Characterization Of Nitrate Reductase Research Articles

Purification and Characterization of Nitrate Reductase (NAR) from Pseudomonas sp. SH7 Isolate

Sixty five soil samples and fifteen water samples were collected from different places in which previously explosions were occurred in Iraq. Seven isolates showed ability to utilize 0.1mM trinitrotoluene (TNT) and/or 0.2mM glycerol trinitrate (GTN) as a sole carbon and nitrogen source and one of these isolates showed the highest nitrate reduction which was classified and coded as Pseudomonas sp. SH7. The highest nitrate reductase activity extracted by sonication while optimum conditions for enzyme production in minimal media pH 7 containing 0.25mM GTN at 35oC for 3 days under aerobic condition. Nitrate reductase was purified by 40-60% ammonium sulphate, ion exchange and gel filtration. Nitrate reductase molecular weight determined by SDS-PAGE was 115 kD. The characterization of purified enzyme activity and stability was higher at a pH between 6.5-7.5 and. Maximum activity was at 35oC and stable at 30-40oC for 15 min., while for heat sensitivity 100% activity observed at 45oC for 20 min. Treatment with 200 µM azide and 500 µM cyanide inhibited the activity by 76 and 91% respectively.

Isolation and characterization of nitrate reductase from the halophilic sulfur-oxidizing bacterium Thioalkalivibrio nitratireducens

A novel nitrate reductase (NR) was isolated from cell extract of the haloalkaliphilic bacterium Thioalkalivibrio nitratireducens strain ALEN 2 and characterized. This enzyme is a classical nitrate reductase containing molybdopterin cofactor in the active site and at least one iron-sulfur cluster per subunit. Mass spectrometric analysis showed high homology of NR with the catalytic subunit NarG of the membrane nitrate reductase from the moderately halophilic bacterium Halomonas halodenitrificans. In solution, NR exists as a monomer with a molecular weight of 130-140 kDa and as a homotetramer of about 600 kDa. The specific nitrate reductase activity of NR is 12 micromol/min per mg protein, the maximal values being observed within the neutral range of pH. Like other membrane nitrate reductases, NR reduces chlorate and is inhibited by azide and cyanide. It exhibits a higher thermal stability than most mesophilic enzymes.

Purification and Characterization of Nitrate Reductase from the Halotolerant Cyanobacterium Aphanothece halophytica

Factors affecting the activity of nitrate reductase (E.C.1.7.7.2) from the halotolerant cyanobacterium Aphanothece halophytica were investigated. Cells grown in nitrate-containing medium exhibited higher nitrate reductase activity than cells grown in medium in which nitrate was replaced by glutamine. When ammonium was present in the medium instead of nitrate, the activity of nitrate reductase was virtually non-detectable, albeit with normal cell growth. The enzyme was localized mainly in the cytoplasm. The enzyme was purified 406-fold with a specific activity of 40.6 µmol/min/mg protein. SDS-PAGE revealed a subunit molecular mass of 58 kDa. Gel filtration experiments revealed a native molecular mass of 61 kDa. The Km value for nitrate was 0.46 mM. Both methyl viologen and ferredoxin could serve as electron donor with Km values of 4.3 mM and 5.2 µM, respectively. The enzyme was strongly inhibited by sulfhydryl-reactive agents and cyanide. Nitrite, the product of the enzyme reaction, showed little inhibition. Chlorate, the substrate analog, could moderately inhibit the enzyme activity. NaCl up to 200 mM stimulated the activity of the enzyme whereas enzyme inhibition was observed at ≥300 mM NaCl.

Isolation, Purification, and Characterization of Nitrate Reductase from a Salt-Tolerant Rhodotorula glutinis Yeast Strain Grown in the Presence of Tungsten

The salt-tolerant Rhodotorula glutinis yeast strain grew in medium containing nitrate, 1 mM tungsten, and trace amounts of molybdenum (as impurities from the reagents used). Isolation of electrophoretically homogenous preparation of nitrate reductase from the Rh. glutinis cells grown under these growth conditions is described. The isolated nitrate reductase is a molybdenum-containing homodimer with molecular mass of 130 kD, containing 0.177 mol of Mo per mol of the enzyme. The activity of the enzyme is maximal at pH 7.0 and 35-45 degrees C and is inhibited by low concentrations of azide and cyanide. The enzyme is almost insensitive to 1 mM tungsten.

Molecular cloning and characterization of nitrate reductase from Ricinus communis L. heterologously expressed in Pichia pastoris.

In a previous paper, we showed that nitrate reductase (NR; EC 1.6.6.1) from leaves of Ricinus communis L. differed from most other higher-plant NRs by an unusually strong Mg2+-sensitivity, a different pH-activity profile and only little ATP-dependent inactivation [A. Kandlbinder et al. (2000) J Exp Bot 51:1099-1105]. In order to elucidate these deviating properties in more detail, the NR gene from R. communis was cloned, expressed heterologously and characterized. The deduced protein sequence showed that Ricinus NR has a serine phosphorylation site and a 14-3-3 binding motif, a common characteristic of NRs. Functional Ricinus NR protein was expressed in the yeast Pichia pastoris and compared with the features of Arabidopsis thaliana NR2 synthesized by the same expression system (AtNR2). The recombinant Ricinus NR (RcNR) itself was not inactivated by incubation with MgATP. As yeast extracts might lack factors required for NR regulation, desalted leaf extracts containing NR kinases and 14-3-3 proteins were prepared from 4-day-darkened (and therefore NR-free) leaves of Ricinus, and added to the assay of RcNR to check for ATP-dependent inactivation and Mg2+-sensitivity. When RcNR was combined with the NR-free extracts described above, its unusually high Mg2+-sensitivity was restored, but it remained unresponsive to ATP. In contrast, AtNR2 became inactive when incubated with the protein mixture and ATP. Thus, insensitivity to ATP appears to be an inherent property of Ricinus NR, whereas the high Mg2+-sensitivity depends on one or several factors in Ricinus leaves. This as yet unknown factor(s) was boiling-sensitive and appeared to interact specifically with recombinant Ricinus NR to provide the Mg2+-sensitivity of the authentic leaf enzyme.

Purification and characterization of nitrate reductase from nodule cytosol of soybean plants

Nodulated soybean (Glycine max [L.] Merr.) plants were grown in a nitrogen‐free liquid culture medium prepared with distilled water. The cytosol fraction from root nodules showed a significant level of NADH‐dependent nitrate reductase activity, even when the root did not show activity. This nitrate reductase was purified by column chromatography and native polyacrylamide gel electrophoresis (PAGE). The purified protein showed a main band at 100 kDa on sodium dodecyl sulfate (SDS)‐PAGE. The Km value for nitrate was 0.16 mM, and the highest activity was obtained at around pH 7.5. These characteristics are very similar to the inducible type of nitrate reductase, previously purified from soybean leaves. The developmental change in activity of this enzyme corresponded to that in nitrogenase activity.

Characterization of Nitrate Reductase from Light- and Dark-Exposed Leaves (Comparison of Different Species and Effects of 14-3-3 Inhibitor Proteins).

Nitrate reductase (NR) was extracted and partially purified from leaves of squash (Curcurbita maxima), spinach (Spinacia oleracea), and three transgenic Nicotiana plumbaginifolia leaves in the presence of phosphatase inhibitors to preserve its phosphorylation state. Purified squash NR showed activation by substrates (hysteresis) when prepared from leaves in the light as well as in darkness. A 14-3-3 protein known to inhibit phosphorylated spinach NR in the presence of Mg2+ decreased by 70 to 85% the activity of purified NR from dark-exposed leaves, whereas NR from light-exposed leaves decreased by 10 to 25%. Apparent lack of posttranslational NR regulation in a transgenic N. plumbaginifolia expressing an NR construct with an N-terminal deletion ([delta]NR) may be explained by more easy dissociation of 14-3-3 proteins from [delta]NR. Partially purified [delta]NR was, however, inhibited by 14-3-3 protein, and the binding constant of 14-3-3 protein (4 x 108 M-1) and the NR-inhibiting protein concentration that results in a 50% reduction of free NR (2.5 nM) were the same for NR and [delta]NR. Regulation of NR activity by phosphorylation and binding of 14-3-3 protein was a general feature for all plants tested, whereas activation by substrates as a possible regulation mechanism was verified only for squash.

Purification and Characterization of Nitrate Reductase from the Halophile Archaebacterium Haloferax mediterranei

Nitrate reductase is induced in cells of Haloferax mediterranei by the presence of nitrate upon anaerobic conditions. This enzyme was purified more than 35-fold with a yield of 49%. Densitograms of polyacrylamide gel electrophoresis show the preparation to be 85% purity. The best enzyme preparation has a specific activity of 13.6 U /m g protein. It is the first halophilic nitrate reductase that has been purified near to homogeneity. The purification consists of five steps: an ammonium sulphate precipitation and four successive gel chromatographies with Sepharose CL-4 B, calcium phosphate, DEAE-Sephacel and Sephacryl S-200. An average Mr of 170,000 was estimated by gel chromatography and non-denaturing gel electrophoresis. Effectiveness of electron donors, cofactors and inhibitors are reported. At low salt concentration the halophilic nitrate reductase was inactivated following first-order kinetics. The Km for nitrate depends on salt concentration and shows values in the range from 2.5 to 6.7 mM.

Immunochemical Characterization of Nitrate Reductase Forms from Wild-Type (cv Williams) and nr1 Mutant Soybean

Soybean (Glycine max L. Merr.) leaves contain two forms of nitrate reductase (NR)-NAD(P)H:NR and NADH:NR. Wild-type (cv Williams), nr(1) mutant and an unrelated cultivar (Prize) were grown with either no N source or with nitrate. Crude extracts were assayed for NR activities and the enzyme forms were purified on blue Sepharose. Analyses were done by polyacrylamide gel electrophoresis and ;Western blotting' using antibodies specific for NR. NAD(P)H:NR was identified as the constitutive NR present in wild-type and Prize, but was absent from the mutant. All three soybean lines contained nitrate-inducible NADH:NR with highest activity at pH 7.5. The results showed that NAD(P)H:NR and constitutive NR were one in the same and confirmed the presence of NADH:NR with pH 7.5 optimum.

Characterization of nitrate reductase from the yeastCandida utilis

The assimilatory nitrate reductase [NAD (P) H: nitrate oxidoreductase, EC 1.6.6.2] of the nitrate-utilizing yeastCandida utilis was prepared in soluble form from cells grown aerobically with nitrate as the nitrogen source, and some of its properties studied. The enzyme is cytoplasmic in intracellular distribution and its activity is NAD (P) H-dependent. The enzyme utilized both NADH and NADPH as electron donors but not reduced viologen dyes, flavins and other reductants. Flavin compounds, at higher concentrations, decreased the enzyme activity, while at very low concentrations, FAD (10−5 M) showed little stimulation. The optimal activity of the enzyme was obtained when the reaction was carried out for 10 min at 37° C at pH 7·0 with 20 mM KNO3, 0·1 mM NAD (P) H, 0.01 mM FAD and 140 µg enzyme protein. Complete loss of enzyme activity was observed on exposure to 50° C for 3 min. pHMB, cyanide, azide and EDTA, in that order, inhibited the enzyme activity effectively. The inhibition caused by pHMB was largely reversed by DTT.