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At least 757 records · Page 42Linked to original sources
PubMed1994

Stereospecificity of hydride transfer and substrate specificity for FMN-containing NAD(P)H-flavin oxidoreductase from the luminescent bacterium, Vibrio fischeri ATCC 7744.

The stereospecificity of the hydride transfer in NAD(P)H-flavin reductase reaction of V. fischeri ATCC 7744 was determined by 1H-NMR spectroscopy using stereospecifically labeled reduced beta-nicotinamide adenine dinucleotide (beta-NADH). The recombinant flavoenzyme, purified from E. coli cells, selectively transferred the pro-R hydrogen at the C-4 position of the nicotinamide ring to flavin and is therefore classified as an A-side specific enzyme. Lumiflavin was used for the reductase reaction, but lumichrome and alpha-NADH were not utilized as electron acceptor and donor, respectively.

FMN Reductase↗
PubMed1987

A convenient radiometric assay for flavin-containing monooxygenase activity.

A rapid, convenient assay to determine the activity of the flavin-containing monooxygenase is described. The method is based on direct analysis of quenched incubation mixtures by thin-layer chromatography and utilizes tritiated dimethylaniline as the substrate. The synthesis of the radiolabeled substrate is described. The usefulness of dimethylaniline N-oxide formation as a measure of flavin-containing monooxygenase activity was assessed using the purified hog liver enzyme, hog liver microsomes, and liver microsomes from untreated and phenobarbital-pretreated rats.

Aniline Compounds↗
PubMed1991

Estimation of flavin-containing monooxygenase activities in crude tissue preparations by thiourea-dependent oxidation of thiocholine.

The activity of flavin-containing monooxygenases in microsomes and whole homogenates is readily estimated by following the thiourea-dependent oxidation of thiocholine. NADPH- and oxygen-dependent flavin-containing monooxygenases catalyze the oxidation of thiourea to formamidine sulfenic acid, which oxidizes thiocholine to thiocholine disulfide. The latter reaction is quite rapid and never rate limiting even at concentrations of thiocholine below 30 microM. The loss of thiocholine in deproteinized aliquots of the reaction medium is measured colorimetrically with the thiol reagent, DTNB [5,5'-dithiobis(2-nitrobenzoate)]. In the absence of thiourea, thiocholine is not oxidized and its disulfide is not reduced at a detectable rate even in reactions containing 4-5 mg of liver or kidney homogenate protein per milliliter. In all tissues where both can be measured, rates of thiocholine oxidation and N,N-dimethylaniline N-oxygenation were virtually identical, which suggests that both activities are catalyzed by the same monooxygenase.

Aniline Compounds↗
PubMed1991

Multiple forms of liver microsomal flavin-containing monooxygenases: complete covalent structure of form 2.

Hepatic flavin-containing monooxygenases catalyze NADPH-dependent oxygenation of a wide variety of drugs that possess a nucleophilic heteroatom. Two forms of these enzymes (form 1 and 2) have been isolated from rabbit liver microsomes and partially characterized (Ozols, J., 1989, Biochem. Biophys. Res. Commun. 163, 49-55). The complete amino acid sequence of form 2 is presented here. Sequence determination was achieved by pulsed liquid-phase and solid-phase sequencing of 40 peptides generated by chemical and enzymatic cleavages, including CNBr cleavage of tryptophanyl residues. Form 2 monooxygenase contains 533 amino acid residues and has a molecular weight of 60,089. The COOH terminus of this enzyme is very hydrophobic and presumably functions to anchor the protein to the membrane. Form 2 is readily degraded, since a form lacking residues 1 to 278 and a form without the COOH-terminal segment were also isolated from solubilized membrane preparations. The amino acid sequence of form 2 is 52% identical to that of form 1 and shows 55% identity to the sequence of rabbit lung monooxygenase derived from the cDNA data. The putative FAD and NADP binding segments around residues 9 and 190 are conserved in all three forms. Three variable segments can also be identified in these isoforms. These are residues 308 to 321, residues 408 to 421, and the membrane binding domain, residues 505 to 533. A comparison of the presently limited amino acid sequence data of flavin-containing monooxygenases (FMOs) implies that a particular FMO in different mammalian species may be very similar, but isozymes within a species may exhibit more extensive variability with respect to homology and catalytic activity. This study documents the structural diversity of a second hepatic FMO from rabbit liver and establishes this class of drug-metabolizing enzymes as a family of related proteins.

Amino Acid Sequence↗
PubMed1980

Resonance Raman and coherent anti-stokes Raman scattering spectra of flavin derivatives. Vibrational assignments and the zwitterionic structure of 8-methylamino-riboflavin.

Resonance Raman and coherent anti-stokes Raman scattering spectra are reported for flavins modified by deprotonation at N3 and by CH substitution for N1, N3 and N5, using laser excitation in resonance with the visible electronic transition. Vibrational shifts are used to make qualitative assignments of the observed vibrational modes. Both 8-CH3NH- and ionized 8-OH-riboflavin were found to give markedly different spectra patterns form flavin itself; this observation supports a partial zwitterionic structure for the 8-CH3NH derivatives.

Chemical Phenomena↗
PubMed1983

Guanethidine N-oxide formation as a measure of cellular flavin-containing monooxygenase activity.

The usefulness of guanethidine N-oxide formation as a measure of cellular flavin-containing monooxygenase activity was assessed using the purified hog liver enzyme, rat liver microsomes and hepatocytes. The apparent Km and Vmax for this reaction in hepatocytes were 0.30 +/- 0.20 mM and 0.81 +/- 0.36 nmole per 10(6) cells min-1 respectively. The Km for the purified enzyme was 0.31 mM and the Vmax was 0.56 nmole per microgram enzyme min-1. 2-Diethylaminoethyl-2,2-diphenyl valerate (SKF-525A) at a concentration of 0.5 mM had no effect on guanethidine N-oxide formation by either rat liver microsomes or the purified enzyme. In contrast 2,4-dichloro-6-phenylphenoxyethylamine (DPEA) at the same concentration caused greater than a 100% increase in the microsomal production of guanethidine N-oxide. The tertiary amines imipramine, chloropromazine and methylpyrilene inhibited N-oxide formation by both hepatocytes and the purified enzyme. These data indicate that guanethidine N-oxide formation can be used as a measure of cellular flavin-containing monooxygenase activity.

Animals↗
PubMed1985

Identification of distinct hepatic and pulmonary forms of microsomal flavin-containing monooxygenase in the mouse and rabbit.

The flavin-containing monooxygenase has been purified from mouse and rabbit lung microsomes and shown to be distinct from the flavin-containing monooxygenase found in the liver of the same species. The mouse and rabbit lung monooxygenases have a unique ability to N-oxidize the primary aliphatic amine, n-octylamine, commonly included in microsomal incubations to inhibit cytochrome P-450. In the mouse lung, this compound not only serves as a substrate but is also a positive effector of metabolism. The mouse and rabbit lung enzymes have unusual pH optimum, near 9.8, compared to the liver enzymes which have peaks near pH 8.8. Using antibodies raised in goats, Ouchterlony immunodiffusion analysis indicates that the liver and lung proteins are immunochemically dissimilar.

Animals↗
PubMed1991

Evidence that the protein components of bovine erythrocyte green heme binding protein and flavin reductase are identical.

Bovine erythrocyte green heme binding protein and bovine erythrocyte flavin reductase have been isolated in highly purified forms and subjected to amino acid analysis and N-terminal amino acid sequence analysis. The two proteins possess similar amino acid compositions and identical N-terminal amino acid sequences. Moreover, the two proteins are immunochemically cross-reactive and are indistinguishable when compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by double diffusion technique. This study provides evidence that the protein components of bovine erythrocyte green heme binding protein and flavin reductase are identical.

Amino Acid Sequence↗
PubMed1990

Metabolism of fluperlapine by cytochrome P450-dependent and flavin-dependent monooxygenases in continuous cultures of rat and human cells.

The metabolism of fluperlapine, a neuroleptic dibenzazepine derivative with a N-methyl-piperazinyl substituent, was investigated in continuous cultures of rat and human cells which express various cytochrome P450-dependent monooxygenase activities. The differentiated rat hepatoma cells H4IIEC3/G- and their variants 2sFou and FGC-5 metabolized fluperlapine predominantly by N-oxygenation and only to a minor degree by N-demethylation or glucuronidation of primary phenolic products. Total fluperlapine metabolism in dedifferentiated rat hepatoma cells H5 and partially differentiated human hepatoma cells HepG2 was much smaller than in the differentiated rat hepatoma lines. This was primarily attributable to their low capacity for N-oxygenation. Human lung adenocarcinoma lines NCI-H322 and NCI-H358 formed only trace amounts of fluperlapine N-oxide. Pretreatment of 2sFou cells with benz(a)anthracene, phenobarbital or dexamethasone markedly increased the formation of N-demethylated and glucuronidated products but did not affect the rate of N-oxide formation. Guanethidine and cysteamine, inhibitors of flavin-dependent monooxygenase activity, reduced fluperlapine N-oxidation more strongly than aldrin epoxidation, a marker for cytochrome P450 activity. In contrast, n-octylamine inhibited aldrin epoxidation but was without effect on fluperlapine N-oxygenation. The results suggest that certain cells in continuous culture are capable of expressing flavin-dependent monooxygenase(s) in addition to cytochrome P450-containing monooxygenases. Such cells may offer useful systems for studying the oxidative metabolism of a broad spectrum of xenobiotics and analysing the importance of the two oxygenation reactions for the biological effects of their substrates.

Animals↗
PubMed1990

Antimalarial action of flavin analogues seems not be due to inhibition of glutathione reductase of host erythrocytes.

A series of 10-(4'-chlorophenyl)-3-substituted flavins (1a-f) were examined with respect to their antimalarial properties. They were tested against Plasmodium falciparum in vitro and Plasmodium vinckei vinckei in vivo. The proposition that they might act through glutathione reductase (GR) (EC 1.6.4.2) inhibition has been studied. Inhibition of P. falciparum in vitro by these compounds shows only slight variation between analogues; in contrast, inhibition of human erythrocyte GR by members of the same series is highly variable, indicating that this is probably not their primary mode of antimalarial action. Results of the P. vinckei vinckei screen showed that 10-(4'-chlorophenyl)-3-methyl,3-ethyl and 3-propyl substituted flavins are active in vivo over the dose range screened (10-70 mg/kg).

Animals↗
PubMed1996

Further characterization of rat brain flavin-containing monooxygenase. Metabolism of imipramine to its N-oxide.

Flavin-containing monooxygenase (FMO) activity was compared in rat liver and brain microsomes by estimating the actual amount of imipramine N-oxide relative to the corresponding activity, measured using substrate-stimulated rates of NADPH oxidation. The activities measured as NADPH oxidation rates were significantly higher than those estimated from the N-oxide formed. The brain FMO activity was detectable only in the presence of detergents (sodium cholate or Lubrol PX) or in microsomes that were freeze-thawed several times. The antibody to rabbit pulmonary FMO selectively inhibited imipramine N-oxidation. The antiserum to the rat liver NADPH cytochrome P-450 reductase had no effect on imipramine N-oxidation, indicating the noninvolvement of cytochrome P-450 in the above metabolic pathway. A flavin-containing monooxygenase was partially purified from the rat brain microsomes using sequential chromatography on n-octylamino-Sepharose 4B, DEAE-Sephacel and 2',5'-ADP agarose. The purified FMO was resolved by SDS-PAGE into two bands (approximately 57 and 61 KDa, respectively) both of which cross-reacted with antibody to rabbit pulmonary FMO. The purified enzyme metabolized imipramine and the model substrate methimazole to their respective N-oxide and S-oxides.

Animals↗
PubMed1995

Flavin-containing monooxygenase mediated metabolism of psychoactive drugs by human brain microsomes.

Flavin-containing monooxygenases (FMO) catalyze the oxidation of certain xenobiotics and drugs which contain a nucleophilic heteroatom. Here we report the first assessment of human brain flavin-containing monooxygenase from tissues obtained at autopsy from seven traffic accident victims. Human brain microsomes catalyzed the S-oxidation or N-oxidation of model substrates methimazole and N,N-dimethylaniline, respectively. The psychoactive drugs chlorpromazine, imipramine and fluoxetine, were also metabolized by human brain FMO. 'Western' immunoblot analyses revealed immunological cross-reactivity of the human brain FMO with rabbit pulmonary FMO. Immunocytochemistry further revealed the localization of the FMO predominantly in the neuronal cell bodies in the magnocellular reticular nuclei, colliculi and substantia nigra. Human brain clearly contains an active FMO system, and it is conceivable that such enzyme(s) are significantly involved in the local metabolism and modulation of pharmacological effects of psychoactive drugs.

Adult↗
PubMed1996

Xenobiotic biotransforming enzymes in the central nervous system: an isoform of flavin-containing monooxygenase (FMO4) is expressed in rabbit brain.

The flavin-containing monooxygenase (FMO, EC 1.14.13.8) is involved in the metabolism of a number of important xenobiotics including many which affect the central nervous system (CNS). Recently, reports in the literature concerning the amount, activity, location, and isozyme characteristics of this enzyme in the brain have presented conflicting evidence. In order to resolve some of the controversy surrounding FMO in the brain, a highly sensitive method for the detection of flavin-containing monooxygenase (FMO) mRNA in whole brain was employed. A poorly conserved region of FMO transcripts was used to design five sets of oligonucleotide primers. Each primer set was specific for one of the five currently known isoforms of FMO. Four and five isoforms, respectively, are expressed in rabbit liver and kidney, as determined by reverse transcription-polymerase chain reaction. However, only one set of primers amplified a specific rabbit brain cDNA fragment. The sequence of the amplification produced affirmed its identity as a segment of FMO4 cDNA. Thus, the FMO of rabbit brain may consist of a single, as yet uncharacterized isozyme and, contrary to several recent reports, is likely to be expressed at low levels.

Animals↗
PubMed1995

Expression of the bile acid-inducible NADH:flavin oxidoreductase gene of Eubacterium sp. VPI 12708 in Escherichia coli.

The intestinal microorganism Eubacterium sp. VPI 12708 synthesizes a bile acid-inducible NADH:flavin oxidoreductase (NADH:FOR) which presumably functions in the 7 alpha-dehydroxylation of cholic acid to deoxycholic acid. The baiH gene encoding NADH:FOR was subcloned into an IPTG-inducible expression vector, pBaiH2.2. Escherichia coli DH5 alpha cells transformed with pBaiH2.2 expressed 10-fold higher levels of NADH:FOR upon induction with IPTG than did Eubacterium sp. VPI 12708 cells induced with cholic acid. The NADH:FOR produced by E. coli DH5 alpha(pBaiH2.2) was purified to > 95% electrophoretic homogeneity in three steps. The purified NADH:FOR was similar to that of Eubacterium sp. VPI 12708 in subunit and native M(r) (ca. 72,000 and 210,000, respectively), pH optimum, sensitivity to inhibitors, and electron acceptor specificity. It contained 1 mol of FAD, up to 2 mol of iron, and 1 mol of copper per mol of subunit. The enzyme reduced synthetic quinones, dyes, flavins, and O2 with NADH as the electron donor, but did not reduce disulfide compounds, various unsaturated bile acids, cytochrome c, physiological quinones, or cell fractions from Eubacterium sp. VPI 12708. Addition of purified NADH:FOR to Eubacterium sp. VPI 12708 cell extracts altered the balance of oxidized and reduced bile acid intermediates produced during cholic acid 7 alpha-dehydroxylation, suggesting that the enzyme may regulate the cellular ratio of NAD to NADH.

Amino Acid Sequence↗
PubMed2005

The flavin reductase ActVB from Streptomyces coelicolor: characterization of the electron transferase activity of the flavoprotein form.

The flavin reductase ActVB is involved in the last step of actinorhodin biosynthesis in Streptomyces coelicolor. Although ActVB can be isolated with some FMN bound, this form was not involved in the flavin reductase activity. By studying the ferric reductase activity of ActVB, we show that its FMN-bound form exhibits a proper enzymatic activity of reduction of iron complexes by NADH. This shows that ActVB active site exhibits a dual property with regard to the FMN. It can use it as a substrate that goes in and off the active site or as a cofactor to provide an electron transferase activity to the polypeptide.

Bacterial Proteins↗
PubMed2006

Mutation, polymorphism and perspectives for the future of human flavin-containing monooxygenase 3.

Flavin-containing monooxygenases (FMOs) catalyze NADPH-dependent monooxygenation of soft-nucleophilic nitrogen, sulfur, and phosphorous atoms contained within various drugs, pesticides, and xenobiotics. Flavin-containing monooxygenase 3 (FMO3) is responsible for the majority of FMO-mediated xenobiotic metabolism in the adult human liver. Mutations in the FMO3 gene can result in defective trimethylamine (TMA) N-oxygenation, which gives rise to the disorder known as trimethylaminuria (TMAU) or "fish-odour syndrome". To date 18 mutations of FMO3 gene have been reported that cause TMAU, and polymorphic variants of the gene have also been identified. Interindividual variability in the expression of FMO3 may affect drug and foreign chemical metabolism in the liver and other tissues. It is important therefore to study how base sequence variation of the FMO3 gene might affect the ability of individuals and different ethnic population groups to deal with the variety of environmental chemicals and pharmaceutical products that are substrates for FMO3.

Adult↗
PubMed1997

Antibodies to a synthetic peptide that react with flavin-containing monooxygenase (HLFMO3) in human hepatic microsomes.

Flavin-containing monooxygenases (FMOs) catalyze the oxidation of a diverse array of xenobiotic compounds. The purpose of this investigation was to develop a specific immunological probe to human hepatic flavin-containing monooxygenase (HLFMO3). An oligopetide corresponding to amino acid residues 257-270 of HLFMO3 was coupled to keyhole limpet hemocyanin (KLH) through the sulfhydryl group of a cysteine residue added to the amino-terminus of the peptide. This peptide-KLH conjugate was used to generate a polyclonal antibody. The resulting immunoglobulin showed specific Western blot reactivity with HLFMO3 protein in human hepatic microsomes, the same protein that is recognized by a polyclonal antibody directed against macaque liver FMO. These findings demonstrate that an antibody directed against a synthetic peptide derived from HLFMO3 can be easily produced in large quantities and used in studies for the immunodetection and immunoquantification of HLFMO3. This is also the first antipeptide antibody directed against an FMO of any species.

Animals↗
PubMed1982

Proximity of reactive cysteine residue and flavin in Escherichia coli pyruvate oxidase as estimated by fluorescence energy transfer.

Pyruvate oxidase of Escherichia coli possesses a reactive cysteine residue believed to be associated with the thiamin pyrophosphate (TPP) binding site. This residue is not reactive in the presence of TPP. Exposure of the enzyme to cysteine-directed fluorescent reagents results in the formation of fluorescent protein conjugates. Although these reagents do not react solely with the TPP-protectable cysteine residue, the fluorescence emission spectrum of a probe attached to this residue can be obtained by a difference technique. It was determined that the fluorescence emission of probes at the TPP-protectable site is very low due to energy transfer to the FAD coenzyme and that this fluorescence is greatly enhanced upon reduction or extraction of the flavin. Application of fluorescence energy transfer theory enabled the determination of an upper limit for the distance between the probes at the TPP-protectable site and the flavin adenine dinucleotide (FAD) (roughly 20 A). Thus, the TPP binding site and the FAD coenzyme are likely in close proximity.

Anilino Naphthalenesulfonates↗