oxidase activates nitrate reductase (NADPH:nitrate oxidoreductase, EC 1.6.6.2) in an extract from Neurospora crassa mutant strain Nit-1; however, FeMo-co is unable to activate nitrate reductase in strain Nit-1. Mo-co from xanthine oxidase is unable to activate nitrogenase in an extract of Azotobacter vinelandii mutant strain UW45. Inactive component I in this extract can be activated by FeMo-co. These results indicate that nitrate reductase and xanthine oxidase share a common molybdenum cofactor, but this cofactor is different from the molybdenum cofactor in nitrogenase.A. vinelandii synthesizes both Mo-co and FeMo-co. Mo-co is produced when the cells fix N(2) and also when they are repressed for nitrogenase synthesis by growth in a medium containing excess ammonium. However, FeMo-co is not produced when cells are grown in an ammonium-containing medium. Partially purified preparations of component I from A. vinelandii and Klebsiella pneumoniae contain both FeMo-co and Mo-co. The presence of both FeMo-co and Mo-co activities in partially purified preparations of component I explains previous reports of activation of inactive nitrate reductase in strain Nit-1 by acid-treated component I of nitrogenase. The Mo-co can be separated from FeMo-co in these preparations by chromatography on Sephadex G-100 in N-methylformamide. Both FeMo-co and Mo-co are sensitive to oxygen.
Proc Natl Acad Sci U S A 1977 Dec
PMID:Molybdenum cofactors from molybdoenzymes and in vitro reconstitution of nitrogenase and nitrate reductase. 14 98

Nitrate reductase (NaR) linked to reduced methyl viologen from Clostridium perfringens was purified by ammonium sulfate precipitation. DEAE-cellulose chromatography, disc electrophoresis on polyacrylamide gel, and triple DEAE-Sephadex chromatography. The specific activity was increased 1,200-fold with a yield of 9%. The purified preparation was nearly homogeneous in disc electrophoresis. It was brown, and its spectrum showed a slight shoulder near 420 nm as well as a peak at 280 nm. The molecular weight was found to be 90,000 based on s020,w (5.8S) and 80,000 by Sephadex G-100 gel filtration. In SDS-polyacrylamide electrophoresis, it showed only a single band with a molecular weight of 90,000; it had no subunit structure. The isoelectric point was pH 5.5, and the optimum pH was 9. Mn2+, Fe2+, Mg2+, and Ca2+ stimulated the activity. Km for nitrate was 0.10 mM, and nitrate was stoichiometrically reduced to nitrite in the presence of 2 mM Mn2+. Ferredoxin fraction obtained from extracts of the bacterium was utilizable as an electron donor at pH 8. Cyanide and azide inhibited the enzyme. The formation of NaR was induced by nitrate and inhibited by 0.5 mM tungstate, but recovered in the presence of 0.1 mM molybdate; NaR of C. perfringens appears to be a molybdo-iron-sulfur protein.
J Biochem 1977 Dec
PMID:Studies on nitrate reductase of Clostridium perfringens. Purification, some properties, and effect of tungstate on its formation. 20 90

The maize root has two main proteinase and carboxypeptidase components. Proteinase I and carboxypeptidase I, which predominate in older plants, appear to have a serine group at their active sites and have been estimated to have molecular weights of approximately 54000 and 77000 respectively. Proteinase I, which has been purified up to 500-fold, degrades haemoglobin and azocasein with maximum activity at pH 4 and 9--10 respectively, while on maize root protein it gives most hydrolysis in the neutral pH range. The main portion of the nitrate-reductase-inactivating activity in the maize root extract is due to proteinase I. Carboxypeptidase I, like several other plant carboxypeptidases such as carboxypeptidase C which have now (IUB Recommendations 1978) been classified as serine carboxypeptidases (EC 3.4.16.1), has maximum activity around pH 5 and has esterase activity. A second group of proteases, proteinase II and carboxypeptidase II, separated from the above on carboxymethyl-cellulose, were shown to have different molecular weight properties and be equally sensitive to serine and thiol group inhibitors. Proteinase II degrades haemoglobin, but not azocasein and does not mediate nitrate reductase inactivation. Associated with this second group of proteases was a macromolecular component which inactivated nitrate reductase but, unlike the action of proteinase I, was not inhibited by phenylmethylsulphonyl fluoride or casein. It was inhibited by metal chelating agents which were without effect on nitrate reductase inactivation due to proteinase I.
Eur J Biochem 1979 Dec 17
PMID:Isolation and characterisation of peptide hydrolases from the maize root. 39 8

In E. coli K12 (F'nif+Kp) hybrids, electron-transport-dependent phosphorylation is not necessary for anaerobic nitrogen fixation, and substrate level phosphorylation can provide sufficient ATP from glucose for nitrogenase activity. The fumarate-reduction system, however, is essential in these hybrids for the transfer of electrons to nitrogenase. This system is probably also involved in maintaining the membrane in the energized state, thereby allowing nitrogen fixation to occur. The nitrate-reduction system, which can energize the membrane like the fumarate-reduction system, is not necessary for nitrogenase activity in the E. coli K12(F'nif+Kp) hybrids. However, two nitrate reductase genes, chlA, and chlB, are essential for inhibition of nitrogen fixation by nitrate. Moreover, nitrate inhibits nitrogenase activity and this inhibition is most probably effected through a regulator factor coded by chlA and chlB.
Aust J Biol Sci 1979 Dec
PMID:Pathways of energy metabolism required for phenotypic expression of nif+Kp genes in Escherichia coli. 39 94

Spinach nitrate reductase complex previously inactivated by treatment with mercurials p-hydroxymercuribenzoate or p-hydroxymercuriphenyl sulphonate can be reactivated by incubation with dithioerythritol. The reactivation of NADH-diaphorase seems to be FAD-dependent, whereas that of FNH2-nitrate reductase is not. The requirement of FAD for NADH-inactivation of nitrate reductase treated with p-hydroxymercuribenzoate disappears after treatment with dithioerythritol.
Rev Esp Fisiol 1977 Dec
PMID:Nitrate reductase from Spinacea oleracea. FAD and the reactivation of the enzyme treated with p-Hydroxymercuribenzoate. 59 86

Mutants of A. nidulans at several loci lack detectable NADPH-nitrate reductase activity. These loci include niaD, the structural gene for the nitrate reductase polypeptide, and five other loci termed cnxABC, E, F, G and H which are presumed to be involved in the formation of a molybdenum-containing component (MCC) necessary for nitrate reductase activity. When forzen mycelia from A. nidulans deletion mutant niaD26 were homogenized in a Ten Broeck homogenizer together with frozen mycelia from either cnxA6, cnxE29, cnsF12, cnxG4 or cnxH3 strains grown on urea + nitrate as the nitrogen source, nitrate reductase activity was detectable in the extract. Similar results were obtained by co-homogenizind niaD mycelia with Neurospora crassa nit-1 mycelia induced on nitrate. Thus, all A. nidulans cnx mutants are similar to the N. crassa nit-1 strain in their capacity to yield NADPH-nitrate reductase in the presence of the presumed MCC. As judged by the amounts of nitrate reductase formed, niaD26 mycelia grown on urea +/- nitrate contained much more available MCC than ammonium-grown mycelia. No NADPH-nitrate reductase activity was found in extracts prepared by co-homogenizing mycelia from all five A. nidulans cnx strains. Wild-type A. nidulans NADPH-nitrate reductase acid dissociated by adjustment to pH 2.0-2.5 AND RE-ADJUSTED TO PH 7 could itself re-assemble to form active nitrate reductase and thus was not a useful source of MCC for these experiments. These results are consistent with the conclusion that the active nitrate reductase complex is composed of polypeptide components which are the niaD gene product, plus the MCC which is formed through the combined action of the cnx gene products. Further, the production of MCC may be regulated in response to the nitrogen nutrition available to the organism.
Mol Gen Genet 1976 Dec 08
PMID:Formation of NADPH-nitrate reductase activity in vitro from Aspergillus nidulans niaD and cnx mutants. 79 78

Nitrate reductase A has been solubilized from purified cytoplasmic membranes by extraction with tert-amyl alcohol. The resulting aqueous solution contained monomeric reductase which polymerized slowly to dimers and tetramers with sedimentation coefficients of respectively 10.5, 16 and 23 Svedbergunits. The polymerization could be stopped to some extent by addition of a small amount of Triton X-100. These distinct entities of nitrate reductase A were separable on electro-focusing, DEAE-column chromatography and polyacrylamide gel electrophoresis, and have been proved to consist of similar subunits with molecular weights of 104000, 63000, and 56000 daltons. The molecular weights of monomeric nitrate reductase A was found to be about 240000 daltons. Chlorate reductase C has been solubilized by a similar procedure, resulting in only monomeric enzyme. Chlorate reductase C exhibited a sedimentation coefficient of 7.7 Svedbergunits, an isoelectric point of pH = 4.55 and a molecular weight of approx. 180000 daltons. It was found to consist of three subunits with molecular weights of 75000, 63000 and 56000 daltons. The latter two subunits are most probably common in nitrate reductase A and chlorate reductase C.
Arch Microbiol 1976 Dec 01
PMID:Characterization of purified nitrate reductase A and chlorate reductase C from Proteus mirabilis. 79 37

Three genotypically different chlorate resistant mutants, chl I, chl II and chl III, appeared to lack completely nitrate reductase A, chlorate reductase C and tetrathionate reductase activity. Fumarate reductase is only partially affected in chl I and chl III and unaffected in chl II. Formate dehydrogenase is only partially diminished in chl II, hydrogenase is diminished in chl I and chl II and completely absent in chl III. Subunits of nitrate reductase A, chlorate reductase C and tetrathionate reductase have been identified in protein profiles of purified cytoplasmic membranes from the wild type and the three mutant strains, grown under various conditions. Only the presence and absence of the largest subunits of these enzymes appeared to be correlated with their repression and derepression in the wild type membranes. On the cytoplasmic membranes of the chl I and chl III mutants these subunits lack for the greater part. In the chl II mutant, however, these subunits are inserted in the membrane all together after anaerobic growth with or without nitrate. A model for the repression/derepression mechanism for the reductases has been proposed. It includes repression by cytochrome b components, whereas the redox-state of the nitrate reductase A molecule itself is also involved in its derepression under anaerobic conditions.
Arch Microbiol 1976 Dec 01
PMID:The correlation between the protein composition of cytoplasmic membranes and the formation of nitrate reductase A, chlorate reductase C and tetrathionate reductase in Proteus mirabilis wild type and some cholate resistant mutants. 79 38


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