Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:APRD00631 (Gel)
 14,881 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A simple three-step method was established for the purification of NAD(P)H dehydrogenase (quinone) ('DT-diaphorase', EC 1.6.99.2) from rat liver by affinity chromatography with a recovery of above 50%. The final enzyme preparation was purified about 750-fold and was electrophoretically homogeneous. Gel filtration showed that the enzyme had a mol.wt. of about 55 000, and one molecule of FAD was found per 55 000 mol.wt. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis gave a mol.wt. of about 27 000. Two N-terminal amino acids, asparagine/aspartic acid and glutamine/glutamic acid, were found in about equal yield, suggesting the presence of two non-identical polypeptide chains in the enzyme. NAD(P)H dehydrogenase was selectively removed by this affinity-chromatographic method from a microsomal carboxylation system. The system, which was solubilized by detergent and is dependent on vitamin K (2-methyl-3-phytyl-1,4-naphthaquinone or analogues with other side chains), lost its activity on the removal of the enzyme. The activity can be completely restored to the system by adding purified cytoplasmic NAD(P)H dehydrogenase or by using the quinol form of vitamin K1 (2-methyl-3-phytyl-1,4-naphthaquinol).
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PMID:NAD(P)H dehydrogenase and its role in the vitamin K (2-methyl-3-phytyl-1,4-naphthaquinone)-dependent carboxylation reaction. 62 56

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

Vanillyl-alcohol oxidase was purified 32-fold from Penicillium simplicissimum, grown on veratryl alcohol as its sole source of carbon and energy. SDS/PAGE of the purified enzyme reveals a single fluorescent band of 65 kDa. Gel filtration and sedimentation-velocity experiments indicate that the purified enzyme exists in solution as an octamer, containing 1 molecule flavin/subunit. The covalently bound prosthetic group of the enzyme was identified as 8 alpha-(N3-histidyl)-FAD from pH-dependent fluorescence quenching (pKa = 4.85) and no decrease in fluorescence upon reduction with sodium borohydride. The enzyme shows a narrow substrate specificity, only vanillyl alcohol and 4-hydroxybenzyl alcohol are substrates for the enzyme. Cinnamyl alcohol is a strong competitive inhibitor of vanillyl-alcohol oxidation. The visible absorption spectrum of the oxidized enzyme shows maxima at 354 nm and 439 nm, and shoulders at 370, 417 and 461 nm. Under anaerobic conditions, the enzyme is easily reduced by vanillyl alcohol to the two-electron reduced form. Upon mixing with air, rapid reoxidation of the flavin occurs. Both with dithionite reduction and photoreduction in the presence of EDTA and 5-deazaflavin the red semiquinone flavin radical is transiently stabilized. Opposite to most flavoprotein oxidases, vanillyl-alcohol oxidase does not form a flavin N5-sulfite adduct. Photoreduction of the enzyme in the presence of the competitive inhibitor cinnamyl alcohol gives rise to a complete, irreversible bleaching of the flavin spectrum.
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PMID:Purification and characterization of vanillyl-alcohol oxidase from Penicillium simplicissimum. A novel aromatic alcohol oxidase containing covalently bound FAD. 139 72

Recently, the purification of nitric oxide synthase (EC 1.14.23) from rat cerebellum has been reported, and the enzyme is a calmodulin-requiring enzyme (Bredt, D. S., and Snyder, S. H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 682-685). In this paper, nitric oxide synthase has been purified to near homogeneity from the cytosol fraction of rat polymorphonuclear neutrophils. The purification procedure involves affinity chromatography with adenosine 2',5'-diphosphate-agarose and an anion exchange column, DEAE-Bio-Gel A. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the enzyme migrated as a single protein band with Mr = 150,000. The molecular weight was estimated to be 150,000 by gel filtration on a Superose 12 HR 10/30. The purified enzyme was unstable with a half-life of 3 h at pH 7.4 and 4 degrees C. The enzyme activity required the presence of Ca2+, NADPH, FAD, and (6R)-5,6,7,8-tetrahydro-L-biopterin. Calmodulin antagonists (W5, W7, W13, and trifluoperazine dihydrochloride) did not inhibit the enzyme activity, and the addition of calmodulin was also ineffective for the increase in the enzyme activity. The neutrophil enzyme appears to be a calmodulin-independent type of nitric oxide synthase.
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PMID:Calmodulin-independent nitric oxide synthase from rat polymorphonuclear neutrophils. 170 88

A soluble nitric oxide (NO) synthase activity was purified 426-fold from a mouse macrophage cell line activated with interferon gamma and bacterial lipopolysaccharide by sequential anion-exchange, affinity, and gel filtration chromatography. SDS/PAGE of the purified NO synthase gave three closely spaced silver-staining protein bands between 125 and 135 kDa. When assayed in the presence of L-arginine, NADPH, tetrahydrobiopterin, FAD, and reduced thiol, purified NO synthase had a specific activity of 1313 nmol of NO2- plus NO3- per min per mg. The apparent Km of the enzyme for L-arginine and NADPH was 2.8 and 0.3 microM, respectively. Addition of calcium ions with or without calmodulin did not increase the activity of the purified enzyme, and NO synthesis was not altered by calmodulin inhibitors. Gel filtration chromatography indicated that the induced NO synthase was catalytically competent as a dimer of approximately 250 kDa but could be dissociated into inactive monomers of approximately 130 kDa in the absence of L-arginine, FAD, and tetrahydrobiopterin. Upon heat denaturation, NO synthase released 1.1 mol of FAD and 0.55 mol of FMN per mol of 130-kDa subunit. Thus, inducible macrophage NO synthase differs in several respects from constitutive NO synthases and is one of very few eukaryotic enzymes containing both FAD and FMN.
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PMID:Purification and characterization of the cytokine-induced macrophage nitric oxide synthase: an FAD- and FMN-containing flavoprotein. 171 79

The synthesis of nitric oxide (.NO) from L-arginine has been demonstrated in a number of cell types and functions either as a cell signaling agent or as a key component of the cell-mediated immune response. Both constitutive and inducible activities have been described. Herein we report the purification of inducible .NO synthase (EC 1.14.23) from activated murine macrophages using a two-column procedure. Crude 100,000 x g supernatant was passed through a 2'-5'-ADP-Sepharose 4B affinity column followed by a DEAE-Bio-Gel A anion exchange column. The .NO synthase ran as a band of Mr = 130,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration experiments using a Superose 6 HR 10/30 column estimated the native molecular weight to be 260 +/- 30 kDa, indicating that the native enzyme exists as a dimer. Activity was dependent upon L-arginine (Km = 16 +/- 1 microM at 37 degrees C and pH 7.5) and NADPH. Both (6R)-tetrahydro-L-biopterin and FAD enhanced activity, whereas Mg2+ and FMN had no effect on activity. Fluorescence studies demonstrated the presence of one bound FAD and one bound FMN per subunit.
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PMID:Purification of the inducible murine macrophage nitric oxide synthase. Identification as a flavoprotein. 172 Jul 73

Aspartic acid 244 that occurs at the putative NAD(+)-binding site of rat liver S-adenosylhomocysteinase was replaced by glutamic acid by oligonucleotide-directed mutagenesis. The mutant enzyme was purified to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel permeation chromatography showed that the purified mutant enzyme was a tetramer as is the wild-type enzyme. In contrast to the wild-type enzyme, which possesses 1 mol of tightly bound NAD+ per mol of enzyme subunit, the mutant enzyme had only 0.05 mol of NAD+ but contained about 0.6 mol each of NADH and adenine per mol of subunit. The mutant enzyme, after removal of the bound compounds by acid-ammonium sulfate treatment, exhibited S-adenosylhomocysteinase activity when assayed in the presence of NAD+. From the appearance of activity as a function of NAD+ concentration, the enzyme was shown to bind NAD+ with a Kd of 23.0 microM at 25 degrees C, a value greater than 280-fold greater than that of the wild-type enzyme. In the presence of a saturating concentration of NAD+, the mutant enzyme showed apparent Km values for substrates similar to those of the wild-type enzyme. Moderate decreases of 8- and 15-fold were observed in Vmax values for the synthetic and hydrolytic directions, respectively. These results indicate the importance of Asp-244 in binding NAD+, and are consistent with the idea that the region of S-adenosylhomocysteinase from residues 213 to 244 is part of the NAD+ binding site. This region has structural features characteristic of the dinucleotide-binding domains of NAD(+)- and FAD-binding proteins (Ogawa, H., Gomi, T., Mueckler, M. M., Fujioka, M., Backlund, P.S., Jr., Aksamit, R.R., Unson, C.G., and Cantoni, G.L. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 719-723).
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PMID:Site-directed mutagenesis of rat liver S-adenosylhomocysteinase. Effect of conversion of aspartic acid 244 to glutamic acid on coenzyme binding. 197 8

The respiratory chain-linked NADH-quinone reductase (NQR) and NADH-ferricyanide dehydrogenase (NFD) were extracted from membranes of Escherichia coli by n-dodecyl octaethyleneglycol monoether detergent and purified by DEAE-Sephacel, DEAE-5PW and Bio-Gel HTP column chromatography. The purified NQR contained FAD as a cofactor, catalyzed the reduction of ubiquinone-1 (Q1) and reacted with NADH, but not with deamino-NADH (d-NADH), with an apparent Km of 48 microM. On the other hand, the purified NFD contained FMN as a cofactor, reacted with both NADH and d-NADH, and catalyzed the reduction of ferricyanide but not Q1. NFD showed a high affinity for both NADH and d-NADH with a Km of 7-9 microM. NFD was inactivated, whereas NQR was rather activated, by preincubation with an electron donor in the absence of electron acceptor. These properties were compared with those of activities observed with inverted membrane vesicles with special reference to the generation of inside-positive membrane potential (delta psi). It was found that d-NADH-reactive FMN-containing NFD is a dehydrogenase part of energy-generating NADH-quinone reductase complex. The FAD-containing NQR was very similar to that purified by Jaworowski et al. (Biochemistry (1981) 20, 2041-2047), and reduced Q1 without generating delta psi.
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PMID:Purification of NADH-ferricyanide dehydrogenase and NADH-quinone reductase from Escherichia coli membranes and their roles in the respiratory chain. 267 83

The presence of flavin compound(s) giving a yellowish-green autofluorescence in rat hepatocyte plasma membrane has recently been reported (Nokubo, M. et al. (1988) Biochim. Biophys. Acta 939, 441-448). The fluorophore can quantitatively be extracted with water at 80 degrees C from isolated plasma membranes. Gel filtration of the extract eluted with water showed two peaks, the fluorescence of which closely resembled that of riboflavin. The major peak comigrated with proteins and the minor one displayed a position identical to authentic riboflavin. When the components of the major peak were rechromatographed after acetic acid treatment and eluted with 20 mM of acetic acid, the fluorescent compound separated from the proteins and eluted at the same position as riboflavin. In paper chromatography and HPLC, the behavior of the fluorescent compound (separated by acid treatment from the proteins) was identical to that of riboflavin. SDS gel filtration of subcellular fractions of rat liver revealed that riboflavin was the dominant flavin, whereas FAD and FMN were not detectable in the plasma membrane. Microsomes and mitochondria contain predominantly FAD and FMN, and only minor quantities of riboflavin. The presence of riboflavin in the plasma membrane is a novel finding, the functional significance of which is still unclear; however, a hypothesis can be forwarded on the basis of the ability of flavins to generate superoxide anion radicals during their autoxidation.
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PMID:Identification of protein-bound riboflavin in rat hepatocyte plasma membrane as a source of autofluorescence. 273 Sep 7

Previous attempts to produce anti-(ADP-ribose) antibodies by immunization of rabbits with ADP-ribose conjugated to serum albumin had resulted in the production of 5'AMP-specific antibodies [Bredehorst et al. (1978) Eur. J. Biochem. 82, 105-113]. To obtain true anti-(ADP-ribose) antibodies an antigen was constructed that was resistant to enzymic degradation at the pyrophosphate group. The enzymically active beta-methylene derivative of NAD (NAD[CH2]) was synthesized from ADP containing a methylene bridge (CH2) instead of an oxygen in the diphosphate group. NAD[CH2] was converted to its N6-[(2-carboxyethyl)thiomethyl] derivative and hydrolyzed to the corresponding ADP[CH2]-ribose derivative which was then coupled to bovine serum albumin. The antibodies obtained with this antigen were specific for free or protein-bound ADP-ribose groups, except for a cross-reaction with FAD, AMP, ADP, ATP or poly(ADP-ribose) interfered with [3H]ADP-ribose tracer binding only at higher concentrations. No interference was observed with poly(A), RNA and DNA at 6000-fold excess. The antibodies were purified on a novel type of affinity matrix. This was formed from NAD and guanidinobutyrate by a cholera-toxin-catalyzed reaction and the product, ADP-ribosyl guanidinobutyrate, was bound to Affi Gel by carbodiimide-aided condensation. The purified antibodies allowed the detection of ADP-ribose conjugated to polypeptides in amounts lower than 1 pmol as demonstrated by immunoblotting of [14C]ADP-ribosylated elongation factor 2. They also could be used to observe in situ, by indirect immunofluorescence, the increased mono(ADP-ribosyl)ation of nuclear proteins in dimethyl-sulfate-treated cells, and to show that histone H2B was the principal histone acceptor of single ADP-ribose groups in alkylated 3T3 cells.
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PMID:Production of anti-(ADP-ribose) antibodies with the aid of a dinucleotide-pyrophosphatase-resistant hapten and their application for the detection of mono(ADP-ribosyl)ated polypeptides. 300 88


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