Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: KEGG:D02011 (FAD)
 5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The NADPH-dependent superoxide-generating oxidase of pig neutrophils is activated by sodium dodecyl sulfate in a cell-free system. The activation requires both membrane and cytosolic components. The membrane component was effectively extracted with 0.75% octyl glucoside and the extract was fractionated by wheat-germ-agglutinin-agarose column chromatography. The chromatography resulted in loss of the O2--generating activity in the cell-free system. The activity, however, was restored by the reconstitution with the fraction which passed through the column (fraction A) and the one eluted with N-acetylglucosamine (fraction B) using an octyl glucose dilution procedure: both fractions were pre-mixed in the presence of 0.75% octyl glucoside and diluted by putting the mixture into the detergent-free assay mixture. The latter fraction was copurified with cytochrome b558, the content of which is 2.12 +/- 0.53 nmol/mg protein (mean +/- SD, n = 5). The potency of fraction B in the reconstitution of the O2--generating activity was lost by heat treatment and decreased by protease treatment, whereas that of fraction A was not affected. Fraction A in the reconstitution of the O2--generating activity was replaced by lipid extracted from fraction A, furthermore, by exogenous phospholipid, azolectin. The O2--generating activity reconstituted with azolectin and the partially purified component in fraction B was dependent on SDS, cytosol and the concentrations of azolectin and FAD. The activity was sensitive to p-chloromercuribenzoate but not to azide. The maximal activity was obtained at pH 7.0-7.5. The Km values for NADPH and NADH were 0.024 mM and 0.57 mM, respectively. These properties were consistent with those of the NADPH oxidase responsible for the respiratory burst. The activity in the reconstitution system was 20.5 +/- 3.5 mumol O2-.min-1.mg-1 membrane-derived protein (mean +/- SD, n = 5) which shows that the membrane component was purified about 100-fold. These findings indicate that cytochrome b558 is probably a membrane component of the O2--generating NADPH oxidase and its activation in the cell-free system requires the reconstitution with phospholipids.
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PMID:Reconstitution of the partially purified membrane component of the superoxide-generating NADPH oxidase of pig neutrophils with phospholipid. 215 45

The methylenetetrahydrofolate reductase from the carbon-monoxide-utilizing homoacetogen Peptostreptococcus productus (strain Marburg) has been purified to apparent homogeneity. The purified enzyme catalyzed the oxidation of NADH with methylenetetrahydrofolate as the electron acceptor at a specific activity of 380 mumols.min-1 mg protein-1 (37 degrees C; pH 5.5). The apparent Km for NADH was near 10 microM. The apparent molecular mass of the enzyme was determined by gel filtration to be approximately 250.0 kDa. The enzyme consists of eight identical subunits with a molecular mass of 32 kDa. It contains 4 FAD/mol octamer which were reduced by the enzyme with NADH as the electron donor; iron could not be detected. Oxygen had no effect on the enzyme. Ultracentrifugation of cell extracts revealed that about 40% of the enzyme activity was recovered in the particulate fraction, suggesting that the enzyme is associated with the membrane. The enzyme also catalyzed the methylenetetrahydrofolate reduction with methylene blue as an artificial electron donor. The oxidation of methyltetrahydrofolate was mediated with methylene blue as the electron acceptor; neither NAD+ nor viologen dyes could replace methylene blue in this reaction. NADP(H) or FAD(H2) were not used to substrates for the reaction in either direction. The activity of the purified enzyme, which was proposed to be involved in sodium translocation across the cytoplasmic membrane, was not affected by the absence or presence of added sodium. The properties of the enzyme differ from those of the ferredoxin-dependent methylenetetrahydrofolate reductase of the homoacetogen Clostridium formicoaceticum and of the NADP(+)-dependent reductase of eucaryotes investigated so far.
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PMID:Purification and properties of a NADH-dependent 5,10-methylenetetrahydrofolate reductase from Peptostreptococcus productus. 220 95

Two distinct forms (FMO-I and FMO-II) of flavin-containing monooxygenase were purified from the liver microsomes of guinea pig. The minimum molecular weights estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 54,000 for FMO-I and 56,000 for FMO-II, respectively. Tryptic digestion of these enzymes gave different electrophoretic patterns, suggesting that FMO-I and -II have distinct amino acid sequences. The amino terminal sequence of FMO-II could not be estimated probably due to its blocking while that of FMO-I was determined to be highly homologous to the rabbit liver flavin-containing monooxygenase (J. Ozols, 1989, Biochem. Biophys. Res. Commun. 163, 49-55). Absorption maxima of FMO-I and -II were recorded at 368 and 440 nm and 381 and 456 nm, respectively. Molar ratios of FAD to both of these apoenzymes were shown to be one to one. Substrate specificity of FMO-I and -II was determined using 15 compounds as the substrate. The results showed two enzymes that exhibited overlapped but different specificity toward these substrates although FMO-I had lower activity than did FMO-II with all compounds except thiobenzamide. Of particular interest, only FMO-II showed considerably high activities for primary amines, n-octylamine, and n-decylamine. Immunoglobulin G raised against FMO-II could recognize FMO-I as well as FMO-II, but the reactivity of FMO-I toward the antibody was obviously lower than that of FMO-II. Electrophoresis followed by immunostaining revealed that microsomes of lung, kidney, urinary bladder, testis, and spleen contain the same protein as FMO-II and/or FMO-I. Only lung was shown to have an additional isozyme of FAD-monooxygenase with a molecular weight apparently higher than those of FMO-I and -II. These results strongly suggest that at least two forms of flavin-containing monooxygenases distinct from the lung-type isozyme are expressed in liver of guinea pigs.
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PMID:Multiplicity of liver microsomal flavin-containing monooxygenase in the guinea pig: its purification and characterization. 236 22

A menadione-stimulated, superoxide-generating enzyme was purified 127-fold from resting bovine polymorphonuclear leukocyte (neutrophil) membranes with a yield of 34%. The enzyme was extracted with Triton X-100 and purified by chromatography on DEAE-Sepharose CL-6B, NAD-agarose, and Sephacryl S-200. The purified enzyme contained FAD and had an apparent molecular mass of 93 kDa by sodium dodecyl sulfate gel electrophoresis. In a nondenaturing gel electrophoresis system, the enzyme was multimeric (Mr greater than 400,000). The oxidase showed 3-4-fold higher activity (Vm) with NADH compared with NADPH, but the Km for both pyridine nucleotides was similar (39 and 47 microM, respectively). The enzyme transferred electrons to cytochrome c, dichlorophenolindophenol, and nitro blue tetrazolium. Cytochrome c reduction was stimulated 4-fold by menadione and was inhibited 70% by superoxide dismutase. Cytochrome c reduction was not inhibited by several mitochondrial respiratory chain inhibitors (azide, cyanide, and rotenone) but was sensitive to thiol-reactive agents (p-chloromercuribenzoate and monoiodo acetate). The catalytic properties of this enzyme distinguish it from the NADPH-dependent superoxide-generating respiratory burst oxidase (NADPH-oxidase) of human neutrophils. Nevertheless, antibodies to this enzyme inhibited not only the purified menadione-stimulated oxidase, but also the respiratory burst oxidase in membranes isolated from activated human neutrophils, indicating similar antigenic determinants are shared by these enzymes. Western blots of human neutrophil membranes visualized a plasma membrane protein of molecular mass 67 kDa, corresponding in size to a protein previously reported in preparations of the human respiratory burst oxidase.
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PMID:A menadione-stimulated pyridine nucleotide oxidase from resting bovine neutrophil membranes. Purification, properties, and immunochemical cross-reactivity with the human neutrophil NADPH oxidase. 245 25

Bacterial plasmids have genes that confer highly specific resistances to As, Bi, Cd, Cu, Cr, Hg, Pb, Te, Zn, and other toxic heavy metals. For each toxic cation or anion, generally a different resistance system exists, and these systems may be "linked" together on multiple resistance plasmids. For Cd2+, AsO2-, AsO4(3)-, Hg2+, and organomercurials, DNA sequence analysis has supplemented direct physiological and biochemical experiments to produce sophisticated understanding. The cadA ATPase of S. aureus plasmids is a 727 amino acid membrane ATPase that pumps Cd2+ from the cells as rapidly as it is accumulated. This polypeptide is related by sequence to other cation translocating ATPases, including the membrane K+ ATPases of Escherichia coli and Streptococcus faecalis, the H+ ATPases of yeast and Neurospora, the Na+/K+ ATPases of vertebrate animals, and the Ca2+ ATPases of rabbit muscle. The conserved residues include the aspartyl residue that is phosphorylated, the lysine involved in ATP binding, and the proline within a membrane translocating region. The arsenate and arsenite translocating ATPase consists of 3 polypeptides (from DNA sequence analysis), including a recognizable ATP binding protein (arsA), an integral membrane protein (arsB gene), and a substrate specificity subunit (arsC gene). Inorganic mercury and organomercurial degradation is carried out by a series of about 6 polypeptides, including 2 soluble intracellular enzymes (organomercurial lyase and mercuric reductase). The latter is related by sequence and function to glutathione reductase and lipoamide dehydrogenase of prokaryotes and eukaryotes. These enzymes are dimeric, FAD-containing, NAD(P)H-dependent oxidoreductases. Other recognizable polypeptides in the mer system include a DNA-binding regulatory protein from the merR gene and a Hg2+ transport system consisting of a periplasmic Hg2(+)-binding protein (merP gene) and a membrane protein (merT gene) in gram negative systems.
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PMID:DNA sequence analysis of bacterial toxic heavy metal resistances. 248 81

Two NADPH-reductase preparations (FAD-containing monooxygenases) were isolated from rabbit liver microsomes, referred to as from 1 and from 2. Purification was achieved by means of anion-exchange, cation-exchange and hydroxylapatite chromatography in the presence of cholate and Nonidet P-40. Affinity chromatography on 2', 5'-ADP Sepharose was used to increase the purity and to concentrate the enzyme. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, form 1 exhibited a single band at Mr 58,500 and form 2 at Mr 58,000. The NH2- terminus of form 1 is blocked, whereas the NH2-terminus of form 2 is homologous to the NADPH-phydroxybenzoate hydrolase from Pseudomonas fluorescens. The latter and the form 2 enzyme share 11 identical residues in the NH2-terminal segment of 15 residues. Both forms were subjected to tryptic cleavages and peptide mapping. Sequence analysis of the peptides obtained indicated that forms 1 and 2 are similar but not identical proteins. A tryptic peptide, homologous to residues 3 to 32 of form 2 enzyme was isolated from the form 1 protein. This segment has 24 residues that are identical to the form 2 and contains the consensus sequence Gly-X-Gly-X-X-Gly, found in most FAD binding proteins. These results indicate that the NADPH-monooxygenase system consists of at least two distinct proteins representing different gene products.
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PMID:Liver microsomes contain two distinct NADPH-Monooxygenases with NH2-terminal segments homologous to the flavin containing NADPH-monooxygenase of Pseudomonas fluorescens. 250 69

A new ESR assay has been developed for the characterization of unilamellar lipid vesicles. It is based on the reduction by photogenerated FADH2 of amphiphilic spin-labels having the spin in the polar group. FADH2 is generated in situ under anaerobic conditions from its oxidized form (FAD) by photoreduction in the presence of excess EDTA as the reducing agent. Photoreduction is induced by exposing the FAD/EDTA mixture to white light of a commercial slide projector. FADH2 as an impermeable agent reduces spin-label molecules located on the outer layer of the bilayer that are readily accessible in a first fast reaction; spin-label located on the inner layer of the bilayer is reduced in a second slow reaction. The ESR assay is suitable for the routine characterization of unilamellar membrane vesicles: it allows the determination of the vesicle size, the entrapped volume, the bilayer asymmetry, the bilayer integrity, and the vesicle stability. The ESR assay developed is of general applicability: it can be used with charged and uncharged bilayers which may be labeled with either neutral or charged spin-labels. An assessment of the new ESR assay is given in comparison to the existing ascorbate method which uses sodium ascorbate as the reducing agent. Various other potential reducing agents for spin-labels have been tested and found unsuitable for the ESR assays discussed here.
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PMID:A new electron spin resonance assay for membrane asymmetry and entrapped volume of unilamellar lipid vesicles based on photoreduced flavin adenine dinucleotide. 254 82

A superoxide-generating NADPH oxidase was solubilized from phorbol 12-myristate 13-acetate-activated human neutrophils with a mixture of sodium deoxycholate (0.125%, w/v) and Lubrol-PX (0.125%, v/v). The solubilized preparation contained FAD (577 pmol/mg of protein) and cytochrome b-245 (479 pmol/mg of protein) and produced 11.61 mol of O2-./s per mol of cytochrome b (340 nmol of O2-./min per mg of protein). On addition of NADPH, the cytochrome b-245 was reduced by 7.9% and the FAD by 38% in the aerobic steady state; NADH addition caused little steady-state reduction of cytochrome b and FAD. In this preparation, and several others, the measured rate of O2-. production correlated with the turnover of cytochrome b calculated from the extent of cytochrome b-245 reduction under aerobic conditions. Addition of diphenyleneiodonium abolished the reduction of both the FAD and cytochrome b-245 components and inhibited O2-. production. The haem ligand imidazole inhibited O2-. generation and cytochrome b reduction while permitting FAD reduction. These results support the suggestion that the human neutrophil NADPH oxidase has the electron-transport sequence: NADPH----FAD----cytochrome b-245----O2.
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PMID:Studies on the electron-transfer mechanism of the human neutrophil NADPH oxidase. 255 3

The flavoprotein D-amino-acid oxidase was purified to homogeneity from the yeast Rhodotorula gracilis by a highly reproducible procedure. The amino acid composition of the protein was determined; the protein monomer had a molecular mass of 39 kDa and contained one molecule of FAD. The ratio between A274/A455 was about 8.2. D-Amino-acid oxidase from yeast showed typical flavin spectral perturbations on binding of the competitive inhibitor benzoate and was reduced by D-alanine under anaerobiosis. The enzyme reacted readily with sulfite to form a covalent reversible adduct and stabilized the red anionic form of the flavin semiquinone on photoreduction in the presence of 5-deazariboflavin; the 3,4-dihydro-FAD form was not detectable after reduction with sodium borohydride. Thus D-amino-acid oxidase from yeast exhibited most of the general properties of the dehydrogenase/oxidase class of flavoproteins; at the same time, the enzyme showed some peculiar features with respect to the same protein from pig kidney.
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PMID:Properties of D-amino-acid oxidase from Rhodotorula gracilis. 256 32

The respiratory chain of a marine bacterium, Vibrio alginolyticus, required Na+ for maximum activity, and the site of Na+ -dependent activation was localized on the NADH-quinone reductase segment. The Na+ -dependent NADH-quinone reductase extruded Na+ as a direct result of redox reaction. It was composed of three subunits, alpha, beta, and gamma, with apparent Mr of 52, 46, and 32 KDa, respectively. The reduction of ubiquinone-1 to ubiquinol proceeded via ubisemiquinone radicals. The former reaction was catalyzed by the FAD-containing beta subunit. This reaction showed no specific requirement for Na+. For the formation of ubiquinol, the presence of the gamma subunit and the FMN-containing alpha subunit was essential. The latter reaction specifically required Na+ for activity and was strongly inhibited by 2-n-heptyl-4-hydroxyquinoline N-oxide. It was assigned to the coupling site for Na+ transport. The mode of energy coupling of redox-driven Na+ pump was compared with those of decarboxylase- and ATP-driven Na+ pumps found in other bacteria.
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PMID:Sodium-transport NADH-quinone reductase of a marine Vibrio alginolyticus. 268 59


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