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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)

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

Escherichia coli trimethylamine N-oxide (TMAO) reductase I, the major enzyme among inducible TMAO reductases, was purified to homogeneity by an improved method including heat treatment, ammonium sulfate precipitation, and chromatographies on Bio-Gel A-1.5m, DEAE-cellulose, and Reactive blue-agarose. The molecular weight was estimated by gel filtration to be approximately 200,000. A single subunit peptide with a molecular weight of 95,000 was found by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This enzyme contained 1.96 atoms of molybdenum, 0.96 atoms of iron, 1.52 atoms of zinc, and less than 0.4 atoms of acid-labile sulfur per molecular weight of 200,000. The absorption spectrum of the enzyme showed a peak at 278 nm and a shoulder at 288 nm, but no characteristic absorption was found from 350 to 700 nm. A fluorescent derivative of molybdenum cofactor was found when the enzyme was boiled with iodine in acidic solution; its fluorescence spectra were almost the same as those of the form A derivative of molybdopterin found in sulfite oxidase. The molybdenum cofactor released from heated TMAO reductase I reconstituted nitrate reductase in the extracts of Neurospora crassa mutant strain nit-1 lacking molybdenum cofactor. Thus, TMAO reductase I contains molybdopterin, which is a common constituent of some molybdenum-containing enzymes. Some kinetic properties were also determined.
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PMID:Further characterization of trimethylamine N-oxide reductase from Escherichia coli, a molybdoprotein. 352 39

Nitrate reductase of Mitsuokella multiacidus (formerly Bacteroides multiacidus) was solublized from the membrane fraction with 1% sodium deoxycholate and purified 40-fold by immunoaffinity chromatography on the antibody-Affi-Gel 10 column. The preparation showed a major band (86% of total protein) with enzyme activity and a minor band on polyacrylamide gel after disc electrophoresis in the presence of 0.1% Triton X-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a major band, the relative mobility of which corresponded to a molecular weight of 160,000, and two minor bands. The molecular weight of the enzyme was determined to be 160,000 by gel filtration on Bio-Gel A-1.5 m in the presence of 0.1% deoxycholate. Molybdenum cofactor was detected in the enzyme by fluorescence spectroscopy and by complementation of nitrate reductase from the nit-1 mutant of Neurospora crassa. The M. multiacidus enzyme catalyzed reduction of nitrate, chlorate, and bromate using methyl viologen as an electron donor. The maximal activity was found at pH 6.2-7.5 for nitrate reduction. Either methyl or benzyl viologen served well as the electron donor, but FAD, FMN, and horse heart cytochrome c were not effective. Ferredoxin from Clostridium pasteurianum supplied electron to the nitrate reductase. The purified enzyme had Km values of 0.13 mM, 0.12 mM, and 0.22 mM for nitrate, methyl viologen, and ferredoxin, respectively. The enzyme activity was inhibited by cyanide (85% at 1 mM), azide (88% at 0.1 mM), and thiocyanate (75% at 10 mM).
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PMID:Purification and properties of nitrate reductase from Mitsuokella multiacidus. 371 Oct 52

NADH:nitrate reductase (EC 1.6.6.1) was isolated from squash cotyledons (Cucurbita maxima L.) by a combination of Blue Sepharose and zinc-chelate affinity chromatographies followed by gel filtration on Bio-Gel A-1.5m. These preparations gave a single protein staining band (Mr = 115,000) on sodium dodecyl sulfate gel electrophoresis, indicating that the enzyme is homogeneous. The native Mr of nitrate reductase was found to be 230,000, with a minor form of Mr = 420,000 also occurring. These results indicate that the native nitrate reductase is a homodimer of Mr = 115,000 subunits. Acidic amino acids predominate over basic amino acids, as shown both by the amino acid composition of the enzyme and an isoelectric point for nitrate reductase of 5.7. The homogeneous nitrate reductase had a UV/visible spectrum typical of a b-type cytochrome. The enzyme was found to contain one each of flavin (as FAD), heme iron, molybdenum, and Mo-pterin/Mr = 115,000 subunit. A model is proposed for squash nitrate reductase in which two Mr = 115,000 subunits are joined to made the native enzyme. Each subunit contains 1 eq of FAD, cytochrome b, and molybdenum/Mo-pterin.
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PMID:Quaternary structure and composition of squash NADH:nitrate reductase. 403 8