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)

The reduced nicotinamide adenine dinucleotide phosphate (NADPH):nitrate oxidoreductase (EC 1.6.6.2) from Aspergillus nidulans wild-type bi-1 was purified by means of salt fractionation, gel filtration, affinity chromatography, and polyacrylamide gel electrophoresis. Enzyme which was adsorbed on Cibacron blue agarose could be eluted with 2 mM NADPH or 2 mM oxidized NADP (NADP(+)), the former being about three times more effective than the latter. About half the total NADPH:nitrate reductase activity adsorbed on agarose required elution with 1 M NaCl. This salt-elutable form remained active with NADPH and was not converted to the NADPH-elutable form after readsorption on Cibacron blue agarose. The NADPH-eluted enzyme exhibited a markedly different electrophoretic mobility than the enzyme eluted with NADP(+) or NaCl. After electrophoresis on polyacrylamide gels, the NADPH-eluted NADPH:nitrate reductase was separated into four proteins, two of which contained nonheme iron and exhibited reduced methyl viologen-nitrate reductase activity. None of these proteins, singly or in combination, reduced nitrate with NADPH as substrate. Difference spectra analyses and specific heme iron stains revealed the presence of cytochrome b(557) in the largest of the proteins. The molecular weights of the four proteins, which were determined from the relationship of their mobilities on varied concentrations of acrylamide gel, were 360,000, 300,000, 240,000, and 118,000. The subunit molecular weights of these, which are determined via sodium dodecyl sulfate slab gel electrophoresis, were 49,000, 50,000, and 75,000. The key role of NADPH in maintenance of the active form of the heteromultimer is further substantiated.
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PMID:Further characterization of the reduced nicotinamide adenine dinucleotide phosphate: nitrate oxidoreductase in Aspergillus nidulans. 3 44

The assimilatory nitrate reductase of the phototrophic bacterium Rhodopseudomonas capsulata strain AD2 was purified to homogeneity by a combination of ammonium sulfate fractionation, chromatography on DEAE-cellulose and isoelectric focusing (isoelectric point of 4.8). The purified enzyme was active only with reduced viologen dyes or reduced flavin as electron donors. Contrary to other bacterial assimilatory nitrate reductases, the enzyme was not inhibited by chlorate, but rather accepted this substance as an alternate substrate. The molecular weight of the enzyme was 185,000 dalton as determined by gelfiltration. Subunit analysis by sodium dodecyl sulfate (SDS) gel electrophoresis yielded a single protein band with a molecular weight of 85,000 dalton,, suggesting that the enzyme was composed of two identical subunits. The nitrate reductase contained 0.8 g-atoms molybdenum per 1.85 x 10(5) g protein and exhibited absorption maxima at 418, 523 and 552 nm in the reduced state (dithionite as reductant). The nitrate reductase of Rps. capsulata AD2 is the first prokaryotic enzyme of the assimilatory type that has been shown to contain heme.
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PMID:Assimilatory nitrate reductase of Rhodopseudomonas capsulata AD2: a molybdo-hemeprotein. 15 48

Membrane vesicles of Veillonella alcalescens, grown in the presence of L-lactate and KNO-3, actively transport amino acids under anaerobic conditions in the presence of several electron donors and the electron acceptor nitrate. The highest initial rates of uptake are obtained with L-lactate, followed by reduced nicotinamide adenine dinucleotide, glycerol-1-phosphate, formate, and L-malate.. The membrane vesicles contain the dehydrogenases for these electron donors, and these enzymes are coupled with nitrate reductase. In membrane vesicles from cells, grown in the presence of nitrate, the dehydrogenases are not coupled with fumarate reducatase, and anaerobic transport of amino acids does not occur with fumarate as electron acceptor. Under aerobic conditions none of the physiological electron donors can energize transport. However, a high rate of uptake is observed with the electron donor system ascorbate-phenazine metho-sulfate. This electron donor system also effectively energizes transport under anaerobicconditions in the presence of the electron acceptor nitrate.
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PMID:Amino acid transport in membrane vesicles of obligately anaerobic Veillonella alcalescens. 16 33

NADH:nitrate reductase (EC 1.6.6.1) from Chlorella vulgaris has been purified 640-fold with an over-all yield of 26% by a combination of protamine sulfate fractionation, ammonium sulfate fractionation, gel chromatography, density gradient centrifugation, and DEAE-chromatography. The purified enzyme is stable for more than 2 months when stored at minus 20 degrees in phosphate buffer (pH 6.9) containing 40% (v/v) glycerol. After the initial steps of the purification, a constant ratio of NADH:nitrate reductase activity to NADH:cytochrome c reductase and reduced methyl viologen:nitrate reductase activities was observed. One band of protein was detected after polyacrylamide gel electrophoresis of the purified enzyme. This band also gave a positive stain for heme, NADH dehydrogenase, and reduced methyl viologen:nitrate reductase. One band, corresponding to a molecular weight of 100, 000, was detected after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme contains FAD, heme, and molybdenum in a 1:1:0.8 ratio. One "cyanide binding site" per molybdenum was found. No non-heme-iron or labile sulfide was detected. From a dry weight determination of the purified enzyme, a minimal molecular weight of 152, 000 per molecule of heme or FAD was calculated. An s20, w of 9.7 S for nitrate reductase was found by the use of sucrose density gradient centrifugation and a Stokes radius of 89 A was estimated by gel filtration techniques. From these values, and the assumption that the partial specific volume is 0.725 cc/g, a molecular weight of 356, 000 was estimated for the native enzyme. These data suggest that the native enzyme contains a minimum of 2 molecules each of FAD, heme, and molybdenum and is composed of at least three subunits.
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PMID:Reduced nicotinamide adenine dinucleotide-nitrate reductase of Chlorella vulgaris. Purification, prosthetic groups, and molecular properties. 16 92

The heterogeneous form of nitrate reductase released from the membrane fraction of Escherichia coli by heat treatment was converted to a new electrophoretic form by incubation with trypsin. As a result of the trypsin treatment, the heat-released enzyme was converted from an associating-dissociating system to a nonassociating monomer (Mr approximately 200,000) which retained full enzymatic activity. Several distinct subunits in the 47,000- to 59,000-dalton range were converted to a single 43,000-dalton subunit during the trypsin treatment, while the other major subunit (155,000 daltons) was unaffected. Nitrate reductase extracted from the membrane fraction with deoxycholate and ammonium sulfate was composed of two apparently homogeneous subunits (155,000 and 59,000 daltons). The detergent-extracted enzyme preparation was converted by trypsin to an electrophoretic form very similar to the product of trypsin treatment of the heat-released enzyme with an identical subunit composition (155,000 and 43,000 daltons). These results demonstrate that the heterogeneous subunits present in the heat-released enzyme are produced during heat treatment by proteolytic cleavage of a single 59,000-dalton subunit. The fragments removed by trypsin treatment are implicated in the self-associating properties of the heat-released enzyme.
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PMID:Limited proteolysis of nitrate reductase purified from membranes of Escherichia coli. 32 Feb 11

The cytoplasmic nitrate reductase in heme mutant H-14 of Staphylococcus aureus was partially purified by steps which included ammonium sulfate fractionation and chromatography on Bio-Gel A 1.5m and ion-exchange columns. The active fractions from the ion-exchange columns showed two forms of the enzyme upon electrophoresis in nondenaturing gels of polyacrylamide; these corresponded to proteins of R(f) 0.16 and 0.28. Each form contained a predominant polypeptide of molecular weight 140,000, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The R(f) 0.16 form contained another major polypeptide of molecular weight 57,000, but the R(f) 0.28 form contained several other polypeptides. The sedimentation properties of the enzyme were examined after partial purification on Bio-Gel A 1.5m. In sucrose gradients containing Triton X-100 the enzyme sedimented as a homogeneous peak with an estimated molecular weight of 225,000; without detergent a heterogeneous profile was observed of molecular weight greater than 250,000. Treatment of the enzyme with trypsin increased the specific activity, and the enzyme sedimented as a homogeneous peak in sucrose gradients without Triton X-100, with an estimated molecular weight of 202,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that trypsin treatment converted the polypeptide of molecular weight 140,000 to a polypeptide of molecular weight 112,000. We conclude that the cytoplasmic nitrate reductase of S. aureus has a large subunit of molecular weight 140,000, which can be modified by trypsin to a polypeptide of molecular weight 112,000 without loss of catalytic activity.
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PMID:Partial purification and some properties of the Staphylococcus aureus cytoplasmic nitrate reductase. 45 98

Assimilatory nitrate reductase (EC 1.6.6.1 NADH:nitrate oxidoreductase) from Chlorella vulgaris purified by affinity chromatography was found to be homogeneous as judged by electrophoresis on sodium dodecyl sulfate-polyacrylamide gel and by analytical ultracentrifugal techniques. The molecular weight of the intact enzyme and that of the enzyme dissociated in 6 M GuHCl, determined by sedimentation equilibrium studies, were 280,000 +/- 10,000 and 90,000 +/- 5,000, respectively. Comparable values were obtained using the S20,w value and the D20,w values in Svedberg's equation. The D20,w values were determined by laser light-scattering measurements. Active enzyme centrifugation showed that the monomer is an active species. A quantitative re-evaluation of the prosthetic groups present (FAD, heme, and molybdenum) was also made and was consistent with the conclusion that the active monomer contains three subunits as previously deduced by Solomonson et al. ((1975) J. Biol. Chem. 250, 4120). Electron micrographs showed images which corresponded to three subunits, supporting the data obtained by hydrodynamic studies. The enzyme is not cigar-shaped, as previously surmised, but has a roughly globular structure.
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PMID:Physical studies on assimilatory nitrate reductase from Chlorella vulgaris. 50 Jun 68

Reduced nicotinamide adenine dinucleotide phosphate-dependent nitrate reductase activity in crude extracts of Trichoderma virde was significantly inhibited by physiological concentrations of ammonium chloride, sodium chloride, and potassium chloride, but not by ammonium or sodium sulfate. The chloride inhibition of nitrate reductase activity increased in a linear manner with chloride concentration.
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PMID:Decrease in nitrate reductase activity in extracts of Trichoderma viride Incubated with chlorides. 55 76

The membrane-bound formate dehydrogenase of Escherichia coli grown anaerobically in the presence of nitrate was solubilized with deoxycholate and purified to near homogeneity. The purification procedure included ammonium sulfate fractionation and chromatography on Bio-Gel A-1.5m and DEAE Bio-Gel A in the presence of the nonionic detergent, Triton X-100. This detergent caused a significant decrease in the molecular weight of the soluble formate dehydrogenase complex and allowed the enzyme then to be resolved from other membrane components. Anaerobic conditions were required throughout due to the sensitivity of the enzyme to oxygen inactivation. Formate dehydrogenase was judged to be at least 93 to 99% pure by the following procedures: polyacrylamide gel electrophoresis in the presence of Triton X-100 and sodium dodecyl sulfate, gel filtration, and sedimentation velocity studies. The purified enzyme exists as a detergent-protein complex (0.20 +/- 0.03 g of Triton X-100/g of protein) which has an S20,w of 18.1 S and a Stokes radius of 76 A. This corresponds to a molecular weight of 590,000 +/- 59,000. The enzyme had an absorbance spectrum of a b-type cytochrome which could be completely reduced by formate. The heme content corresponds to an equivalent weight of 154,000 which suggests a tetrameric structure for the enzyme. Formate dehydrogenase was found to contain (in relative molar amounts): 1.0 heme, 0.95 molybdenum, 0.96 selenium, 14 non-heme iron, and 13 acid-labile sulfide. Neither FAD nor FMN could be detected. The enzyme contains three polypeptides, designated alpha, beta, and gamma, whose molecular weights were estimated by gel electrophoresis in the presence of sodium dodecyl sulfate to be 110,000, 32,000, and 20,000, respectively. After separation of the polypeptides by gel filtration in the presence of sodium dodecyl sulfate alpha, beta, and gamma were found in 1:1.2:0.55 molar ratios. A study of the enzyme obtained from cells grown with [75Se]selenite showed that only the alpha polypeptide contained significant amounts of selenium. The enzyme will catalyze the formate-dependent reduction of phenazine methosulfate, dichlorophenolindophenol, methylene blue, nitroblue tetrazolium, benzyl viologen, methyl viologen, ferricyanide, and coenzyme Q6. Cyanide, azide, p-hydroxymercuribenzoate, iodoacetamide, and oxygen inhibit the enzyme. The procedure which was designed for the purification of formate dehydrogenase also yields a highly purified preparation of nitrate reductase. This nitrate reductase has been shown to contain significant amounts of heme (Enoch, H. G., and Lester, R. L. (1974) Biochem. Biophys. Res Commun. 61,1234-1241). The enzyme contains three polypeptides with molecular weights of 155,000, 63,000, and 19,000. When measured in the presence of Trition X-100 the Stokes radius of nitrate reductase is 75 A and the S20,w is 16 S which corresponds to a molecular weight of 498,000.
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PMID:The purification and properties of formate dehydrogenase and nitrate reductase from Escherichia coli. 109 93

A mutation in a new gene, molR, prevented the synthesis in Escherichia coli of molybdoenzymes, including the two formate dehydrogenase isoenzymes, nitrate reductase and trimethylamine-N-oxide reductase. This phenotype was suppressed by supplementing the media with molybdate. Thus, the molR mutant was phenotypically similar to previously described chlD mutants, thought to be defective in molybdate transport. The molR gene is located at 65.3 min in the E. coli chromosome, in contrast to the chlD gene, which maps at 17 min and thus can be readily distinguished. The molR gene is also cotransducible with a hitherto unidentified gene essential for the production of 2-oxoglutarate from isocitrate, designated icdB (located at 66 min). The molR mutant strain SE1100 also failed to produce the hydrogenase component of formate hydrogenlyase (HYD3) in molybdate-unsupplemented media. The amount of molybdate required by strain SE1100 for the production of parental levels of formate hydrogenlyase activity was dependent on the growth medium. In Luria-Bertani medium, this value was about 100 microM, and in glucose-minimal medium, 1.0 microM was sufficient. In low-sulfur medium, this value decreased to about 50 nM. The addition of sulfate or selenite increased the amount of molybdate needed for the production of formate hydrogenlyase activity. These data suggest that in the absence of the high-affinity molybdate transport system, E. coli utilizes sulfate and selenite transport systems for transporting molybdate, preferring sulfate transport over the selenite transport system.
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PMID:Identification of a new gene, molR, essential for utilization of molybdate by Escherichia coli. 215 10


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