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)

A particulate fraction prepared from Mycobacterium phlei grown in a metal-deficient medium exhibited a greatly reduced activity of stearoyl-CoA desaturase compared to that from normally grown cells. Metal deficiency, however, had no effect on the FAD-dependent NADPH-cytochrome C reductase activity, which has been suggested to participate in the desaturation process. When the cells were grown in the deficient medium supplemented with both Fe2+ and Mg2+, the desaturase activity was restored to the normal level. Supplementation with Mg2+ alone promoted growth but did not restore the desaturase activity, whereas Fe2+ alone did cause a significant restoration. Among the various metal ions tested, only Fe2+ and Fe3+ enhanced the formation of desaturase activity in the deficient medium. When added to the assay medium in vitro, Fe2+ and Fe3+ did not stimulate the desaturase activity of the particulate fraction from the deficient cells. Cultivation in the metal-deficient medium had essentially no effect on the levels of cytochromes in the particulate fraction, but dramatically decreased the non-heme iron content and the amount of a high-spin ferric species exhibiting an ESR signal at g=4.3. No labile sulfur could be detected in the normal or metal-deficient particulate fractions. It is concluded that the presence of iron ions in the culture medium is necessary for the synthesis and/or assembly of the terminal portion of the desaturase system.
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PMID:Effect of metal ions in the culture medium on the stearoyl-coenzyme A desaturase activity of Mycobacterium phlei. 0 87

Isolated hepatocytes and liver microsomes incubated with monomethyl-1,1 dimethyl- and 1,2 dimethyl-hydrazines produced free radical intermediates which were detected by ESR spectroscopy by using 4-pyridyl-1-oxide-t-butyl nitrone (4-POBN) as spin trapping agent. The spectral features of the spin adducts derived from all three hydrazine derivatives corresponded to the values reported for the methyl free radical adduct of 4-POBN. In the microsomal preparations inhibitors of the mixed function oxidase system and the destruction of cytochrome P450 by pretreating the rats with CoCl2 all decreased the free radical formation. Methimazole, an inhibitor of FAD-containing monoxygenase system, similarly decreased the activation of 1,1 dimethyl-hydrazine, but not that of monomethyl- and 1,2 dimethyl-hydrazines. The addition to liver microsomes of physiological concentrations of glutathione (GSH) lowered by approx. 80% the intensities of the ESR signals. Consistently, incubation of isolated hepatocytes with methyl-hydrazines decreased the intracellular GSH content, suggesting that GSH can effectively scavenge the methyl free radicals. The results obtained suggest that methyl free radicals could be the alkylating species responsible for the toxic and/or carcinogenic effect of methyl-hydrazines.
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PMID:Free radical activation of monomethyl and dimethyl hydrazines in isolated hepatocytes and liver microsomes. 253 41

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

Two hydrazine spin labels, 1-oxyl-2,2,5,5-tetramethylpyrroline-3-carbonyl ethyl hydrazine and 1-oxyl-2,2,6,6-tetramethylpiperidino-4-hydrazine, were synthesized as probes of the FAD binding site of monoamine oxidase. The reporter nitroxide moiety showed an ESR spectrum classified as partially immobilized which is indicative of FAD near the surface of the enzyme. Attempts to pick up flavin semiquinone or free radical intermediates during substrate oxidation with the spin traps 5,5-dimethyl-1-pyrroline-1-oxidase and phenyl-t-butylnitrone were not successful.
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PMID:ESR analysis of the FAD in bovine liver monoamine oxidase. 631 61

Ferredoxin-dependent glutamate synthase (native enzyme) [EC 1.4.7.1] of spinach has been purified to homogeneity in the presence of 2-oxoglutarate and sodium chloride and the properties of the enzyme have been studied. The molecular weight of the enzyme was estimated to be 140,000 by gel filtration. Subunit analysis by SDS-gel electrophoresis yielded a single protein band whose molecular weight was about 170,000. This purified enzyme showed a flavo-protein-like absorption spectrum having maxima at 279 and 438 nm with shoulders at 415 and 460 nm and a broad band around 360 nm. Fluorometric data indicated the presence of 2 mol of flavin per mol of the enzyme. Preliminary paper chromatography results indicated the presence of FAD and FMN in the purified enzyme. The enzyme also contained 4 mol of acid-labile sulfide and 4 g-atoms iron per mol of enzyme. In the absence of 2-oxoglutarate and/or sodium chloride, the purified enzyme was separated by either DE-52 cellulose chromatography or gel filtration with Ultrogel AcA 34 into two molecular forms (modified enzymes) with considerable inactivation. When reduced methyl viologen plus ferredoxin was used as the electron donor, the purified (native) enzyme showed high ferredoxin-dependent activity with a specific activity of 100 units/mg protein. Methyl viologen-dependent activity was negligible in the absence of ferredoxin. Kinetic properties and results of ESR studies were described. The results indicate that ferredoxin-linked glutamate synthase of spinach leaves is an iron-sulfur flavoprotein.
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PMID:Flavin and iron-sulfur containing ferredoxin-linked glutamate synthase from spinach leaves. 674 4

The spatial localization of the coenzyme FAD in the quaternary structure of the alcohol oxidase from the yeast Pichia pinus was studied by tritium planigraphy and ESR methods. In the present paper we measured the specific radioactivity of FAD labelled as a part of the alcohol oxidase complex. The specific-radioactivity ratio for two FAD portions (FMN and AMP) was calculated. ESR experiments show 4 A (0.4 nm) to be the depth of immersion of paramagnetic isoalloxazines into alcohol oxidase octamer molecules. It is suggested that FAD molecules are bound to the surface of the octamer, rather than to the subunit interfaces. The orientation of the prosthetic group FAD in the alcohol oxidase protein is discussed.
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PMID:Flavin-dependent alcohol oxidase from the yeast Pichia pinus. Spatial localization of the coenzyme FAD in the protein structure: hot-tritium bombardment and ESR experiments. 765 1

The flavins of ferredoxin-NADP+ reductase (FNR) and flavodoxin from the cyanobacterium Anabaena PCC 7119 were obtained in their semiquinone states at pH 7 by photoreduction of the pure proteins in the presence of EDTA and 5-deazariboflavin. For FNR, the ESR signal of the FAD semiquinone was centred at g = 2.005 with linewidths 2.0 mT in H2O and 1.48 mT in D2O. These data are in agreement with those reported for other neutral flavin semiquinones. The linewidths were the same when measured either at X-band (9.35 GHz) or at S-band (4 GHz), indicating that line broadening is due to unresolved nuclear hyperfine couplings, caused in part by exchangeable protons. When the substrate, NADP+, was added to the semiquinone form of the protein no changes in the linewidth or shape of the spectra were detected, but a decrease in the ESR signal due to the FNR semiquinone was observed, consistent with the reduction of NADP+ to NADPH by reduced FNR and, subsequent displacement of the equilibrium. No changes in the shape or linewidth of the FNR ESR signals were observed when photoreduction of FNR was performed in the presence of either flavodoxin or ferredoxin. Electron nuclear double resonance (ENDOR) spectroscopy of FNR semiquinone from Anabaena PCC 7119 provided further information about the interactions of the flavin radical with protons. A group of signals, with couplings of 5-9.5 MHz, is attributed to protons on C6 and on 8-CH3 of the flavin ring. No change in these hyperfine couplings was detected when the protein was studied in D2O, but the coupling Aiso attributed to protons on 8-CH3 decreased from 8.12 MHz to 7.72 MHz in the presence of NADP+. The decrease in the electron spin density distribution on this part of the flavin ring system was attributed to binding of the substrate, polarising the electron density distribution of the flavin towards the pyrimidine ring. A second group of signals was observed, with hyperfine couplings less than 3 MHz, some of which disappeared when the protein was transferred into D2O. Effects of NADP+ binding to the protein were also observed in these weak couplings. These signals are attributed to displaced water protons, or to exchangeable protons from amino acid residues on the protein near the flavin-binding site, involved in substrate stabilization.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Electron spin resonance and electron nuclear double resonance studies of flavoproteins involved in the photosynthetic electron transport in the cyanobacterium Anabaena sp. PCC 7119. 785 33

Choline oxidase catalyzes the four-electron oxidation of choline to glycine betaine, with molecular oxygen acting as primary electron acceptor. Recently, the recombinant enzyme expressed in Escherichia coli was purified to homogeneity and shown to contain FAD in a mixture of oxidized and anionic semiquinone redox states [Fan et al. (2003) Arch. Biochem. Biophys., in press]. In this study, methods have been devised to convert the enzyme-bound flavin semiquinone to oxidized FAD and vice versa, allowing characterization of the resulting forms of choline oxidase. The enzyme-bound oxidized flavin showed typical UV-vis absorbance peaks at 359 and 452 nm (with epsilon(452) = 11.4 M(-1) cm(-1)) and emitted light at 530 nm (with lambda(ex) at 452 nm). The affinity of the enzyme for sulfite was high (with a K(d) value of approximately 50 microM at pH 7 and 15 degrees C), suggesting the presence of a positive charge near the N(1)C(2)=O locus of the flavin. The enzyme-bound anionic flavin semiquinone was unusually insensitive to oxygen or ferricyanide at pH 8 and showed absorbance peaks at 372 and 495 nm (with epsilon(372) = 19.95 M(-1) cm(-1)), maximal fluorescence emission at 454 nm (with lambda(ex) at 372 nm), circular dichroic signals at 370 and 406 nm, and an ESR peak-to-peak line width of 13.9 G. Both UV-vis absorbance studies on the enzyme under turnover with choline and steady-state kinetic data with either choline or betaine aldehyde were consistent with the flavin semiquinone being not involved in catalysis. The pH dependence of the kinetic parameters at varying concentrations of both choline and oxygen indicated that a catalytic base is required for choline oxidation but not for oxygen reduction and that the order of the kinetic steps involving substrate binding and product release is not affected by pH.
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PMID:Spectroscopic and kinetic properties of recombinant choline oxidase from Arthrobacter globiformis. 1469 Apr 28

Mammalian thioredoxin reductase (TrxR) is an NADPH-dependent homodimer with three redox-active centers per subunit: a FAD, an N-terminal domain dithiol (Cys(59)/Cys(64)), and a C-terminal cysteine/selenocysteine motif (Cys(497)/Sec(498)). TrxR has multiple roles in antioxidant defense. Opposing these functions, it may also assume a pro-oxidant role under some conditions. In the absence of its main electron-accepting substrates (e.g. thioredoxin), wild-type TrxR generates superoxide (O ), which was here detected and quantified by ESR spin trapping with 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO). The peroxidase activity of wild-type TrxR efficiently converted the O adduct (DEPMPO/HOO(*)) to the hydroxyl radical adduct (DEPMPO/HO(*)). This peroxidase activity was Sec-dependent, although multiple mutants lacking Sec could still generate O . Variants of TrxR with C59S and/or C64S mutations displayed markedly reduced inherent NADPH oxidase activity, suggesting that the Cys(59)/Cys(64) dithiol is required for O generation and that O is not derived directly from the FAD. Mutations in the Cys(59)/Cys(64) dithiol also blocked the peroxidase and disulfide reductase activities presumably because of an inability to reduce the Cys(497)/Sec(498) active site. Although the bulk of the DEPMPO/HO(*) signal generated by wild-type TrxR was due to its combined NADPH oxidase and Sec-dependent peroxidase activities, additional experiments showed that some free HO(*) could be generated by the enzyme in an H(2)O(2)-dependent and Sec-independent manner. The direct NADPH oxidase and peroxidase activities of TrxR characterized here give insights into the full catalytic potential of this enzyme and may have biological consequences beyond those solely related to its reduction of thioredoxin.
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PMID:The selenium-independent inherent pro-oxidant NADPH oxidase activity of mammalian thioredoxin reductase and its selenium-dependent direct peroxidase activities. 2045 4

Flagellated bacteria modulate their swimming behavior in response to environmental cues through the CheA/CheY signaling pathway. In addition to responding to external chemicals, bacteria also monitor internal conditions that reflect the availability of oxygen, light, and reducing equivalents, in a process termed "energy taxis." In Escherichia coli, the transmembrane receptor Aer is the primary energy sensor for motility. Genetic and physiological data suggest that Aer monitors the electron transport chain through the redox state of its FAD cofactor. However, direct biochemical data correlating FAD redox chemistry with CheA kinase activity have been lacking. Here, we test this hypothesis via functional reconstitution of Aer into nanodiscs. As purified, Aer contains fully oxidized FAD, which can be chemically reduced to the anionic semiquinone (ASQ). Oxidized Aer activates CheA, whereas ASQ Aer reversibly inhibits CheA. Under these conditions, Aer cannot be further reduced to the hydroquinone, in contrast to the proposed Aer signaling model. Pulse ESR spectroscopy of the ASQ corroborates a potential mechanism for signaling in that the resulting distance between the two flavin-binding PAS (Per-Arnt-Sim) domains implies that they tightly sandwich the signal-transducing HAMP domain in the kinase-off state. Aer appears to follow oligomerization patterns observed for related chemoreceptors, as higher loading of Aer dimers into nanodiscs increases kinase activity. These results provide a new methodological platform to study Aer function along with new mechanistic details into its signal transduction process.
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PMID:Bacterial Energy Sensor Aer Modulates the Activity of the Chemotaxis Kinase CheA Based on the Redox State of the Flavin Cofactor. 2780 57


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