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Sample records for two-domain multicopper oxidase

  1. Diversity of Two-Domain Laccase-Like Multicopper Oxidase Genes in Streptomyces spp.: Identification of Genes Potentially Involved in Extracellular Activities and Lignocellulose Degradation during Composting of Agricultural Waste

    Science.gov (United States)

    Lu, Lunhui; Zhang, Jiachao; Chen, Anwei; Chen, Ming; Jiang, Min; Yuan, Yujie; Wu, Haipeng; Lai, Mingyong; He, Yibin

    2014-01-01

    Traditional three-domain fungal and bacterial laccases have been extensively studied for their significance in various biotechnological applications. Growing molecular evidence points to a wide occurrence of more recently recognized two-domain laccase-like multicopper oxidase (LMCO) genes in Streptomyces spp. However, the current knowledge about their ecological role and distribution in natural or artificial ecosystems is insufficient. The aim of this study was to investigate the diversity and composition of Streptomyces two-domain LMCO genes in agricultural waste composting, which will contribute to the understanding of the ecological function of Streptomyces two-domain LMCOs with potential extracellular activity and ligninolytic capacity. A new specific PCR primer pair was designed to target the two conserved copper binding regions of Streptomyces two-domain LMCO genes. The obtained sequences mainly clustered with Streptomyces coelicolor, Streptomyces violaceusniger, and Streptomyces griseus. Gene libraries retrieved from six composting samples revealed high diversity and a rapid succession of Streptomyces two-domain LMCO genes during composting. The obtained sequence types cluster in 8 distinct clades, most of which are homologous with Streptomyces two-domain LMCO genes, but the sequences of clades III and VIII do not match with any reference sequence of known streptomycetes. Both lignocellulose degradation rates and phenol oxidase activity at pH 8.0 in the composting process were found to be positively associated with the abundance of Streptomyces two-domain LMCO genes. These observations provide important clues that Streptomyces two-domain LMCOs are potentially involved in bacterial extracellular phenol oxidase activities and lignocellulose breakdown during agricultural waste composting. PMID:24657870

  2. Multi-Copper Oxidases and Human Iron Metabolism

    Science.gov (United States)

    Vashchenko, Ganna; MacGillivray, Ross T. A.

    2013-01-01

    Multi-copper oxidases (MCOs) are a small group of enzymes that oxidize their substrate with the concomitant reduction of dioxygen to two water molecules. Generally, multi-copper oxidases are promiscuous with regards to their reducing substrates and are capable of performing various functions in different species. To date, three multi-copper oxidases have been detected in humans—ceruloplasmin, hephaestin and zyklopen. Each of these enzymes has a high specificity towards iron with the resulting ferroxidase activity being associated with ferroportin, the only known iron exporter protein in humans. Ferroportin exports iron as Fe2+, but transferrin, the major iron transporter protein of blood, can bind only Fe3+ effectively. Iron oxidation in enterocytes is mediated mainly by hephaestin thus allowing dietary iron to enter the bloodstream. Zyklopen is involved in iron efflux from placental trophoblasts during iron transfer from mother to fetus. Release of iron from the liver relies on ferroportin and the ferroxidase activity of ceruloplasmin which is found in blood in a soluble form. Ceruloplasmin, hephaestin and zyklopen show distinctive expression patterns and have unique mechanisms for regulating their expression. These features of human multi-copper ferroxidases can serve as a basis for the precise control of iron efflux in different tissues. In this manuscript, we review the biochemical and biological properties of the three human MCOs and discuss their potential roles in human iron homeostasis. PMID:23807651

  3. Biocatalytic potential of laccase-like multicopper oxidases from Aspergillus niger

    NARCIS (Netherlands)

    Tamayo Ramos, J.A.; Berkel, van W.J.H.; Graaff, de L.H.

    2012-01-01

    BACKGROUND: Laccase-like multicopper oxidases have been reported in several Aspergillus species but they remain uncharacterized. The biocatalytic potential of the Aspergillus niger fungal pigment multicopper oxidases McoA and McoB and ascomycete laccase McoG was investigated. RESULTS: The

  4. Biocatalytic potential of laccase-like multicopper oxidases from Aspergillus niger

    Directory of Open Access Journals (Sweden)

    Tamayo-Ramos Juan Antonio

    2012-12-01

    Full Text Available Abstract Background Laccase-like multicopper oxidases have been reported in several Aspergillus species but they remain uncharacterized. The biocatalytic potential of the Aspergillus niger fungal pigment multicopper oxidases McoA and McoB and ascomycete laccase McoG was investigated. Results The laccase-like multicopper oxidases McoA, McoB and McoG from the commonly used cell factory Aspergillus niger were homologously expressed, purified and analyzed for their biocatalytic potential. All three recombinant enzymes were monomers with apparent molecular masses ranging from 80 to 110 kDa. McoA and McoG resulted to be blue, whereas McoB was yellow. The newly obtained oxidases displayed strongly different activities towards aromatic compounds and synthetic dyes. McoB exhibited high catalytic efficiency with N,N-dimethyl-p-phenylenediamine (DMPPDA and 2,2-azino-di(3-ethylbenzthiazoline sulfonic acid (ABTS, and appeared to be a promising biocatalyst. Besides oxidizing a variety of phenolic compounds, McoB catalyzed successfully the decolorization and detoxification of the widely used textile dye malachite green. Conclusions The A. niger McoA, McoB, and McoG enzymes showed clearly different catalytic properties. Yellow McoB showed broad substrate specificity, catalyzing the oxidation of several phenolic compounds commonly present in different industrial effluents. It also harbored high decolorization and detoxification activity with the synthetic dye malachite green, showing to have an interesting potential as a new industrial biocatalyst.

  5. TtMCO: A highly thermostable laccase-like multicopper oxidase from the thermophilic Thermobaculum terrenum

    DEFF Research Database (Denmark)

    Brander, Søren; Mikkelsen, Jørn Dalgaard; Kepp, Kasper Planeta

    2015-01-01

    This paper reports the identification, heterologous expression in Escherichia coli and characterization of TtMCO from the thermophilic bacterium Thermobaculum terrenum, the first laccase-like multi-copper oxidase (LMCO) from the distinct Phylum Chloroflexi. TtMCO has only 39% identity to its...... closest characterized homologue, CotA from Bacillus subtilis, but sequence and spectrophotometry confirmed copper coordination similar to that of LMCOs. TtMCO is extremely thermophilic with a half-time of inactivation of 2.24 days at 70 degrees C and 350 min at 80°C and pH 7, consistent...

  6. A multicopper oxidase-related protein is essential for insect viability, longevity and ovary development.

    Science.gov (United States)

    Peng, Zeyu; Green, Peter G; Arakane, Yasuyuki; Kanost, Michael R; Gorman, Maureen J

    2014-01-01

    Typical multicopper oxidases (MCOs) have ten conserved histidines and one conserved cysteine that coordinate four copper atoms. These copper ions are required for oxidase activity. During our studies of insect MCOs, we discovered a gene that we named multicopper oxidase-related protein (MCORP). MCORPs share sequence similarity with MCOs, but lack many of the copper-coordinating residues. We identified MCORP orthologs in many insect species, but not in other invertebrates or vertebrates. We predicted that MCORPs would lack oxidase activity due to the absence of copper-coordinating residues. To test this prediction, we purified recombinant Tribolium castaneum (red flour beetle) MCORP and analyzed its enzymatic activity using a variety of substrates. As expected, no oxidase activity was detected. To study MCORP function in vivo, we analyzed expression profiles of TcMCORP and Anopheles gambiae (African malaria mosquito) MCORP, and assessed RNAi-mediated knockdown phenotypes. We found that both MCORPs are constitutively expressed at a low level in all of the tissues we analyzed. Injection of TcMCORP dsRNA into larvae resulted in 100% mortality prior to adult eclosion, with death occurring mainly during the pharate pupal stage or late pharate adult stage. Injection of TcMCORP dsRNA into pharate pupae resulted in the death of approximately 20% of the treated insects during the pupal to adult transition and a greatly shortened life span for the remaining insects. In addition, knockdown of TcMCORP in females prevented oocyte maturation and, thus, greatly decreased the number of eggs laid. These results indicate that TcMCORP is an essential gene and that its function is required for reproduction. An understanding of the role MCORP plays in insect physiology may help to develop new strategies for controlling insect pests.

  7. Catalytic Cycle of Multicopper Oxidases Studied by Combined Quantum- and Molecular-Mechanical Free-Energy Perturbation Methods

    Czech Academy of Sciences Publication Activity Database

    Li, J.; Farrokhnia, M.; Rulíšek, Lubomír; Ryde, U.

    2015-01-01

    Roč. 119, č. 26 (2015), s. 8268-8284 ISSN 1520-6106 R&D Projects: GA ČR(CZ) GA14-31419S Institutional support: RVO:61388963 Keywords : multi-copper oxidases * QM/MM calculations * reduction potentials * pKa acidity constants Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.187, year: 2015

  8. Theoretical studies of the active-site structure, spectroscopic and thermodynamic properties, and reaction mechanism of multicopper oxidases

    Czech Academy of Sciences Publication Activity Database

    Rulíšek, Lubomír; Ryde, U.

    2013-01-01

    Roč. 257, č. 2 (2013), s. 445-458 ISSN 0010-8545 Institutional support: RVO:61388963 Keywords : multi-copper oxidases * QM/MM calculations * multireference calculations * the oretical spectroscopy Subject RIV: CA - Inorganic Chemistry Impact factor: 12.098, year: 2013

  9. Evolution of multicopper oxidase genes in coprophilous and non-coprophilous members of the order sordariales.

    Science.gov (United States)

    Pöggeler, Stefanie

    2011-04-01

    Multicopper oxidases (MCO) catalyze the biological oxidation of various aromatic substrates and have been identified in plants, insects, bacteria, and wood rotting fungi. In nature, they are involved in biodegradation of biopolymers such as lignin and humic compounds, but have also been tested for various industrial applications. In fungi, MCOs have been shown to play important roles during their life cycles, such as in fruiting body formation, pigment formation and pathogenicity. Coprophilous fungi, which grow on the dung of herbivores, appear to encode an unexpectedly high number of enzymes capable of at least partly degrading lignin. This study compared the MCO-coding capacity of the coprophilous filamentous ascomycetes Podospora anserina and Sordaria macrospora with closely related non-coprophilous members of the order Sordariales. An increase of MCO genes in coprophilic members of the Sordariales most probably occurred by gene duplication and horizontal gene transfer events.

  10. Biogenic manganese oxide nanoparticle formation by a multimeric multicopper oxidase Mnx

    Energy Technology Data Exchange (ETDEWEB)

    Romano, Christine A.; Zhou, Mowei; Song, Yang; Wysocki, Vicki H.; Dohnalkova, Alice C.; Kovarik, Libor; Paša-Tolić, Ljiljana; Tebo, Bradley M.

    2017-09-29

    Bacteria that produce Mn oxides are extraordinarily skilled engineers of nanomaterials that contribute significantly to global biogeochemical cycles. Their enzyme-based reaction mechanisms may be genetically tailored for environmental remediation applications or bioenergy production. However, significant challenges exist for structural characterization of the enzymes responsible for biomineralization. The active Mn oxidase, Mnx, in Bacillus sp. PL-12 is a complex composed of a multicopper oxidase (MCO), MnxG, and two accessory proteins MnxE and MnxF. MnxG shares sequence similarity with other, structurally characterized MCOs. However, MnxE and MnxF have no similarity to any characterized proteins. The ~200 kDa complex has been recalcitrant to crystallization, so its structure is unknown. In this study, native mass spectrometry defines the subunit topology and copper binding of the Mnx complex, while high resolution electron microscopy visualizes the protein and nascent Mn oxide minerals. These data provide critical structural information for conceptualizing how Mnx produces nanoparticulate Mn oxides.

  11. Biogenic manganese oxide nanoparticle formation by a multimeric multicopper oxidase Mnx.

    Science.gov (United States)

    Romano, Christine A; Zhou, Mowei; Song, Yang; Wysocki, Vicki H; Dohnalkova, Alice C; Kovarik, Libor; Paša-Tolić, Ljiljana; Tebo, Bradley M

    2017-09-29

    Bacteria that produce Mn oxides are extraordinarily skilled engineers of nanomaterials that contribute significantly to global biogeochemical cycles. Their enzyme-based reaction mechanisms may be genetically tailored for environmental remediation applications or bioenergy production. However, significant challenges exist for structural characterization of the enzymes responsible for biomineralization. The active Mn oxidase in Bacillus sp. PL-12, Mnx, is a complex composed of a multicopper oxidase (MCO), MnxG, and two accessory proteins, MnxE and MnxF. MnxG shares sequence similarity with other, structurally characterized MCOs. MnxE and MnxF have no similarity to any characterized proteins. The ~200 kDa complex has been recalcitrant to crystallization, so its structure is unknown. Here, we show that native mass spectrometry defines the subunit topology and copper binding of Mnx, while high-resolution electron microscopy visualizes the protein and nascent Mn oxide minerals. These data provide critical structural information for understanding Mn biomineralization by such unexplored enzymes.Significant challenges exist for structural characterization of enzymes responsible for biomineralization. Here the authors show that native mass spectrometry and high resolution electron microscopy can define the subunit topology and copper binding of a manganese oxidizing complex, and describe early stage formation of its mineral products.

  12. Characterization of a multicopper oxidase gene cluster in Phanerochaete chrysosporium and evidence of altered splicing of the mco transcripts

    Science.gov (United States)

    Luis F. Larrondo; Bernardo Gonzalez; Dan Cullen; Rafael Vicuna

    2004-01-01

    A cluster of multicopper oxidase genes (mco1, mco2, mco3, mco4) from the lignin-degrading basidiomycete Phanerochaete chrysosporium is described. The four genes share the same transcriptional orientation within a 25 kb region. mco1, mco2 and mco3 are tightly grouped, with intergenic regions of 2.3 and 0.8 kb, respectively, whereas mco4 is located 11 kb upstream of mco1...

  13. Isolation and genome sequencing of four Arctic marine Psychrobacter strains exhibiting multicopper oxidase activity.

    Science.gov (United States)

    Moghadam, Morteza Shojaei; Albersmeier, Andreas; Winkler, Anika; Cimmino, Lorenzo; Rise, Kjersti; Hohmann-Marriott, Martin Frank; Kalinowski, Jörn; Rückert, Christian; Wentzel, Alexander; Lale, Rahmi

    2016-02-16

    Marine cold-temperature environments are an invaluable source of psychrophilic microbial life for new biodiscoveries. An Arctic marine bacterial strain collection was established consisting of 1448 individual isolates originating from biota, water and sediment samples taken at a various depth in the Barents Sea, North of mainland Norway, with an all year round seawater temperature of 4 °C. The entire collection was subjected to high-throughput screening for detection of extracellular laccase activity with guaiacol as a substrate. In total, 13 laccase-positive isolates were identified, all belonging to the Psychrobacter genus. From the most diverse four strains, based on 16S rRNA gene sequence analysis, all originating from the same Botryllus sp. colonial ascidian tunicate sample, genomic DNA was isolated and genome sequenced using a combined approach of whole genome shotgun and 8 kb mate-pair library sequencing on an Illumina MiSeq platform. The genomes were assembled and revealed genome sizes between 3.29 and 3.52 Mbp with an average G + C content of around 42%, with one to seven plasmids present in the four strains. Bioinformatics based genome mining was performed to describe the metabolic potential of these four strains and to identify gene candidates potentially responsible for the observed laccase-positive phenotype. Up to two different laccase-like multicopper oxidase (LMCO) encoding gene candidates were identified in each of the four strains. Heterologous expression of P11F6-LMCO and P11G5-LMCO2 in Escherichia coli BL21 (DE3) resulted in recombinant proteins exhibiting 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) and guaiacol oxidizing activity. Thirteen Psychrobacter species with laccase-positive phenotype were isolated from a collection of Arctic marine bacteria. Four of the isolates were genome sequenced. The overall genome features were similar to other publicly available Psychrobacter genome sequences except for P11G5 harboring seven

  14. Engineering Klebsiella sp. 601 multicopper oxidase enhances the catalytic efficiency towards phenolic substrates.

    Science.gov (United States)

    Li, Yadong; Gong, Zijun; Li, Xin; Li, Yang; Wang, Xing-Guo

    2011-05-31

    Structural comparison between bacterial CueO and fungal laccases has suggested that a charged residue Glu (E106) in CueO replaces the corresponding residue Phe in fungal laccases at the gate of the tunnel connecting type II copper to the protein surface and an extra α-helix (L351-G378) near the type I copper site covers the substrate binding pocket and might compromise the electron transfer from substrate to type I copper. To test this hypothesis, several mutants were made in Klebsiella sp. 601 multicopper oxidase, which is highly homologous to E. coli CueO with a similarity of 90% and an identity of 78%. The E106F mutant gave smaller K(m) (2.4-7 fold) and k(cat) (1-4.4 fold) values for all three substrates DMP, ABTS and SGZ as compared with those for the wild-type enzyme. Its slightly larger k(cat)/K(m) values for three substrates mainly come from the decreased K(m). Deleting α-helix (L351-G378) resulted in the formation of inactive inclusion body when the mutant (Δ)α351-378 was expressed in E. coli. Another mutant α351-380M was then made via substitution of seven amino acid residues in the α-helix (L351-G378) region. The α351-380M mutant was active, and displayed a far-UV CD spectrum markedly different from that for wild-type enzyme. Kinetic studies showed the α351-380M mutant gave very low K(m) values for DMP, ABTS and SGZ, 4.5-, 1.9- and 7-fold less than those for the wild type. In addition, k(cat)/K(m) values were increased, 9.4-fold for DMP, similar for ABTS and 3-fold for SGZ. The Glu residue at position 106 appears not to be the only factor affecting the copper binding, and it may also play a role in maintaining enzyme conformation. The α-helix (L351-G378) may not only block access to the type I copper site but also play a role in substrate specificities of bacterial MCOs. The α351-380M mutant catalyzing oxidation of the phenolic substrate DMP effectively would be very useful in green chemistry.

  15. Magnetic Ganoderma lucidum spore microspheres: A novel material to immobilize CotA multicopper oxidase for dye decolorization

    Energy Technology Data Exchange (ETDEWEB)

    Fan, Lili [School of Chemistry and Chemical Engineering, Harbin Institute of Technology, Harbin 150001 (China); Wang, Yan, E-mail: wangy_msn@hit.edu.cn [School of Chemistry and Chemical Engineering, Harbin Institute of Technology, Harbin 150001 (China); Zhao, Min [College of Life Science, Northeast Forestry University, Harbin 150040 (China); Song, Jinzhu [School of Life Science and Technology, Harbin Institute of Technology, Harbin 150001 (China); Wang, Jueyu; Jin, Zijing [College of Life Science, Northeast Forestry University, Harbin 150040 (China)

    2016-08-05

    Highlights: • Hollow microspheres were obtained from Ganoderma lucidum spores. • Novel magnetic microspheres were prepared by load hollow spore microspheres with Fe{sub 3}O{sub 4} nanoparticles. • CotA multicopper oxidase was immobilized on the magnetic spore microspheres for indigo carmine decolorization. • The immobilized CotA displayed higher decolorization capability and reusability. - Abstract: In this study, hollow microspheres were obtained from Ganoderma lucidum spores. Then the hollow microspheres were loaded with Fe{sub 3}O{sub 4} nanoparticles to prepare novel magnetic spore microspheres. TEM images and X-ray diffractometry demonstrated that the Fe{sub 3}O{sub 4} nanoparticles were incorporated throughout the spore microsphere. CotA multicopper oxidase was chosen as biomacromolecule to study the loading ability of the magnetic spore microspheres. The combination of the CotA enzyme with the microsphere was observed by laser scanning confocal microscope. The loaded amount of CotA on the microspheres was 75 mg/g when the CotA concentration was 1.2 mg/mL and the activity recovery of the immobilized CotA was 81%. The magnetic microspheres loaded with CotA, which can be easily and quickly recovered by an external magnetic field, were used for dye decolorization. After 1 h decolorization, 99% of the indigo carmine has been removed by 10 mg microspheres. In addition, the immobilized CotA retained 75% of activity after 10 consecutive cycles, which indicated that the magnetic spore microspheres are good support material for immobilization of the enzyme.

  16. Magnetic Ganoderma lucidum spore microspheres: A novel material to immobilize CotA multicopper oxidase for dye decolorization

    International Nuclear Information System (INIS)

    Fan, Lili; Wang, Yan; Zhao, Min; Song, Jinzhu; Wang, Jueyu; Jin, Zijing

    2016-01-01

    Highlights: • Hollow microspheres were obtained from Ganoderma lucidum spores. • Novel magnetic microspheres were prepared by load hollow spore microspheres with Fe_3O_4 nanoparticles. • CotA multicopper oxidase was immobilized on the magnetic spore microspheres for indigo carmine decolorization. • The immobilized CotA displayed higher decolorization capability and reusability. - Abstract: In this study, hollow microspheres were obtained from Ganoderma lucidum spores. Then the hollow microspheres were loaded with Fe_3O_4 nanoparticles to prepare novel magnetic spore microspheres. TEM images and X-ray diffractometry demonstrated that the Fe_3O_4 nanoparticles were incorporated throughout the spore microsphere. CotA multicopper oxidase was chosen as biomacromolecule to study the loading ability of the magnetic spore microspheres. The combination of the CotA enzyme with the microsphere was observed by laser scanning confocal microscope. The loaded amount of CotA on the microspheres was 75 mg/g when the CotA concentration was 1.2 mg/mL and the activity recovery of the immobilized CotA was 81%. The magnetic microspheres loaded with CotA, which can be easily and quickly recovered by an external magnetic field, were used for dye decolorization. After 1 h decolorization, 99% of the indigo carmine has been removed by 10 mg microspheres. In addition, the immobilized CotA retained 75% of activity after 10 consecutive cycles, which indicated that the magnetic spore microspheres are good support material for immobilization of the enzyme.

  17. Design of carbon nanotube-based gas-diffusion cathode for O{sub 2} reduction by multicopper oxidases

    Energy Technology Data Exchange (ETDEWEB)

    Lau, Carolin; Adkins, Emily R.; Atanassov, Plamen [University of New Mexico, Center for Emerging Energy Technologies, Albuquerque, NM (United States); Ramasamy, Ramaraja P. [Microbiology and Applied Biochemistry, Airbase Sciences, Air Force Research Laboratory, Tyndall Air Force Base, FL (United States); Nano-Electrochemistry Laboratory, Faculty of Engineering, University of Georgia, Athens, GA (United States); Luckarift, Heather R.; Johnson, Glenn R. [Microbiology and Applied Biochemistry, Airbase Sciences, Air Force Research Laboratory, Tyndall Air Force Base, FL (United States)

    2012-01-15

    Multicopper oxidases, such as laccase or bilirubin oxidase, are known to reduce molecular oxygen at very high redox potentials, which makes them attractive biocatalysts for enzymatic cathodes in biological fuel cells. By designing an enzymatic gas-diffusion electrode, molecular oxygen can be supplied through the gaseous phase, avoiding solubility and diffusion limitations typically associated with liquid electrolytes. In doing so, the current density of enzymatic cathodes can theoretically be enhanced. This publication presents a material study of carbon/Teflon composites that aim to optimize the functionality of the gas-diffusion and catalytic layers for application in enzymatic systems. The modification of the catalytic layer with multiwalled carbon nanotubes, for example, creates the basis for stronger {pi}-{pi} stacking interactions through tethered enzymatic linkers, such as pyrenes or perylene derivates. Cyclic voltammograms show the effective direct electron contact of laccase with carbon nanotube-modified electrodes via tethered crosslinking molecules as a model system. The polarization behavior of laccase-modified gas-diffusion electrodes reveals open-circuit potentials of +550 mV (versus Ag/AgCl) and current densities approaching 0.5 mA cm{sup 2} (at zero potential) in air-breathing mode. (Copyright copyright 2012 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  18. Diffusion-controlled oxygen reduction on multi-copper oxidase-adsorbed carbon aerogel electrodes without mediator

    Energy Technology Data Exchange (ETDEWEB)

    Tsujimura, S.; Kamitaka, Y.; Kano, K. [Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto (Japan)

    2007-12-15

    Bioelectrocatalytic reduction of O{sub 2} into water was archived at diffusion-controlled rate by using enzymes (laccase from Trametes sp. and bilirubin oxidase from Myrothecium verrucaria, which belong to the family of multi-copper oxidase) adsorbed on mesoporous carbon aerogel particle without a mediator. The current density was predominantly controlled by the diffusion of dissolved O{sub 2} in rotating-disk electrode experiments, and reached a value as large as 10 mA cm{sup -2} at 1 atm O{sub 2}, 25 C, and 8,000 rpm on the laccase-adsorbed electrode. The overpotential of the bioelectrocatalytic reduction of O{sub 2} was 0.4-0.55 V smaller than that observed on a Pt disk electrode. Without any optimization, the laccase-adsorbed biocathode showed stable current intensity of the O{sub 2} reduction in an air-saturated buffer at least for 10 days under continuous flow system. (Abstract Copyright [2007], Wiley Periodicals, Inc.)

  19. A multicopper oxidase is essential for manganese oxidation and laccase-like activity in Pedomicrobium sp. ACM 3067.

    Science.gov (United States)

    Ridge, Justin P; Lin, Marianne; Larsen, Eloise I; Fegan, Mark; McEwan, Alastair G; Sly, Lindsay I

    2007-04-01

    Pedomicrobium sp. ACM 3067 is a budding-hyphal bacterium belonging to the alpha-Proteobacteria which is able to oxidize soluble Mn2+ to insoluble manganese oxide. A cosmid, from a whole-genome library, containing the putative genes responsible for manganese oxidation was identified and a primer-walking approach yielded 4350 bp of novel sequence. Analysis of this sequence showed the presence of a predicted three-gene operon, moxCBA. The moxA gene product showed homology to multicopper oxidases (MCOs) and contained the characteristic four copper-binding motifs (A, B, C and D) common to MCOs. An insertion mutation of moxA showed that this gene was essential for both manganese oxidation and laccase-like activity. The moxB gene product showed homology to a family of outer membrane proteins which are essential for Type I secretion in Gram-negative bacteria. moxBA has not been observed in other manganese-oxidizing bacteria but homologues were identified in the genomes of several bacteria including Sinorhizobium meliloti 1021 and Agrobacterium tumefaciens C58. These results suggest that moxBA and its homologues constitute a family of genes encoding an MCO and a predicted component of the Type I secretion system.

  20. Mn(II,III) oxidation and MnO2 mineralization by an expressed bacterial multicopper oxidase

    Science.gov (United States)

    Butterfield, Cristina N.; Soldatova, Alexandra V.; Lee, Sung-Woo; Spiro, Thomas G.; Tebo, Bradley M.

    2013-01-01

    Reactive Mn(IV) oxide minerals are ubiquitous in the environment and control the bioavailability and distribution of many toxic and essential elements and organic compounds. Their formation is thought to be dependent on microbial enzymes, because spontaneous Mn(II) to Mn(IV) oxidation is slow. Several species of marine Bacillus spores oxidize Mn(II) on their exosporium, the outermost layer of the spore, encrusting them with Mn(IV) oxides. Molecular studies have identified the mnx (Mn oxidation) genes, including mnxG, encoding a putative multicopper oxidase (MCO), as responsible for this two-electron oxidation, a surprising finding because MCOs only catalyze single-electron transfer reactions. Characterization of the enzymatic mechanism has been hindered by the lack of purified protein. By purifying active protein from the mnxDEFG expression construct, we found that the resulting enzyme is a blue (absorption maximum 590 nm) complex containing MnxE, MnxF, and MnxG proteins. Further, by analyzing the Mn(II)- and (III)-oxidizing activity in the presence of a Mn(III) chelator, pyrophosphate, we found that the complex facilitates both electron transfers from Mn(II) to Mn(III) and from Mn(III) to Mn(IV). X-ray absorption spectroscopy of the Mn mineral product confirmed its similarity to Mn(IV) oxides generated by whole spores. Our results demonstrate that Mn oxidation from soluble Mn(II) to Mn(IV) oxides is a two-step reaction catalyzed by an MCO-containing complex. With the purification of active Mn oxidase, we will be able to uncover its mechanism, broadening our understanding of Mn mineral formation and the bioinorganic capabilities of MCOs. PMID:23818588

  1. Substrate specificity and copper loading of the manganese-oxidizing multicopper oxidase Mnx from Bacillus sp. PL-12.

    Science.gov (United States)

    Butterfield, Cristina N; Tebo, Bradley M

    2017-02-22

    Manganese(ii) oxidation in the environment is thought to be driven by bacteria because enzymatic catalysis is many orders of magnitude faster than the abiotic processes. The heterologously purified Mn oxidase (Mnx) from marine Bacillus sp. PL-12 is made up of the multicopper oxidase (MCO) MnxG and two small Cu and heme-binding proteins of unknown function, MnxE and MnxF. Mnx binds Cu and oxidizes both Mn(ii) and Mn(iii), generating Mn(iv) oxide minerals that resemble those found on the Bacillus spore surface. Spectroscopic techniques have illuminated details about the metallo-cofactors of Mnx, but very little is known about their requirement for catalytic activity, and even less is known about the substrate specificity of Mnx. Here we quantify the canonical MCO Cu and persistent peripheral Cu bound to Mnx, and test Mnx oxidizing ability toward different substrates at varying pH. Mn(ii) appears to be the best substrate in terms of k cat , but its oxidation does not follow Michaelis-Menten kinetics, instead showing a sigmoidal cooperative behavior. Mnx also oxidizes Fe(ii) substrate, but in a Michaelis-Menten manner and with a decreased activity, as well as organic substrates. The reduced metals are more rapidly consumed than the larger organic substrates, suggesting the hypothesis that the Mnx substrate site is small and tuned for metal oxidation. Of biological relevance is the result that Mnx has the highest catalytic efficiency for Mn(ii) at the pH of sea water, especially when the protein is loaded with greater than the requisite four MCO copper atoms, suggesting that the protein has evolved specifically for Mn oxidation.

  2. On the Possibility of Uphill Intramolecular Electron Transfer in Multicopper Oxidases: Electrochemical and Quantum Chemical Study of Bilirubin Oxidase

    Czech Academy of Sciences Publication Activity Database

    Shleev, S.; Andoralov, V.; Falk, M.; Reimann, C. T.; Ruzgas, T.; Srnec, Martin; Ryde, U.; Rulíšek, Lubomír

    2012-01-01

    Roč. 24, č. 7 (2012), s. 1524-1540 ISSN 1040-0397 Grant - others:7th Framework Program(XE) NMP4-SL-2009-229255 Institutional research plan: CEZ:AV0Z40550506 Keywords : bilirubin oxidase * intramolecular electron transfer * rate-limiting catalytic step * reorganization energy * QM/MM calculations Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 2.817, year: 2012

  3. Characterization of three multicopper oxidases in the filamentous fungus Podospora anserina: A new role of an ABR1-like protein in fungal development?

    Science.gov (United States)

    Xie, Ning; Ruprich-Robert, Gwenaël; Silar, Philippe; Herbert, Eric; Ferrari, Roselyne; Chapeland-Leclerc, Florence

    2018-07-01

    The Podospora anserina genome contains a large family of 15 multicopper oxidases (MCOs), including three genes encoding a FET3-like protein, an ABR1-like protein and an ascorbate oxidase (AO)-like protein. FET3, ABR1 and AO1 are involved in global laccase-like activity since deletion of the relevant genes led to a decrease of activity when laccase substrate (ABTS) was used as substrate. However, contrary to the P. anserina MCO proteins previously characterized, none of these three MCOs seemed to be involved in lignocellulose degradation and in resistance to phenolic compounds and oxidative stress. We showed that the bulk of ferroxidase activity was clearly due to ABR1, and only in minor part to FET3, although ABR1 does not contain all the residues typical of FET3 proteins. Moreover, we showed that ABR1, related to the Aspergillus fumigatus ABR1 protein, was clearly and specifically involved in pigmentation of ascospores. Surprisingly, phenotypes were more severe in mutants lacking both abr1 and ao1. Deletion of the ao1 gene led to an almost total loss of AO activity. No direct involvement of AO1 in fungal developmental process in P. anserina was evidenced, except in a abr1 Δ background. Overall, unlike other previously characterized MCOs, we thus evidence a clear involvement of ABR1 protein in fungal development. Copyright © 2018 Elsevier Inc. All rights reserved.

  4. Surface Mn(II) oxidation actuated by a multicopper oxidase in a soil bacterium leads to the formation of manganese oxide minerals.

    Science.gov (United States)

    Zhang, Zhen; Zhang, Zhongming; Chen, Hong; Liu, Jin; Liu, Chang; Ni, Hong; Zhao, Changsong; Ali, Muhammad; Liu, Fan; Li, Lin

    2015-06-03

    In this manuscript, we report that a bacterial multicopper oxidase (MCO266) catalyzes Mn(II) oxidation on the cell surface, resulting in the surface deposition of Mn(III) and Mn(IV) oxides and the gradual formation of bulky oxide aggregates. These aggregates serve as nucleation centers for the formation of Mn oxide micronodules and Mn-rich sediments. A soil-borne Escherichia coli with high Mn(II)-oxidizing activity formed Mn(III)/Mn(IV) oxide deposit layers and aggregates under laboratory culture conditions. We engineered MCO266 onto the cell surfaces of both an activity-negative recipient and wild-type strains. The results confirmed that MCO266 governs Mn(II) oxidation and initiates the formation of deposits and aggregates. By contrast, a cell-free substrate, heat-killed strains, and intracellularly expressed or purified MCO266 failed to catalyze Mn(II) oxidation. However, purified MCO266 exhibited Mn(II)-oxidizing activity when combined with cell outer membrane component (COMC) fractions in vitro. We demonstrated that Mn(II) oxidation and aggregate formation occurred through an oxygen-dependent biotic transformation process that requires a certain minimum Mn(II) concentration. We propose an approximate electron transfer pathway in which MCO266 transfers only one electron to convert Mn(II) to Mn(III) and then cooperates with other COMC electron transporters to transfer the other electron required to oxidize Mn(III) to Mn(IV).

  5. Surface Induced Dissociation Coupled with High Resolution Mass Spectrometry Unveils Heterogeneity of a 211 kDa Multicopper Oxidase Protein Complex

    Science.gov (United States)

    Zhou, Mowei; Yan, Jing; Romano, Christine A.; Tebo, Bradley M.; Wysocki, Vicki H.; Paša-Tolić, Ljiljana

    2018-01-01

    Manganese oxidation is an important biogeochemical process that is largely regulated by bacteria through enzymatic reactions. However, the detailed mechanism is poorly understood due to challenges in isolating and characterizing these unknown enzymes. A manganese oxidase, Mnx, from Bacillus sp. PL-12 has been successfully overexpressed in active form as a protein complex with a molecular mass of 211 kDa. We have recently used surface induced dissociation (SID) and ion mobility-mass spectrometry (IM-MS) to release and detect folded subcomplexes for determining subunit connectivity and quaternary structure. The data from the native mass spectrometry experiments led to a plausible structural model of this multicopper oxidase, which has been difficult to study by conventional structural biology methods. It was also revealed that each Mnx subunit binds a variable number of copper ions. Becasue of the heterogeneity of the protein and limited mass resolution, ambiguities in assigning some of the observed peaks remained as a barrier to fully understanding the role of metals and potential unknown ligands in Mnx. In this study, we performed SID in a modified Fourier transform-ion cyclotron resonance (FTICR) mass spectrometer. The high mass accuracy and resolution offered by FTICR unveiled unexpected artificial modifications on the protein that had been previously thought to be iron bound species based on lower resolution spectra. Additionally, isotopically resolved spectra of the released subcomplexes revealed the metal binding stoichiometry at different structural levels. This method holds great potential for in-depth characterization of metalloproteins and protein-ligand complexes. [Figure not available: see fulltext.

  6. Promoter isolation and characterization of GhAO-like1, a Gossypium hirsutum gene similar to multicopper oxidases that is highly expressed in reproductive organs.

    Science.gov (United States)

    Lambret-Frotté, Julia; Artico, Sinara; Muniz Nardeli, Sarah; Fonseca, Fernando; Brilhante Oliveira-Neto, Osmundo; Grossi-de-Sá, Maria Fatima; Alves-Ferreira, Marcio

    2016-01-01

    Cotton is one of the most economically important cultivated crops. It is the major source of natural fiber for the textile industry and an important target for genetic modification for both biotic stress and herbicide tolerance. Therefore, the characterization of genes and regulatory regions that might be useful for genetic transformation is indispensable. The isolation and characterization of new regulatory regions is of great importance to drive transgene expression in genetically modified crops. One of the major drawbacks in cotton production is pest damage; therefore, the most promising, cost-effective, and sustainable method for pest control is the development of genetically resistant cotton lines. Considering this scenario, our group isolated and characterized the promoter region of a MCO (multicopper oxidase) from Gossypium hirsutum, named GhAO-like1 (ascorbate oxidase-like1). The quantitative expression, together with the in vivo characterization of the promoter region reveals that GhAO-like1 has a flower- and fruit-specific expression pattern. The GUS activity is mainly observed in stamens, as expected considering that the GhAO-like1 regulatory sequence is enriched in cis elements, which have been characterized as a target of reproductive tissue specific transcription factors. Both histological and quantitative analyses in Arabidopsis thaliana have confirmed flower (mainly in stamens) and fruit expression of GhAO-like1. In the present paper, we isolated and characterized both in silico and in vivo the promoter region of the GhAO-like1 gene. The regulatory region of GhAO-like1 might be useful to confer tissue-specific expression in genetically modified plants.

  7. Spectroscopy of Cu(II)-PcoC and the multicopper oxidase function of PcoA, two essential components of Escherichia coli pco copper resistance operon.

    Science.gov (United States)

    Huffman, David L; Huyett, Jennifer; Outten, F Wayne; Doan, Peter E; Finney, Lydia A; Hoffman, Brian M; O'Halloran, Thomas V

    2002-08-06

    The plasmid-encoded pco copper resistance operon in Escherichia coli consists of seven genes that are expressed from two pco promoters in response to elevated copper; however, little is known about how they mediate resistance to excess environmental copper. Two of the genes encode the soluble periplasmic proteins PcoA and PcoC. We show here that inactivation of PcoC, and PcoA to a lesser extent, causes cells to become more sensitive to copper than wild-type nonresistant strains, consistent with a tightly coupled detoxification pathway. Periplasmic extracts show copper-inducible oxidase activity, attributed to the multicopper oxidase function of PcoA. PcoC, a much smaller protein than PcoA, binds one Cu(II) and exhibits a weak electronic transition characteristic of a type II copper center. ENDOR and ESEEM spectroscopy of Cu(II)-PcoC and the (15)N- and Met-CD(3)-labeled samples are consistent with a tetragonal ligand environment of three nitrogens and one aqua ligand "in the plane". A weakly associated S-Met and aqua are likely axial ligands. At least one N is a histidine and is likely trans to the in-plane aqua ligand. The copper chemistry of PcoC and the oxidase function of PcoA are consistent with the emerging picture of the chromosomally encoded copper homeostasis apparatus in the E. coli cell envelope [Outten, F. W., Huffman, D. L., Hale, J. A., and O'Halloran, T. V. (2001) J. Biol. Chem. 276, 30670-30677]. We propose a model for the plasmid system in which Cu(I)-PcoC functions in this copper efflux pathway as a periplasmic copper binding protein that docks with the multiple repeats of Met-rich domains in PcoA to effect oxidation of Cu(I) to the less toxic Cu(II) form. The solvent accessibility of the Cu(II) in PcoC may allow for metal transfer to other plasmid and chromosomal factors and thus facilitate removal of Cu(II) from the cell envelope.

  8. Structural changes caused by radiation-induced reduction and radiolysis: the effect of X-ray absorbed dose in a fungal multicopper oxidase

    International Nuclear Information System (INIS)

    De la Mora, Eugenio; Lovett, Janet E.; Blanford, Christopher F.; Garman, Elspeth F.; Valderrama, Brenda; Rudino-Pinera, Enrique

    2012-01-01

    Radiation-induced reduction, radiolysis of copper sites and the effect of pH value together with the concomitant geometrical distortions of the active centres were analysed in several fungal (C. gallica) laccase structures collected at cryotemperature. This study emphasizes the importance of careful interpretation when the crystallographic structure of a metalloprotein is described. X-ray radiation induces two main effects at metal centres contained in protein crystals: radiation-induced reduction and radiolysis and a resulting decrease in metal occupancy. In blue multicopper oxidases (BMCOs), the geometry of the active centres and the metal-to-ligand distances change depending on the oxidation states of the Cu atoms, suggesting that these alterations are catalytically relevant to the binding, activation and reduction of O 2 . In this work, the X-ray-determined three-dimensional structure of laccase from the basidiomycete Coriolopsis gallica (Cg L), a high catalytic potential BMCO, is described. By combining spectroscopic techniques (UV–Vis, EPR and XAS) and X-ray crystallography, structural changes at and around the active copper centres were related to pH and absorbed X-ray dose (energy deposited per unit mass). Depletion of two of the four active Cu atoms as well as low occupancies of the remaining Cu atoms, together with different conformations of the metal centres, were observed at both acidic pH and high absorbed dose, correlating with more reduced states of the active coppers. These observations provide additional evidence to support the role of flexibility of copper sites during O 2 reduction. This study supports previous observations indicating that interpretations regarding redox state and metal coordination need to take radiation effects explicitly into account

  9. Diazonium Functionalisation of Carbon Nanotubes for Specific Orientation of Multicopper Oxidases: Controlling Electron Entry Points and Oxygen Diffusion to the Enzyme.

    Science.gov (United States)

    Lalaoui, Noémie; Holzinger, Michael; Le Goff, Alan; Cosnier, Serge

    2016-07-18

    We report the controlled orientation of bilirubin oxidases (BOD) from Myrothecium verrucaria on multiwalled carbon nanotubes (MWCNTs) functionalised by electrografting of 6-carboxynaphthalenediazonium and 4-(2-aminoethyl)benzenediazonium salts. On negatively charged naphthoate-modified MWCNTs, a high-potential (0.44 V vs. SCE) oxygen reduction electrocatalysis is observed, occurring via the T1 copper centre. On positively charged ammonium-modified MWCNTs, a low-potential (0.15 V) oxygen reduction electrocatalysis is observed, occurring through a partially oxidised state of the T2/T3 trinuclear copper cluster. Finally, chemically modified naphthoate MWCNTs exhibit high bioelectrocatalytic current densities of 3.9 mA cm(-2) under air at gas-diffusion electrode. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Catalytic aspects of a copper(II) complex: biological oxidase to ...

    Indian Academy of Sciences (India)

    BISWAJIT CHOWDHURY

    2017-10-03

    Oct 3, 2017 ... made with a Jasco model V-730 UV-Vis spectrophotometer. ..... Ligand-induced coordination changes ... Fet3 protein from yeast, a multinuclear copper oxidase ... of mutants of the multicopper oxidase Fet3p Biochem-.

  11. X-ray analysis of bilirubin oxidase from Myrothecium verrucaria at 2.3 Å resolution using a twinned crystal

    International Nuclear Information System (INIS)

    Mizutani, Kimihiko; Toyoda, Mayuko; Sagara, Kenta; Takahashi, Nobuyuki; Sato, Atsuko; Kamitaka, Yuji; Tsujimura, Seiya; Nakanishi, Yuji; Sugiura, Toshiyuki; Yamaguchi, Shotaro; Kano, Kenji; Mikami, Bunzo

    2010-01-01

    The crystal structure of bilirubin oxidase (BOD) from M. verrucaria has been determined at 2.3 Å resolution using a merohedrally twinned crystal. BOD has four copper-coordination sites that are almost identical to those of other multicopper oxidases and is also very similar to them in overall structure. Bilirubin oxidase (BOD), a multicopper oxidase found in Myrothecium verrucaria, catalyzes the oxidation of bilirubin to biliverdin. Oxygen is the electron acceptor and is reduced to water. BOD is used for diagnostic analysis of bilirubin in serum and has attracted considerable attention as an enzymatic catalyst for the cathode of biofuel cells that work under neutral conditions. Here, the crystal structure of BOD is reported for the first time. Blue bipyramid-shaped crystals of BOD obtained in 2-methyl-2,4-pentanediol (MPD) and ammonium sulfate solution were merohedrally twinned in space group P6 3 . Structure determination was achieved by the single anomalous diffraction (SAD) method using the anomalous diffraction of Cu atoms and synchrotron radiation and twin refinement was performed in the resolution range 33–2.3 Å. The overall organization of BOD is almost the same as that of other multicopper oxidases: the protein is folded into three domains and a total of four copper-binding sites are found in domains 1 and 3. Although the four copper-binding sites were almost identical to those of other multicopper oxidases, the hydrophilic Asn residue (at the same position as a hydrophobic residue such as Leu in other multicopper oxidases) very close to the type I copper might contribute to the characteristically high redox potential of BOD

  12. Intramolecular electron transfer in ascorbate oxidase is enhanced in the presence of oxygen

    DEFF Research Database (Denmark)

    Farver, O; Wherland, S; Pecht, I

    1994-01-01

    Intramolecular electron transfer from the type 1 copper center to the type 3 copper(II) pair is induced in the multi-copper enzyme, ascorbate oxidase, following pulse radiolytic reduction of the type 1 Cu(II) ion. In the presence of a slight excess of dioxygen over ascorbate oxidase, interaction...... between the trinuclear copper center and O2 is observed even with singly reduced ascorbate oxidase molecules. Under these conditions, the rate constant for intramolecular electron transfer from type 1 Cu(I) to type 3 Cu(II) increases 5-fold to 1100 +/- 300 s-1 (20 degrees C, pH 5.8) as compared...

  13. Voltammetry and single-molecule in situ scanning tunneling microscopy of laccases and bilirubin oxidase in electrocatalytic dioxygen reduction on Au(111) single-crystal electrodes

    DEFF Research Database (Denmark)

    Climent, Victor; Zhang, Jingdong; Friis, Esben Peter

    2012-01-01

    Laccases (E.C. 1.10.3.2) are multicopper oxidases catalytically active in the oxidation of diphenolics and related compounds by molecular dioxygen. The laccases contain a single-copper type I center and a trinuclear cluster of a single-copper type II and a dinuclear type III center. The oxidation...

  14. Characterization of novel thermostable bacterial Laccase-like multi-copper oxidases

    DEFF Research Database (Denmark)

    Brander, Søren; Mikkelsen, Jørn Dalgaard

    decolorization, bleaching of paper pulp, delignification of biomass and remediation of polluted water. In order to widen the applicability of LMCOs, it is important to establish the properties and substrate specificities of naturally occurring LMCOs. This is especially true for LMCOs from bacteria, whose role...

  15. Two-domain laccase from Streptomyces coelicolor: a link between laccases and nitrite reductases

    Czech Academy of Sciences Publication Activity Database

    Skálová, Tereza; Dohnálek, Jan; Ostergaard, L. H.; Ostergaard, P. R.; Kolenko, Petr; Dušková, Jarmila; Štěpánková, Andrea; Hašek, Jindřich

    2009-01-01

    Roč. 16, 3a - Special Issue (2009), s. 3-4 ISSN 1211-5894. [Heart of European Crystallographic Meeting /12./. 24.09.2009-26.09.2009, Třešt´] R&D Projects: GA AV ČR IAA500500701; GA ČR GA305/07/1073 Institutional research plan: CEZ:AV0Z40500505 Keywords : laccase * oxidoreductase * multicopper blue protein Subject RIV: CD - Macromolecular Chemistry

  16. Study on dioxygen reduction by mutational modifications of the hydrogen bond network leading from bulk water to the trinuclear copper center in bilirubin oxidase

    Energy Technology Data Exchange (ETDEWEB)

    Morishita, Hirotoshi; Kurita, Daisuke; Kataoka, Kunishige; Sakurai, Takeshi, E-mail: tsakurai@se.kanazawa-u.ac.jp

    2014-07-18

    Highlights: • Proton transport pathway in bilirubin oxidase was mutated. • Two intermediates in the dioxygen reduction steps were trapped and characterized. • A specific glutamate for dioxygen reduction by multicopper oxidases was identified. - Abstract: The hydrogen bond network leading from bulk water to the trinuclear copper center in bilirubin oxidase is constructed with Glu463 and water molecules to transport protons for the four-electron reduction of dioxygen. Substitutions of Glu463 with Gln or Ala were attributed to virtually complete loss or significant reduction in enzymatic activities due to an inhibition of the proton transfer steps to dioxygen. The single turnover reaction of the Glu463Gln mutant afforded the highly magnetically interacted intermediate II (native intermediate) with a broad g = 1.96 electron paramagnetic resonance signal detectable at cryogenic temperatures. Reactions of the double mutants, Cys457Ser/Glu463Gln and Cys457Ser/Glu463Ala afforded the intermediate I (peroxide intermediate) because the type I copper center to donate the fourth electron to dioxygen was vacant in addition to the interference of proton transport due to the mutation at Glu463. The intermediate I gave no electron paramagnetic resonance signal, but the type II copper signal became detectable with the decay of the intermediate I. Structural and functional similarities between multicopper oxidases are discussed based on the present mutation at Glu463 in bilirubin oxidase.

  17. Study on dioxygen reduction by mutational modifications of the hydrogen bond network leading from bulk water to the trinuclear copper center in bilirubin oxidase

    International Nuclear Information System (INIS)

    Morishita, Hirotoshi; Kurita, Daisuke; Kataoka, Kunishige; Sakurai, Takeshi

    2014-01-01

    Highlights: • Proton transport pathway in bilirubin oxidase was mutated. • Two intermediates in the dioxygen reduction steps were trapped and characterized. • A specific glutamate for dioxygen reduction by multicopper oxidases was identified. - Abstract: The hydrogen bond network leading from bulk water to the trinuclear copper center in bilirubin oxidase is constructed with Glu463 and water molecules to transport protons for the four-electron reduction of dioxygen. Substitutions of Glu463 with Gln or Ala were attributed to virtually complete loss or significant reduction in enzymatic activities due to an inhibition of the proton transfer steps to dioxygen. The single turnover reaction of the Glu463Gln mutant afforded the highly magnetically interacted intermediate II (native intermediate) with a broad g = 1.96 electron paramagnetic resonance signal detectable at cryogenic temperatures. Reactions of the double mutants, Cys457Ser/Glu463Gln and Cys457Ser/Glu463Ala afforded the intermediate I (peroxide intermediate) because the type I copper center to donate the fourth electron to dioxygen was vacant in addition to the interference of proton transport due to the mutation at Glu463. The intermediate I gave no electron paramagnetic resonance signal, but the type II copper signal became detectable with the decay of the intermediate I. Structural and functional similarities between multicopper oxidases are discussed based on the present mutation at Glu463 in bilirubin oxidase

  18. Monoamine Oxidase Inhibitors (MAOIs)

    Science.gov (United States)

    ... health-medications/index.shtml. Accessed May 16, 2016. Hirsch M, et al. Monoamine oxidase inhibitors (MAOIs) for ... www.uptodate.com/home. Accessed May 16, 2016. Hirsch M, et al. Discontinuing antidepressant medications in adults. ...

  19. Single-domain versus two-domain configuration in thin ferromagnetic prisms

    International Nuclear Information System (INIS)

    Pini, Maria Gloria; Politi, Paolo

    2007-01-01

    Thin ferromagnetic elements in the form of rectangular prisms are theoretically investigated in order to study the transition from single-domain to two-domain state, with changing the in-plane aspect ratio p. We address two main questions: first, how general is the transition; second, how the critical value p c depends on the physical parameters. We use two complementary methods: discrete-lattice calculations and a micromagnetic continuum approach. Ultrathin films do not appear to split in two domains. Instead, thicker films may undergo the above transition. We have used the continuum approach to analyze recent magnetic force microscopy observations in 30nm-thick patterned permalloy elements, finding a good agreement for p c

  20. Isolated sulfite oxidase deficiency.

    Science.gov (United States)

    Rupar, C A; Gillett, J; Gordon, B A; Ramsay, D A; Johnson, J L; Garrett, R M; Rajagopalan, K V; Jung, J H; Bacheyie, G S; Sellers, A R

    1996-12-01

    Isolated sulfite oxidase (SO) deficiency is an autosomal recessively inherited inborn error of sulfur metabolism. In this report of a ninth patient the clinical history, laboratory results, neuropathological findings and a mutation in the sulfite oxidase gene are described. The data from this patient and previously published patients with isolated sulfite oxidase deficiency and molybdenum cofactor deficiency are summarized to characterize this rare disorder. The patient presented neonatally with intractable seizures and did not progress developmentally beyond the neonatal stage. Dislocated lenses were apparent at 2 months. There was increased urine excretion of sulfite and S-sulfocysteine and a decreased concentration of plasma cystine. A lactic acidemia was present for 6 months. Liver sulfite oxidase activity was not detectable but xanthine dehydrogenase activity was normal. The boy died of respiratory failure at 32 months. Neuropathological findings of cortical necrosis and extensive cavitating leukoencephalopathy were reminiscent of those seen in severe perinatal asphyxia suggesting an etiology of energy deficiency. A point mutation that resulted in a truncated protein missing the molybdenum-binding site has been identified.

  1. A two-domain elevator mechanism for sodium/proton antiport.

    Science.gov (United States)

    Lee, Chiara; Kang, Hae Joo; von Ballmoos, Christoph; Newstead, Simon; Uzdavinys, Povilas; Dotson, David L; Iwata, So; Beckstein, Oliver; Cameron, Alexander D; Drew, David

    2013-09-26

    Sodium/proton (Na(+)/H(+)) antiporters, located at the plasma membrane in every cell, are vital for cell homeostasis. In humans, their dysfunction has been linked to diseases, such as hypertension, heart failure and epilepsy, and they are well-established drug targets. The best understood model system for Na(+)/H(+) antiport is NhaA from Escherichia coli, for which both electron microscopy and crystal structures are available. NhaA is made up of two distinct domains: a core domain and a dimerization domain. In the NhaA crystal structure a cavity is located between the two domains, providing access to the ion-binding site from the inward-facing surface of the protein. Like many Na(+)/H(+) antiporters, the activity of NhaA is regulated by pH, only becoming active above pH 6.5, at which point a conformational change is thought to occur. The only reported NhaA crystal structure so far is of the low pH inactivated form. Here we describe the active-state structure of a Na(+)/H(+) antiporter, NapA from Thermus thermophilus, at 3 Å resolution, solved from crystals grown at pH 7.8. In the NapA structure, the core and dimerization domains are in different positions to those seen in NhaA, and a negatively charged cavity has now opened to the outside. The extracellular cavity allows access to a strictly conserved aspartate residue thought to coordinate ion binding directly, a role supported here by molecular dynamics simulations. To alternate access to this ion-binding site, however, requires a surprisingly large rotation of the core domain, some 20° against the dimerization interface. We conclude that despite their fast transport rates of up to 1,500 ions per second, Na(+)/H(+) antiporters operate by a two-domain rocking bundle model, revealing themes relevant to secondary-active transporters in general.

  2. The Leptospiral Antigen Lp49 is a Two-Domain Protein with Putative Protein Binding Function

    Energy Technology Data Exchange (ETDEWEB)

    Oliveira Giuseppe,P.; Oliveira Neves, F.; Nascimento, A.; Gomes Guimaraes, B.

    2008-01-01

    Pathogenic Leptospira is the etiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. Currently available vaccines have limited effectiveness and therapeutic interventions are complicated by the difficulty in making an early diagnosis of leptospirosis. The genome of Leptospira interrogans was recently sequenced and comparative genomic analysis contributed to the identification of surface antigens, potential candidates for development of new vaccines and serodiagnosis. Lp49 is a membrane-associated protein recognized by antibodies present in sera from early and convalescent phases of leptospirosis patients. Its crystal structure was determined by single-wavelength anomalous diffraction using selenomethionine-labelled crystals and refined at 2.0 Angstroms resolution. Lp49 is composed of two domains and belongs to the all-beta-proteins class. The N-terminal domain folds in an immunoglobulin-like beta-sandwich structure, whereas the C-terminal domain presents a seven-bladed beta-propeller fold. Structural analysis of Lp49 indicates putative protein-protein binding sites, suggesting a role in Leptospira-host interaction. This is the first crystal structure of a leptospiral antigen described to date.

  3. Bilirubin oxidase-like proteins from Podospora anserina: promising thermostable enzymes for application in transformation of plant biomass.

    Science.gov (United States)

    Xie, Ning; Ruprich-Robert, Gwenaël; Silar, Philippe; Chapeland-Leclerc, Florence

    2015-03-01

    Plant biomass degradation by fungi is a critical step for production of biofuels, and laccases are common ligninolytic enzymes envisioned for ligninolysis. Bilirubin oxidases (BODs)-like are related to laccases, but their roles during lignocellulose degradation have not yet been fully investigated. The two BODs of the ascomycete fungus Podospora anserina were characterized by targeted gene deletions. Enzymatic assay revealed that the bod1(Δ) and bod2(Δ) mutants lost partly a thermostable laccase activity. A triple mutant inactivated for bod1, bod2 and mco, a previously investigated multicopper oxidase gene distantly related to laccases, had no thermostable laccase activity. The pattern of fruiting body production in the bod1(Δ) bod2(Δ) double mutant was changed. The bod1(Δ) and bod2(Δ) mutants were reduced in their ability to grow on ligneous and cellulosic materials. Furthermore, bod1(Δ) and bod2(Δ) mutants were defective towards resistance to phenolic substrates and H2 O2 , which may also impact lignocellulose breakdown. Double and triple mutants were more affected than single mutants, evidencing redundancy of function among BODs and mco. Overall, the data show that bod1, bod2 and mco code for non-canonical thermostable laccases that participate in the degradation of lignocellulose. Thanks to their thermal stability, these enzymes may be more promising candidate for biotechnological application than canonical laccases. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

  4. Oxidase-based biocatalytic processes

    DEFF Research Database (Denmark)

    Ramesh, Hemalata; Woodley, John; Krühne, Ulrich

    interestingbiocatalystsbecause they use a mild oxidant (oxygen) as a substrateas opposed to their chemical counterparts which use strong oxidants such as permanganates. A class of oxidases calledmonoamine oxidases has been used as the central case study for the thesis. The rationale for choosing thissystemis that it has been...

  5. Oxidases as Breast Cancer Oncogens

    National Research Council Canada - National Science Library

    Yeldandi, Anjana

    2000-01-01

    ...) in a non-tumorigenic human mammary epithelial cell line to ascertain whether oxidase overexpressing cells undergo transformation when exposed to substrate xanthine for XOX and uric acid for UOX...

  6. Glucose oxidase variants with improved properities

    OpenAIRE

    Fischer, Rainer; Ostafe, Raluca; Prodanovic, Radivoje

    2014-01-01

    Source: WO14173822A3 [EN] The technology provided herein relates to novel variants of microbial glucose oxidase with improved properties, more specifically to polypeptides having glucose oxidase activity as their major enzymatic activity; to nucleic acid molecules encoding said glucose oxidases; vectors and host cells containing the nucleic acids and methods for producing the glucose oxidase; compositions comprising said glucose oxidase; methods for the preparation and production of such enzy...

  7. NADPH Oxidases: Progress and Opportunities

    OpenAIRE

    San Martin, Alejandra; Griendling, Kathy K.

    2014-01-01

    From the initial discovery in 1999 that NADPH oxidases comprise a family of enzymes to our current focus on drug development to treat multiple pathologies related to this enzyme family, progress has been swift and impressive. We have expanded our understanding of the extent of the family, the basic enzymatic biochemistry, the multiple cellular functions controlled by NADPH oxidases, and their varied roles in physiology and diseases. We have developed numerous cell culture tools, animal models...

  8. Protein translocation channel of mitochondrial inner membrane and matrix-exposed import motor communicate via two-domain coupling protein.

    Science.gov (United States)

    Banerjee, Rupa; Gladkova, Christina; Mapa, Koyeli; Witte, Gregor; Mokranjac, Dejana

    2015-12-29

    The majority of mitochondrial proteins are targeted to mitochondria by N-terminal presequences and use the TIM23 complex for their translocation across the mitochondrial inner membrane. During import, translocation through the channel in the inner membrane is coupled to the ATP-dependent action of an Hsp70-based import motor at the matrix face. How these two processes are coordinated remained unclear. We show here that the two domain structure of Tim44 plays a central role in this process. The N-terminal domain of Tim44 interacts with the components of the import motor, whereas its C-terminal domain interacts with the translocation channel and is in contact with translocating proteins. Our data suggest that the translocation channel and the import motor of the TIM23 complex communicate through rearrangements of the two domains of Tim44 that are stimulated by translocating proteins.

  9. Molecular Dissection of the Homotrimeric Sliding Clamp of T4 Phage: Two Domains of a Subunit Display Asymmetric Characteristics.

    Science.gov (United States)

    Singh, Manika Indrajit; Jain, Vikas

    2016-01-26

    Sliding clamp proteins are circular dimers or trimers that encircle DNA and serve as processivity factors during DNA replication. Their presence in all the three domains of life and in bacteriophages clearly indicates their high level of significance. T4 gp45, besides functioning as the DNA polymerase processivity factor, also moonlights as the late promoter transcription determinant. Here we report a detailed biophysical analysis of gp45. The chemical denaturation of gp45 probed by circular dichroism spectroscopy, tryptophan fluorescence anisotropy, and blue-native polyacrylamide gel electrophoresis suggests that the protein follows a three-state denaturation profile and displays an intermediate molten globule-like state. The three-state transition was found to be the result of the sequential unfolding of the two domains, the N-terminal domain (NTD) and the C-terminal domain (CTD), of gp45. The experiments involving Trp fluorescence quenching by acrylamide demonstrate that the CTD undergoes substantial changes in conformation during formation of the intermediate state. Further biophysical dissection of the individual domain reveals contrasting properties of the two domains. The NTD unfolds at low urea concentrations and is also susceptible to protease cleavage, whereas the CTD resists urea-mediated denaturation and is not amenable to protease digestion even at higher urea concentrations. These experiments allow us to conclude that the two domains of gp45 differ in their dynamics. While the CTD shows stability and rigidity, we find that the NTD is unstable and flexible. We believe that the asymmetric characteristics of the two domains and the interface they form hold significance in gp45 structure and function.

  10. Lysyl oxidase in colorectal cancer

    DEFF Research Database (Denmark)

    Cox, Thomas R; Erler, Janine T

    2013-01-01

    Colorectal cancer is the third most prevalent form of cancer worldwide and fourth-leading cause of cancer-related mortality, leading to ~600,000 deaths annually, predominantly affecting the developed world. Lysyl oxidase is a secreted, extracellular matrix-modifying enzyme previously suggested...... to act as a tumor suppressor in colorectal cancer. However, emerging evidence has rapidly implicated lysyl oxidase in promoting metastasis of solid tumors and in particular colorectal cancer at multiple stages, affecting tumor cell proliferation, invasion, and angiogenesis. This emerging research has...... advancements in the field of colorectal cancer....

  11. Lysyl oxidase in cancer research

    DEFF Research Database (Denmark)

    Perryman, Lara; Erler, Janine Terra

    2014-01-01

    Metastasis is the main reason for cancer-associated deaths and therapies are desperately needed to target the progression of cancer. Lysyl oxidase (LOX) plays a pivotal role in cancer progression, including metastasis, and is therefore is an attractive therapeutic target. In this review we...

  12. Direct electrochemistry and intramolecular electron transfer of ascorbate oxidase confined on L-cysteine self-assembled gold electrode.

    Science.gov (United States)

    Patil, Bhushan; Kobayashi, Yoshiki; Fujikawa, Shigenori; Okajima, Takeyoshi; Mao, Lanqun; Ohsaka, Takeo

    2014-02-01

    A direct electrochemistry and intramolecular electron transfer of multicopper oxidases are of a great importance for the fabrication of these enzyme-based bioelectrochemical-devices. Ascorbate oxidase from Acremonium sp. (ASOM) has been successfully immobilized via a chemisorptive interaction on the l-cysteine self-assembled monolayer modified gold electrode (cys-SAM/AuE). Thermodynamics and kinetics of adsorption of ASOM on the cys-SAM/AuE were studied using cyclic voltammetry. A well-defined redox wave centered at 166±3mV (vs. Ag│AgCl│KCl(sat.)) was observed in 5.0mM phosphate buffer solution (pH7.0) at the fabricated ASOM electrode, abbreviated as ASOM/cys-SAM/AuE, confirming a direct electrochemistry, i.e., a direct electron transfer (DET) between ASOM and cys-SAM/AuE. The direct electrochemistry of ASOM was further confirmed by taking into account the chemical oxidation of ascorbic acid (AA) by O2 via an intramolecular electron transfer in the ASOM as well as the electrocatalytic oxidation of AA at the ASOM/cys-SAM/AuE. Thermodynamics and kinetics of the adsorption of ASOM on the cys-SAM/AuE have been elaborated along with its direct electron transfer at the modified electrodes on the basis of its intramolecular electron transfer and electrocatalytic activity towards ascorbic acid oxidation and O2 reduction. ASOM saturated surface area was obtained as 2.41×10(-11)molcm(-2) with the apparent adsorption coefficient of 1.63×10(6)Lmol(-1). The ASOM confined on the cys-SAM/AuE possesses its essential enzymatic function. © 2013.

  13. Professor Tyndale John Rendle-Short (1919-2010), British and Australian paediatrician: A life in two domains.

    Science.gov (United States)

    Pearn, John

    2014-05-01

    Professor Tyndale John Rendle-Short (1919-2010), a British and Australian paediatrician, lived a professional life of considerable influence in two domains - academic paediatrics and fundamentalist theology. A Cambridge medical graduate (1943) and doctor-soldier, he was appointed as the Foundation Professor of Child Health at the University of Queensland (1961). In Australia, he was a pioneer in three paediatric developments ('rooming-in' for mothers in hospitals, autism research and cystic fibrosis). His A Synopsis of Children's Diseases was published in six editions, was translated into three languages and was used as a standard paediatric textbook on four Continents. Distinct from this clinical domain, as a passionate anti-Darwinist his fundamentalist theology was that variously self-described as 'theistic evolution' (believing in 'progressive Creationism') and later that of 'six-literal day young-earth Creation'. He established and was the Foundation Chairman of the Creation Science Foundation (UK) and was World Chairman of the US-based Creation Ministries International. This biography is a record of this perhaps paradoxical and unique life. © The Author(s) 2013 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

  14. Interpretation of NMR relaxation properties of Pin1, a two-domain protein, based on Brownian dynamic simulations

    International Nuclear Information System (INIS)

    Bernado, Pau; Fernandes, Miguel X.; Jacobs, Doris M.; Fiebig, Klaus; Garcia de la Torre, Jose; Pons, Miquel

    2004-01-01

    Many important proteins contain multiple domains connected by flexible linkers. Inter-domain motion is suggested to play a key role in many processes involving molecular recognition. Heteronuclear NMR relaxation is sensitive to motions in the relevant time scales and could provide valuable information on the dynamics of multi-domain proteins. However, the standard analysis based on the separation of global tumbling and fast local motions is no longer valid for multi-domain proteins undergoing internal motions involving complete domains and that take place on the same time scale than the overall motion.The complexity of the motions experienced even for the simplest two-domain proteins are difficult to capture with simple extensions of the classical Lipari-Szabo approach. Hydrodynamic effects are expected to dominate the motion of the individual globular domains, as well as that of the complete protein. Using Pin1 as a test case, we have simulated its motion at the microsecond time scale, at a reasonable computational expense, using Brownian Dynamic simulations on simplified models. The resulting trajectories provide insight on the interplay between global and inter-domain motion and can be analyzed using the recently published method of isotropic Reorientational Mode Dynamics which offer a way of calculating their contribution to heteronuclear relaxation rates. The analysis of trajectories computed with Pin1 models of different flexibility provides a general framework to understand the dynamics of multi-domain proteins and explains some of the observed features in the relaxation rate profile of free Pin1

  15. A two-domain real-time algorithm for optimal data reduction: A case study on accelerator magnet measurements

    CERN Document Server

    Arpaia, P; Inglese, V

    2010-01-01

    A real-time algorithm of data reduction, based on the combination a two lossy techniques specifically optimized for high-rate magnetic measurements in two domains (e.g. time and space), is proposed. The first technique exploits an adaptive sampling rule based on the power estimation of the flux increments in order to optimize the information to be gathered for magnetic field analysis in real time. The tracking condition is defined by the target noise level in the Nyquist band required by post-processing procedure of magnetic analysis. The second technique uses a data reduction algorithm in order to improve the compression ratio while preserving the consistency of the measured signal. The allowed loss is set equal to the random noise level in the signal in order to force the loss and the noise to cancel rather than to add, by improving the signal-to-noise ratio. Numerical analysis and experimental results of on-field performance characterization and validation for two case studies of magnetic measurement syste...

  16. A two-domain real-time algorithm for optimal data reduction: a case study on accelerator magnet measurements

    International Nuclear Information System (INIS)

    Arpaia, Pasquale; Buzio, Marco; Inglese, Vitaliano

    2010-01-01

    A real-time algorithm of data reduction, based on the combination of two lossy techniques specifically optimized for high-rate magnetic measurements in two domains (e.g. time and space), is proposed. The first technique exploits an adaptive sampling rule based on the power estimation of the flux increments in order to optimize the information to be gathered for magnetic field analysis in real time. The tracking condition is defined by the target noise level in the Nyquist band required by the post-processing procedure of magnetic analysis. The second technique uses a data reduction algorithm in order to improve the compression ratio while preserving the consistency of the measured signal. The allowed loss is set equal to the random noise level in the signal in order to force the loss and the noise to cancel rather than to add, by improving the signal-to-noise ratio. Numerical analysis and experimental results of on-field performance characterization and validation for two case studies of magnetic measurement systems for testing magnets of the Large Hadron Collider at the European Organization for Nuclear Research (CERN) are reported

  17. Interpretation of NMR relaxation properties of Pin1, a two-domain protein, based on Brownian dynamic simulations

    Energy Technology Data Exchange (ETDEWEB)

    Bernado, Pau [Institut de Biologie Structurale, Jean Pierre Ebel (France); Fernandes, Miguel X. [Universidad de Murcia, Departamento de Quimica Fisica, Facultad de Quimica (Spain); Jacobs, Doris M. [Johann Wolfgang Goethe-Universitaet Frankfurt, Institut fuer Organische Chemie und Chemische Biologie (Germany); Fiebig, Klaus [Affinium Pharmaceuticals (Canada); Garcia de la Torre, Jose [Universidad de Murcia, Departamento de Quimica Fisica, Facultad de Quimica (Spain); Pons, Miquel [Laboratori de RMN de Biomolecules, Parc Cientific de Barcelona (Spain)], E-mail: mpons@ub.edu

    2004-05-15

    Many important proteins contain multiple domains connected by flexible linkers. Inter-domain motion is suggested to play a key role in many processes involving molecular recognition. Heteronuclear NMR relaxation is sensitive to motions in the relevant time scales and could provide valuable information on the dynamics of multi-domain proteins. However, the standard analysis based on the separation of global tumbling and fast local motions is no longer valid for multi-domain proteins undergoing internal motions involving complete domains and that take place on the same time scale than the overall motion.The complexity of the motions experienced even for the simplest two-domain proteins are difficult to capture with simple extensions of the classical Lipari-Szabo approach. Hydrodynamic effects are expected to dominate the motion of the individual globular domains, as well as that of the complete protein. Using Pin1 as a test case, we have simulated its motion at the microsecond time scale, at a reasonable computational expense, using Brownian Dynamic simulations on simplified models. The resulting trajectories provide insight on the interplay between global and inter-domain motion and can be analyzed using the recently published method of isotropic Reorientational Mode Dynamics which offer a way of calculating their contribution to heteronuclear relaxation rates. The analysis of trajectories computed with Pin1 models of different flexibility provides a general framework to understand the dynamics of multi-domain proteins and explains some of the observed features in the relaxation rate profile of free Pin1.

  18. Exploring flavin-containing carbohydrate oxidases

    NARCIS (Netherlands)

    Ferrari, Alessandro Renato

    2017-01-01

    Oxidases are enzymes capable of removing one or more electrons from their substrate and transfer them to molecular oxygen, forming hydrogen peroxide. Due to their high regio- and enantioselectivity, their use is preferred over traditional organic chemistry methods. Among the oxidases, flavoprotein

  19. The terminal oxidases of Paracoccus denitrificans

    NARCIS (Netherlands)

    de Gier, J.-W.; Lübben, M; Reijnders, W N; Tipker, C A; Slotboom, D.J.; van Spanning, R J; Stouthamer, A.H.; van der Oost, J.

    Three distinct types of terminal oxidases participate in the aerobic respiratory pathways of Paracoccus denitrificans. Two alternative genes encoding subunit I of the aa3-type cytochrome c oxidase have been isolated before, namely ctaDI and ctaDII. Each of these genes can be expressed separately to

  20. Cotton Ascorbate Oxidase Promotes Cell Growth in Cultured Tobacco Bright Yellow-2 Cells through Generation of Apoplast Oxidation

    Science.gov (United States)

    Li, Rong; Xin, Shan; Tao, Chengcheng; Jin, Xiang; Li, Hongbin

    2017-01-01

    Ascorbate oxidase (AO) plays an important role in cell growth through the modulation of reduction/oxidation (redox) control of the apoplast. Here, a cotton (Gossypium hirsutum) apoplastic ascorbate oxidase gene (GhAO1) was obtained from fast elongating fiber tissues. GhAO1 belongs to the multicopper oxidase (MCO) family and includes a signal peptide and several transmembrane regions. Analyses of quantitative real-time polymerase chain reaction (QRT-PCR) and enzyme activity showed that GhAO1 was expressed abundantly in 15-day post-anthesis (dpa) wild-type (WT) fibers in comparison with fuzzless-lintless (fl) mutant ovules. Subcellular distribution analysis in onion cells demonstrated that GhAO1 is localized in the cell wall. In transgenic tobacco bright yellow-2 (BY-2) cells with ectopic overexpression of GhAO1, the enhancement of cell growth with 1.52-fold increase in length versus controls was indicated, as well as the enrichment of both total ascorbate in whole-cells and dehydroascorbate acid (DHA) in apoplasts. In addition, promoted activities of AO and monodehydroascorbate reductase (MDAR) in apoplasts and dehydroascorbate reductase (DHAR) in whole-cells were displayed in transgenic tobacco BY-2 cells. Accumulation of H2O2, and influenced expressions of Ca2+ channel genes with the activation of NtMPK9 and NtCPK5 and the suppression of NtTPC1B were also demonstrated in transgenic tobacco BY-2 cells. Finally, significant induced expression of the tobacco NtAO gene in WT BY-2 cells under indole-3-acetic acid (IAA) treatment appeared; however, the sensitivity of the NtAO gene expression to IAA disappeared in transgenic BY-2 cells, revealing that the regulated expression of the AO gene is under the control of IAA. Taken together, these results provide evidence that GhAO1 plays an important role in fiber cell elongation and may promote cell growth by generating the oxidation of apoplasts, via the auxin-mediated signaling pathway. PMID:28644407

  1. EXACT SOLUTION OF HEAT CONDUCTION IN A TWO-DOMAIN COMPOSITE CYLINDER WITH AN ORTHOTROPIC OUTER LAYER

    International Nuclear Information System (INIS)

    AVILES-RAMOS, C.; RUDY, C.

    2000-01-01

    The transient exact solution of heat conduction in a two-domain composite cylinder is developed using the separation of variables technique. The inner cylinder is isotropic and the outer cylindrical layer is orthotropic. Temperature solutions are obtained for boundary conditions of the first and second kinds at the outer surface of the orthotropic layer. These solutions are applied to heat flow calorimeters modeling assuming that there is heat generation due to nuclear reactions in the inner cylinder. Heat flow calorimeter simulations are carried out assuming that the inner cylinder is filled with plutonium oxide powder. The first objective in these simulations is to predict the onset of thermal equilibrium of the calorimeter with its environment. Two types of boundary conditions at the outer surface of the orthotropic layer are used to predict thermal equilibrium. The procedure developed to carry out these simulations can be used as a guideline for the design of calorimeters. Another important application of these solutions is on the estimation of thermophysical properties of orthotropic cylinders. The thermal conductivities in the vertical, radial and circumferential directions of the orthotropic outer layer can be estimated using this exact solution and experimental data. Simultaneous estimation of the volumetric heat capacity and thermal conductivities is also possible. Furthermore, this solution has potential applications to the solution of the inverse heat conduction problem in this cylindrical geometry. An interesting feature of the construction of this solution is that two different sets of eigenfunctions need to be considered in the eigenfunction expansion. These eigenfunctions sets depend on the relative values of the thermal diffusivity of the inner cylinder and the thermal diffusivity in the vertical direction of the outer cylindrical layer

  2. Symmetrical refolding of protein domains and subunits: example of the dimeric two-domain 3-isopropylmalate dehydrogenases.

    Science.gov (United States)

    Gráczer, Eva; Varga, Andrea; Melnik, Bogdan; Semisotnov, Gennady; Závodszky, Péter; Vas, Mária

    2009-02-10

    The refolding mechanism of the homodimeric two-domain 3-isopropylmalate dehydrogenase (IPMDH) from the organisms adapted to different temperatures, Thermus thermophilus (Tt), Escherichia coli (Ec), and Vibrio sp. I5 (Vib), is described. In all three cases, instead of a self-template mechanism, the high extent of symmetry and cooperativity in folding of subunits and domains have been concluded from the following experimental findings: The complex time course of refolding, monitored by Trp fluorescence, consists of a fast (the rate constant varies as 16.5, 25.0, and 11.7 min-1 in the order of Tt, Ec, and Vib IPMDHs) and a slow (the rate constants are 0.11, 0.80, and 0.23 min-1 for the three different species) first-order process. However, a burst increase of Trp fluorescence anisotropy to the value of the native states indicates that in all three cases the association of the two polypeptide chains occurs at the beginning of refolding. This dimeric species binds the substrate IPM, but the native-like interactions of the tertiary and quaternary structures are only formed during the slow phase of refolding, accompanied by further increase of protein fluorescence and appearance of FRET between Trp side chain(s) and the bound NADH. Joining the contacting arms of each subunit also takes place exclusively during this slow phase. To monitor refolding of each domain within the intact molecule of T. thermophilus IPMDH, Trp's (located in separate domains) were systematically replaced with Phe's. The refolding processes of the mutants were followed by measuring changes in Trp fluorescence and in FRET between the particular Trp and NADH. The high similarity of time courses (both in biphasicity and in their rates) strongly suggests cooperative folding of the domains during formation of the native three-dimensional structure of IPMDH.

  3. Immobilization of oxidases and their analytical applications

    International Nuclear Information System (INIS)

    Yasinzai, M.

    2007-01-01

    Immobilized enzymes are replacing their soluble counter-parts in nearly every field of application. These enzyme modifications have evolved from a research curiosity into an entire branch of Biotechnology. An immobilization method for flavin containing oxidases and their use in flow injection system is described. An electrochemical detector for H/sub 2/O/sub 2/ is assembled which is used effectively for the determination of glucose using more common glucose oxidase and the simultaneous determination of sugars. The combination of oxidases with hydrolases have been used for the determination of maltose and starch. (author)

  4. Artificial proteins as allosteric modulators of PDZ3 and SH3 in two-domain constructs: A computational characterization of novel chimeric proteins

    Czech Academy of Sciences Publication Activity Database

    Palani, K.; Pfeiferová, L.; Boušová, Kristýna; Bednárová, L.; Obšilová, Veronika; Vondrášek, J.

    2016-01-01

    Roč. 84, č. 10 (2016), s. 1358-1374 ISSN 0887-3585 Institutional support: RVO:67985823 Keywords : protein design * fusion proteins * PDZ3 * SH3 * Trp-cage * two domain proteins * molecular dynamics simulation * circular dichroism Subject RIV: BO - Biophysics Impact factor: 2.289, year: 2016

  5. Artificial proteins as allosteric modulators of PDZ3 and SH3 in two-domain constructs: A computational characterization of novel chimeric proteins

    Czech Academy of Sciences Publication Activity Database

    Palani, Kirubakaran; Pfeiferová, Lucie; Boušová, Kristýna; Bednárová, Lucie; Obšilová, V.; Vondrášek, Jiří

    2016-01-01

    Roč. 84, č. 10 (2016), s. 1358-1374 ISSN 0887-3585 Institutional support: RVO:61388963 Keywords : protein design * fusion proteins * PDZ3 * SH3 * Trp-cage * two domain proteins Subject RIV: CE - Biochemistry Impact factor: 2.289, year: 2016

  6. The antioxidant properties, cytotoxicity and monoamine oxidase ...

    African Journals Online (AJOL)

    ajl yemi

    2011-11-28

    Nov 28, 2011 ... Department of Pharmaceutical Chemistry, North-West University, Private Bag X6001, Potchefstroom 2520, ..... on the inhibition of the catabolism of serotonin, .... Structure of human monoamine oxidase B, a drug target for.

  7. Vanillyl-alcohol oxidase, a tasteful biocatalyst

    NARCIS (Netherlands)

    Heuvel, van den R.H.H.; Fraaije, M.W.; Mattevi, A.; Laane, C.; Berkel, van W.J.H.

    2001-01-01

    The covalent flavoenzyme vanillyl-alcohol oxidase (VAO) is a versatile biocatalyst. It converts a wide range of phenolic compounds by catalysing oxidation, deamination, demethylation, dehydrogenation and hydroxylation reactions. The production of natural vanillin, 4-hydroxybenzaldehyde, coniferyl

  8. Polyphenol Oxidase Enzyme and Inactivation Methods

    Directory of Open Access Journals (Sweden)

    Leman Yılmaz

    2018-03-01

    Full Text Available Polyphenol oxidase enzyme is found in vegetables and fruits, as well as in some animal organs and microorganisms. Polyphenol oxidase enzyme responsible for enzymatic browning is a group of copper proteins that catalyses the oxidation of phenolic compounds to quinones, which produce brown pigments, commonly found in fruits and vegetables. During the industrial preparation of fruits and vegetables, results of catalytic effect of polyphenol oxidase causes enzymatic browning. Enzymatic browning impairs the appearance of products containing phenolic compounds along with undesirable colour, odor and taste formation and significant loss of nutritional value of the products. This affects the acceptability of the products by the consumers and causes economic losses. In this review, some characteristics of polyphenol oxidase enzyme in different fruits and vegetables have been reviewed and information about chemical antibrowning agents, thermal applications, irradiation applications and alternative methods such as high pressure processing, pulse electric field, supercritical carbon dioxide and ultrasound applications to inactivate this enzyme has been presented.

  9. Genetics Home Reference: isolated sulfite oxidase deficiency

    Science.gov (United States)

    ... and Management Resources (1 link) GeneReview: Isolated Sulfite Oxidase Deficiency General Information from MedlinePlus (5 links) Diagnostic Tests Drug Therapy Genetic Counseling Palliative Care Surgery and ...

  10. Cooperativity in the two-domain arginine kinase from the sea anemone Anthopleura japonicus. II. Evidence from site-directed mutagenesis studies.

    Science.gov (United States)

    Tada, Hiroshi; Suzuki, Tomohiko

    2010-08-01

    The arginine kinase (AK) from the sea anemone Anthopleura japonicus has an unusual two-domain structure (contiguous dimer; denoted by D1-D2). In a previous report, we suggested cooperativity in the contiguous dimer, which may be a result of domain-domain interactions, using MBP-fused enzymes. To further understand this observation, we inserted six-Lys residues into the linker region of the two-domain AK (D1-K6-D2 mutant) using His-tagged enzyme. The dissociation constants, K(a) and K(ia), of the mutant were similar to those of the wild-type enzyme but the catalytic constant, k(cat), was decreased to 28% that of the wild-type, indicating that some of the domain-domain interactions are lost due to the six-Lys insertion. Y68 plays a major role in arginine binding in the catalytic pocket in Limulus AK, and introduction of mutation at the Y68 position virtually abolishes catalytic activity. Thus, the constructed D1(Y68G)-D2 and D1-D2(Y68G) mutants mimic the D1(inactive)-D2(active) and D1(active)-D2(inactive) enzymes, respectively. The k(cat) values of both Y68 mutants were decreased to 13-18% that of the wild-type enzyme, which is much less than the 50% level of the two-domain enzyme. Thus, it is clear that substrate-binding to both domains is necessary for full expression of activity. In other words, substrate-binding appears to act as the trigger of the functional cooperativity in two-domain AK. Copyright 2010 Elsevier B.V. All rights reserved.

  11. Crystallization of carbohydrate oxidase from Microdochium nivale

    Czech Academy of Sciences Publication Activity Database

    Dušková, Jarmila; Dohnálek, Jan; Skálová, Tereza; Ostergaard, L. H.; Fuglsang, C. C.; Kolenko, Petr; Štěpánková, Andrea; Hašek, Jindřich

    2009-01-01

    Roč. 65, č. 6 (2009), s. 638-640 ISSN 1744-3091 R&D Projects: GA AV ČR IAA500500701; GA ČR GA305/07/1073 Institutional research plan: CEZ:AV0Z40500505 Keywords : carbohydrate oxidase * crystallization * data processing Subject RIV: CD - Macromolecular Chemistry Impact factor: 0.551, year: 2009

  12. Investigation of antihemolytic, xanthine oxidase inhibition ...

    African Journals Online (AJOL)

    Abbreviations: SVEs: Salvia Verbenaca L. aerial part Extracts; CrE: Crud Extract; ChE: Chloroform Extract ; EAE: Ethyl Acetate Extract; AqE : Aqueous Extract ; ROS: Reactive Oxygen Spices; AAPH : 2,2, -Azobis (2-AmidinoPropane) Dihydrochloride ; DPPH: DiPhenyl- Picryl-Hydrazyl; XO: Xanthine Oxidase; Gen: Gentamicin ...

  13. Genetic defects of cytochrome c oxidase assembly

    Czech Academy of Sciences Publication Activity Database

    Pecina, Petr; Houšťková, H.; Hansíková, H.; Zeman, J.; Houštěk, Josef

    2004-01-01

    Roč. 53, Suppl. 1 (2004), s. S213-S223 ISSN 0862-8408 R&D Projects: GA ČR GA303/03/0749 Institutional research plan: CEZ:AV0Z5011922 Keywords : cytochrome c oxidase * mitochondrial disorders Subject RIV: FB - Endocrinology, Diabetology, Metabolism, Nutrition Impact factor: 1.140, year: 2004

  14. The antioxidant properties, cytotoxicity and monoamine oxidase ...

    African Journals Online (AJOL)

    Tarchonanthus camphoratus (camphor bush) has been widely used for numerous medicinal purposes. The aim of the present study was to evaluate the antioxidant properties, cytotoxicity and monoamine oxidase inhibition activities of the crude dichloromethane leaf extract of T. camphoratus. The antioxidant activities were ...

  15. N-Terminal Domains in Two-Domain Proteins Are Biased to Be Shorter and Predicted to Fold Faster Than Their C-Terminal Counterparts

    Directory of Open Access Journals (Sweden)

    Etai Jacob

    2013-04-01

    Full Text Available Computational analysis of proteomes in all kingdoms of life reveals a strong tendency for N-terminal domains in two-domain proteins to have shorter sequences than their neighboring C-terminal domains. Given that folding rates are affected by chain length, we asked whether the tendency for N-terminal domains to be shorter than their neighboring C-terminal domains reflects selection for faster-folding N-terminal domains. Calculations of absolute contact order, another predictor of folding rate, provide additional evidence that N-terminal domains tend to fold faster than their neighboring C-terminal domains. A possible explanation for this bias, which is more pronounced in prokaryotes than in eukaryotes, is that faster folding of N-terminal domains reduces the risk for protein aggregation during folding by preventing formation of nonnative interdomain interactions. This explanation is supported by our finding that two-domain proteins with a shorter N-terminal domain are much more abundant than those with a shorter C-terminal domain.

  16. A role for NADPH oxidase in antigen presentation

    Directory of Open Access Journals (Sweden)

    Gail J Gardiner

    2013-09-01

    Full Text Available The nicotinamide adenine dinucleotide phosphate (NADPH oxidase expressed in phagocytes is a multi-subunit enzyme complex that generates superoxide (O2.-. This radical is an important precursor of hydrogen peroxide (H2O2 and other reactive oxygen species (ROS needed for microbicidal activity during innate immune responses. Inherited defects in NADPH oxidase give rise to chronic granulomatous disease (CGD, a primary immunodeficiency characterized by recurrent infections and granulomatous inflammation. Interestingly, CGD, CGD carrier status, and oxidase gene polymorphisms have all been associated with autoinflammatory and autoimmune disorders, suggesting a potential role for NADPH oxidase in regulating adaptive immune responses. Here, NADPH oxidase function in antigen processing and presentation is reviewed. NADPH oxidase influences dendritic cell (DC crosspresentation by major histocompatibility complex class I molecules (MHC-I through regulation of the phagosomal microenvironment, while in B lymphocytes, NADPH oxidase alters epitope selection by major histocompatibility complex class II molecules (MHC-II.

  17. Role of pH in oxidase variability of Aeromonas hydrophila.

    OpenAIRE

    Hunt, L K; Overman, T L; Otero, R B

    1981-01-01

    Some strains of Aeromonas hydrophila may be oxidase negative or only weakly oxidase positive by the Kovacs method taken from the surface of a differential medium, such as MacConkey agar. Six strains of A. hydrophila, two oxidase variable, one oxidase constant, and three weakly oxidase positive on MacConkey agar, were studied to determine the cause of oxidase variability. The bacteriostatic dyes in MacConkey agar were considered possible inhibitors of the oxidase reaction. The concentration of...

  18. Plasma diamine oxidase activity in asthmatic children

    Directory of Open Access Journals (Sweden)

    Kyoichiro Toyoshima

    1996-01-01

    Full Text Available Histamine plays an important role in the development of asthmatic symptoms. Diamine oxidase (DAO histaminase, which inactivates histamine, is located in the intestine and kidney and is released into plasma. Plasma DAO activity in asthmatic children was measured by a recently developed high performance liquid chromatographic method using histamine as the DAO substrate. Diamine oxidase activity was higher in severely asthmatic children than in those with mild asthma. A time course study during the acute exacerbation phase revealed that DAO activity rose during acute asthmatic attacks and then decreased gradually over several days. Although the mechanisms of plasma DAO activity increase during acute asthmatic attacks could not be explained, data showed that plasma DAO activity is an important index of histamine metabolism in asthmatics and may relate to some mechanisms of acute exacerbation of airway inflammation. Consequently, fluctuations in plasma DAO can be used as one of various indices of instability in management of asthma.

  19. Lysyl Oxidase and the Tumor Microenvironment

    Directory of Open Access Journals (Sweden)

    Tong-Hong Wang

    2016-12-01

    Full Text Available The lysyl oxidase (LOX family of oxidases contains a group of extracellular copper-dependent enzymes that catalyze the cross-linking of collagen and elastin by oxidation, thus maintaining the rigidity and structural stability of the extracellular matrix (ECM. Aberrant expression or activation of LOX alters the cellular microenvironment, leading to many diseases, including atherosclerosis, tissue fibrosis, and cancer. Recently, a number of studies have shown that LOX is overexpressed in most cancers and that it is involved in the regulation of tumor progression and metastasis. In contrast, a few reports have also indicated the tumor-suppressing role of LOX. In this short review, we discuss recent research on the correlations between LOX and cancer. Further, the role of LOX in tumor microenvironment remodeling, tumorigenesis, and metastasis and the underlying mechanisms have also been elucidated.

  20. Monoamine oxidase inhibitors from Gentiana lutea.

    Science.gov (United States)

    Haraguchi, Hiroyuki; Tanaka, Yasumasa; Kabbash, Amal; Fujioka, Toshihiro; Ishizu, Takashi; Yagi, Akira

    2004-08-01

    Three monoamine oxidase (MAO) inhibitors were isolated from Gentiana lutea. Their structures were elucidated to be 3-3''linked-(2'-hydroxy-4-O-isoprenylchalcone)-(2'''-hydroxy-4''-O-isoprenyldihydrochalcone) (1), 2-methoxy-3-(1,1'-dimethylallyl)-6a,10a-dihydrobenzo(1,2-c)chroman-6-one and 5-hydroxyflavanone. These compounds, and the hydrolysis product of 1, displayed competitive inhibitory properties against MAO-B which was more effective than MAO-A.

  1. Imaging Monoamine Oxidase in the Human Brain

    Energy Technology Data Exchange (ETDEWEB)

    Fowler, J. S.; Volkow, N. D.; Wang, G-J.; Logan, Jean

    1999-11-10

    Positron emission tomography (PET) studies mapping monoamine oxidase in the human brain have been used to measure the turnover rate for MAO B; to determine the minimum effective dose of a new MAO inhibitor drug lazabemide and to document MAO inhibition by cigarette smoke. These studies illustrate the power of PET and radiotracer chemistry to measure normal biochemical processes and to provide information on the effect of drug exposure on specific molecular targets.

  2. Imaging Monoamine Oxidase in the Human Brain

    International Nuclear Information System (INIS)

    Fowler, J. S.; Volkow, N. D.; Wang, G-J.; Logan, Jean

    1999-01-01

    Positron emission tomography (PET) studies mapping monoamine oxidase in the human brain have been used to measure the turnover rate for MAO B; to determine the minimum effective dose of a new MAO inhibitor drug lazabemide and to document MAO inhibition by cigarette smoke. These studies illustrate the power of PET and radiotracer chemistry to measure normal biochemical processes and to provide information on the effect of drug exposure on specific molecular targets

  3. Identification of aldehyde oxidase 1 and aldehyde oxidase homologue 1 as dioxin-inducible genes

    International Nuclear Information System (INIS)

    Rivera, Steven P.; Choi, Hyun Ho; Chapman, Brett; Whitekus, Michael J.; Terao, Mineko; Garattini, Enrico; Hankinson, Oliver

    2005-01-01

    Aldehyde oxidases are a family of highly related molybdo-flavoenzymes acting upon a variety of compounds of industrial and medical importance. We have identified aldehyde oxidase 1 (AOX1) as a 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) inducible gene in the mouse hepatoma cell line Hepa-1. AOX1 mRNA levels were not increased by dioxin in mutant derivatives of the Hepa-1 cell line lacking either functional aryl hydrocarbon receptor (AHR) or aryl hydrocarbon receptor nuclear translocator (ARNT) proteins, thus demonstrating that transcriptional induction of AOX1 in response to dioxin occurs through the AHR pathway. Dioxin induction of AOX1 mRNA was also observed in mouse liver. In addition, levels of AOX1 protein as well as those of aldehyde oxidase homologue 1 (AOH1), a recently identified homolog of AOX1, were elevated in mouse liver in response to dioxin. Employing an aldehyde oxidase specific substrate, AOX1/AOH1 activity was shown to be induced by dioxin in mouse liver. This activity was inhibited by a known inhibitor of aldehyde oxidases, and eliminated by including tungstate in the mouse diet, which is known to lead to inactivation of molybdoflavoenzymes, thus confirming that the enzymatic activity was attributable to AOX1/AOH1. Our observations thus identify two additional xenobiotic metabolizing enzymes induced by dioxin

  4. Modulation of NADPH oxidase activity by known uraemic retention solutes

    DEFF Research Database (Denmark)

    Schulz, Anna Marta; Terne, Cindy; Jankowski, Vera

    2014-01-01

    chloride (DPI), an inhibitor of NADPH oxidase. The effect on enzymatic activity of NADPH oxidase was quantified within an incubation time of 120 min. RESULTS: Thirty-nine of the 48 uraemic retention solutes tested had a significant decreasing effect on NADPH oxidase activity. Oxalate has been characterized......BACKGROUND: Uraemia and cardiovascular disease appear to be associated with an increased oxidative burden. One of the key players in the genesis of reactive oxygen species (ROS) is nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Based on initial experiments demonstrating a decreased...... inhibitory effect on NADPH oxidase activity in the presence of plasma from patients with CKD-5D after dialysis compared with before dialysis, we investigated the effect of 48 known and commercially available uraemic retention solutes on the enzymatic activity of NADPH oxidase. METHODS: Mononuclear leucocytes...

  5. Chemoenzymatic combination of glucose oxidase with titanium silicalite -1

    DEFF Research Database (Denmark)

    Vennestrøm, Peter Nicolai Ravnborg; Taarning, Esben; Christensen, Claus H.

    2010-01-01

    Zeozymes: A proof-of-concept is presented for the chemoenzymatic combination of titanium silicalite-1 zeolite with glucose oxidase. In this combination, glucose is oxidized to gluconic acid and the H2O2 byproduct formed in situ is used for the simultaneous oxidation of chemical substrates. Both...... a soluble glucose oxidase and a truly integrated heterogeneous combination whereby the oxidase enzyme is anchored onto the zeolite surface are reported....

  6. Plasma diamine oxidase levels in pregnancy complicated by threatened abortion.

    Science.gov (United States)

    Legge, M; Duff, G B

    1981-02-01

    Plasma diamine oxidase levels were assayed in 66 patients who presented with pregnancy complicated by threatened abortion. Levels within the normal range were associated with continuing pregnancies, whereas levels below the normal range were associated with subsequent abortion. Among those patients in whom gestation was greater than eight weeks, 66.6% of diamine oxidase levels correctly predicted the pregnancy outcome. Assay of the diamine oxidase levels at eight weeks of gestation or less gave little useful information.

  7. Plasma diamine oxidase levels in pregnancy complicated by threatened abortion.

    OpenAIRE

    Legge, M; Duff, G B

    1981-01-01

    Plasma diamine oxidase levels were assayed in 66 patients who presented with pregnancy complicated by threatened abortion. Levels within the normal range were associated with continuing pregnancies, whereas levels below the normal range were associated with subsequent abortion. Among those patients in whom gestation was greater than eight weeks, 66.6% of diamine oxidase levels correctly predicted the pregnancy outcome. Assay of the diamine oxidase levels at eight weeks of gestation or less ga...

  8. Gravity Responsive NADH Oxidase of the Plasma Membrane

    Science.gov (United States)

    Morre, D. James (Inventor)

    2002-01-01

    A method and apparatus for sensing gravity using an NADH oxidase of the plasma membrane which has been found to respond to unit gravity and low centrifugal g forces. The oxidation rate of NADH supplied to the NADH oxidase is measured and translated to represent the relative gravitational force exerted on the protein. The NADH oxidase of the plasma membrane may be obtained from plant or animal sources or may be produced recombinantly.

  9. Transfer of C-terminal residues of human apolipoprotein A-I to insect apolipophorin III creates a two-domain chimeric protein with enhanced lipid binding activity.

    Science.gov (United States)

    Horn, James V C; Ellena, Rachel A; Tran, Jesse J; Beck, Wendy H J; Narayanaswami, Vasanthy; Weers, Paul M M

    2017-08-01

    Apolipophorin III (apoLp-III) is an insect apolipoprotein (18kDa) that comprises a single five-helix bundle domain. In contrast, human apolipoprotein A-I (apoA-I) is a 28kDa two-domain protein: an α-helical N-terminal domain (residues 1-189) and a less structured C-terminal domain (residues 190-243). To better understand the apolipoprotein domain organization, a novel chimeric protein was engineered by attaching residues 179 to 243 of apoA-I to the C-terminal end of apoLp-III. The apoLp-III/apoA-I chimera was successfully expressed and purified in E. coli. Western blot analysis and mass spectrometry confirmed the presence of the C-terminal domain of apoA-I within the chimera. While parent apoLp-III did not self-associate, the chimera formed oligomers similar to apoA-I. The chimera displayed a lower α-helical content, but the stability remained similar compared to apoLp-III, consistent with the addition of a less structured domain. The chimera was able to solubilize phospholipid vesicles at a significantly higher rate compared to apoLp-III, approaching that of apoA-I. The chimera was more effective in protecting phospholipase C-treated low density lipoprotein from aggregation compared to apoLp-III. In addition, binding interaction of the chimera with phosphatidylglycerol vesicles and lipopolysaccharides was considerably improved compared to apoLp-III. Thus, addition of the C-terminal domain of apoA-I to apoLp-III created a two-domain protein, with self-association, lipid and lipopolysaccharide binding properties similar to apoA-I. The apoA-I like behavior of the chimera indicate that these properties are independent from residues residing in the N-terminal domain of apoA-I, and that they can be transferred from apoA-I to apoLp-III. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. NADPH oxidase: an enzyme for multicellularity?

    Science.gov (United States)

    Lalucque, Hervé; Silar, Philippe

    2003-01-01

    Multicellularity has evolved several times during the evolution of eukaryotes. One evolutionary pressure that permits multicellularity relates to the division of work, where one group of cells functions as nutrient providers and the other in specialized roles such as defence or reproduction. This requires signalling systems to ensure harmonious development of multicellular structures. Here, we show that NADPH oxidases are specifically present in organisms that differentiate multicellular structures during their life cycle and are absent from unicellular life forms. The biochemical properties of these enzymes make them ideal candidates for a role in intercellular signalling.

  11. The two-domain LysX protein of Mycobacterium tuberculosis is required for production of lysinylated phosphatidylglycerol and resistance to cationic antimicrobial peptides.

    Directory of Open Access Journals (Sweden)

    Erin Maloney

    2009-07-01

    Full Text Available The well-recognized phospholipids (PLs of Mycobacterium tuberculosis (Mtb include several acidic species such as phosphatidylglycerol (PG, cardiolipin, phosphatidylinositol and its mannoside derivatives, in addition to a single basic species, phosphatidylethanolamine. Here we demonstrate that an additional basic PL, lysinylated PG (L-PG, is a component of the PLs of Mtb H37Rv and that the lysX gene encoding the two-domain lysyl-transferase (mprF-lysyl-tRNA synthetase (lysU protein is responsible for L-PG production. The Mtb lysX mutant is sensitive to cationic antibiotics and peptides, shows increased association with lysosome-associated membrane protein-positive vesicles, and it exhibits altered membrane potential compared to wild type. A lysX complementing strain expressing the intact lysX gene, but not one expressing mprF alone, restored the production of L-PG and rescued the lysX mutant phenotypes, indicating that the expression of both proteins is required for LysX function. The lysX mutant also showed defective growth in mouse and guinea pig lungs and showed reduced pathology relative to wild type, indicating that LysX activity is required for full virulence. Together, our results suggest that LysX-mediated production of L-PG is necessary for the maintenance of optimal membrane integrity and for survival of the pathogen upon infection.

  12. Bioconjugates of luminescent CdSe-ZnS quantum dots with an engineered two-domain protein G for use in fluoroimmunoassays

    Science.gov (United States)

    Tran, Phan T.; Goldman, Ellen R.; Mattoussi, Hedi M.; Anderson, George P.; Mauro, J. Matthew

    2001-06-01

    Colloidal semiconductor quantum dots (QDs) seem suitable for labeling certain biomolecules for use in fluorescent tagging applications, such as fluoro-immunoassays. Compared to organic dye labels, Qds are resistant to photo-degradation, and these luminescent nanoparticles have size-dependent emission spectra spanning a wide range of wavelengths in the visible and near IR. We previously described an electrostatic self-assembly approach for conjugating highly luminescent colloidal CdSe-ZnS core-shell Qds with engineered two-domain recombinant proteins. Here we describe the application of this approach to prepare QD conjugates with the (Beta) 2 immunoglobin G (IgG) binding domain of streptococcal protein G (PG) appended with a basic lucine zipper attachment domain (PG-zb). We also demonstrate that the QD/PG conjugates retain their ability to bind IgG antibodies, and that a specific antibody coupled to QD via the PG functional domain efficiently binds its antigen. These preliminary results indicate that electrostatically self-assembled QD/PG-zb/IgG bioconjugates can be used in fluoro-immunoassays.

  13. Crystallization of carbohydrate oxidase from Microdochium nivale

    International Nuclear Information System (INIS)

    Dušková, Jarmila; Dohnálek, Jan; Skálová, Tereza; Østergaard, Lars Henrik; Fuglsang, Claus Crone; Kolenko, Petr; Štěpánková, Andrea; Hašek, Jindřich

    2009-01-01

    Industrially used carbohydrate oxidase was successfully crystallized in several forms, diffraction data suitable for structural analysis were collected. Microdochium nivale carbohydrate oxidase was produced by heterologous recombinant expression in Aspergillus oryzae, purified and crystallized. The enzyme crystallizes with varying crystal morphologies depending on the crystallization conditions. Several different crystal forms were obtained using the hanging-drop vapour-diffusion method, two of which were used for diffraction measurements. Hexagon-shaped crystals (form I) diffracted to 2.66 Å resolution, with unit-cell parameters a = b = 55.7, c = 610.4 Å and apparent space group P6 2 22. Analysis of the data quality showed almost perfect twinning of the crystals. Attempts to solve the structure by molecular replacement did not give satisfactory results. Recently, clusters of rod-shaped crystals (form II) were grown in a solution containing PEG MME 550. These crystals belonged to the monoclinic system C2, with unit-cell parameters a = 132.9, b = 56.6, c = 86.5 Å, β = 95.7°. Data sets were collected to a resolution of 2.4 Å. The structure was solved by the molecular-replacement method. Model refinement is currently in progress

  14. Cytochemical Localization of Glucose Oxidase in Peroxisomes of Aspergillus niger

    NARCIS (Netherlands)

    Veenhuis, Marten; Dijken, Johannes Pieter van

    1980-01-01

    The subcellular localization of glucose oxidase (E.C. 1.1.3.4) in mycelia of Aspergillus niger has been investigated using cytochemical staining techniques. Mycelia from fermenter cultures, which produced gluconic acid from glucose, contained elevated levels of glucose oxidase and catalase. Both

  15. Xanthine oxidase in human skeletal muscle following eccentric exercise

    DEFF Research Database (Denmark)

    Hellsten, Ylva; Frandsen, Ulrik; Orthenblad, N.

    1997-01-01

    the increase in xanthine oxidase in the muscle there were no detectable changes in the levels of muscle malondialdehyde or in plasma antioxidant capacity up to 4 days post-exercise. 5. It is concluded that eccentric exercise leads to an increased level of xanthine oxidase in human muscle and that the increase...

  16. Laboratory-evolved vanillyl-alcohol oxidase produces natural vanillin

    NARCIS (Netherlands)

    Heuvel, van den R.H.H.; Berg, van den W.A.M.; Rovida, S.; Berkel, van W.J.H.

    2004-01-01

    The flavoenzyme vanillyl-alcohol oxidase was subjected to random mutagenesis to generate mutants with enhanced reactivity to creosol (2-methoxy-4-methylphenol). The vanillyl-alcohol oxidase-mediated conversion of creosol proceeds via a two-step process in which the initially formed vanillyl alcohol

  17. Optimization of glucose oxidase production by Aspergillus niger

    African Journals Online (AJOL)

    user

    2011-02-28

    Feb 28, 2011 ... manganese, cobalt, thioglycolic acid, and gluconic acid according to (Liu et al., .... In this experiment duplicate media of glucose 10% were adjusted at different ... Glucose oxidase as a pharmaceutical anti oxidant Drug. Devt. ... Plush KS, Hellmuth K, Rinas U (1996). kinetics of glucose oxidase excretion by ...

  18. 21 CFR 866.2420 - Oxidase screening test for gonorrhea.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Oxidase screening test for gonorrhea. 866.2420... screening test for gonorrhea. (a) Identification. An oxidase screening test for gonorrhea is an in vitro... of gonorrhea. (b) Classification. Class III (premarket approval) (transitional device). (c) Date PMA...

  19. Modulation of NADPH oxidase activity by known uraemic retention solutes.

    Science.gov (United States)

    Schulz, Anna Marta; Terne, Cindy; Jankowski, Vera; Cohen, Gerald; Schaefer, Mandy; Boehringer, Falko; Tepel, Martin; Kunkel, Desiree; Zidek, Walter; Jankowski, Joachim

    2014-08-01

    Uraemia and cardiovascular disease appear to be associated with an increased oxidative burden. One of the key players in the genesis of reactive oxygen species (ROS) is nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Based on initial experiments demonstrating a decreased inhibitory effect on NADPH oxidase activity in the presence of plasma from patients with CKD-5D after dialysis compared with before dialysis, we investigated the effect of 48 known and commercially available uraemic retention solutes on the enzymatic activity of NADPH oxidase. Mononuclear leucocytes isolated from buffy coats of healthy volunteers were isolated, lysed and incubated with NADH in the presence of plasma from healthy controls and patients with CKD-5D. Furthermore, the leucocytes were lysed and incubated in the presence of uraemic retention solute of interest and diphenyleneiodonium chloride (DPI), an inhibitor of NADPH oxidase. The effect on enzymatic activity of NADPH oxidase was quantified within an incubation time of 120 min. Thirty-nine of the 48 uraemic retention solutes tested had a significant decreasing effect on NADPH oxidase activity. Oxalate has been characterized as the strongest inhibitor of NADPH oxidase (90% of DPI inhibition). Surprisingly, none of the uraemic retention solutes we investigated was found to increase NADPH oxidase activity. Furthermore, plasma from patients with CKD-5D before dialysis caused significantly higher inhibitory effect on NADPH oxidase activity compared with plasma from healthy subjects. However, this effect was significantly decreased in plasma from patients with CKD-5D after dialysis. The results of this study show that uraemic retention solutes modulated the activity of the NADPH oxidase. The results of this study might be the basis for the development of inhibitors applicable as drug in the situation of increased oxidative stress. © 2014 Stichting European Society for Clinical Investigation Journal Foundation.

  20. In Situ Enzymatically Generated Photoswitchable Oxidase Mimetics and Their Application for Colorimetric Detection of Glucose Oxidase.

    Science.gov (United States)

    Cao, Gen-Xia; Wu, Xiu-Ming; Dong, Yu-Ming; Li, Zai-Jun; Wang, Guang-Li

    2016-07-09

    In this study, a simple and amplified colorimetric assay is developed for the detection of the enzymatic activity of glucose oxidase (GOx) based on in situ formation of a photoswitchable oxidase mimetic of PO₄(3-)-capped CdS quantum dots (QDs). GOx catalyzes the oxidation of 1-thio-β-d-glucose to give 1-thio-β-d-gluconic acid which spontaneously hydrolyzes to β-d-gluconic acid and H₂S; the generated H₂S instantly reacts with Cd(2+) in the presence of Na₃PO₄ to give PO₄(3-)-stabilized CdS QDs in situ. Under visible-light (λ ≥ 400 nm) stimulation, the PO₄(3-)-capped CdS QDs are a new style of oxidase mimic derived by producing some active species, such as h⁺, (•)OH, O₂(•-) and a little H₂O₂, which can oxidize the typical substrate (3,3,5,5-tetramethylbenzydine (TMB)) with a color change. Based on the GOx-triggered growth of the oxidase mimetics of PO₄(3-)-capped CdS QDs in situ, we developed a simple and amplified colorimetric assay to probe the enzymatic activity of GOx. The proposed method allowed the detection of the enzymatic activity of GOx over the range from 25 μg/L to 50 mg/L with a low detection limit of 6.6 μg/L. We believe the PO₄(3-)-capped CdS QDs generated in situ with photo-stimulated enzyme-mimicking activity may find wide potential applications in biosensors.

  1. Visualization of monoamine oxidase in human brain

    Energy Technology Data Exchange (ETDEWEB)

    Fowler, J.S.; Volkow, N.D.; Wang, G.J.; Pappas, N.; Shea, C.; MacGregor, R.R.; Logan, J.

    1996-12-31

    Monoamine oxidase is a flavin enzyme which exists in two subtypes, MAO A and MAO B. In human brain MAO B predominates and is largely compartmentalized in cell bodies of serotonergic neurons and glia. Regional distribution of MAO B was determined by positron computed tomography with volunteers after the administration of deuterium substituted [11C]L-deprenyl. The basal ganglia and thalamus exhibited the greatest concentrations of MAO B with intermediate levels in the frontal cortex and cingulate gyrus while lowest levels were observed in the parietal and temporal cortices and cerebellum. We observed that brain MAO B increases with are in health normal subjects, however the increases were generally smaller than those revealed with post-mortem studies.

  2. An ultrafiltration assay for lysyl oxidase

    International Nuclear Information System (INIS)

    Shackleton, D.R.; Hulmes, D.J.

    1990-01-01

    A modification of the original microdistillation assay for lysyl oxidase is described in which Amicon C-10 microconcentrators are used to separate, by ultrafiltration, the 3H-labeled products released from a [4,5-3H]-lysine-labeled elastin substrate. Enzyme activity is determined by scintillation counting of the ultrafiltrate, after subtraction of radioactivity released in the presence of beta-aminopropionitrile, a specific inhibitor of the enzyme. Conditions are described which optimize both the sensitivity and the efficient use of substrate. The assay shows linear inhibition of activity in up to 1 M urea; hence, as the enzyme is normally diluted in the assay, samples in 6 M urea can be assayed directly, without prior dialysis, and corrected for partial inhibition. Comparable results are obtained when enzyme activity is assayed by ultrafiltration or microdistillation. The assay is simple and convenient and, by using disposable containers throughout, it eliminates the need for time-consuming decontamination of radioactive glassware

  3. Evaluation of oxalate decarboxylase and oxalate oxidase for industrial applications.

    Science.gov (United States)

    Cassland, Pierre; Sjöde, Anders; Winestrand, Sandra; Jönsson, Leif J; Nilvebrant, Nils-Olof

    2010-05-01

    Increased recirculation of process water has given rise to problems with formation of calcium oxalate incrusts (scaling) in the pulp and paper industry and in forest biorefineries. The potential in using oxalate decarboxylase from Aspergillus niger for oxalic acid removal in industrial bleaching plant filtrates containing oxalic acid was examined and compared with barley oxalate oxidase. Ten different filtrates from chemical pulping were selected for the evaluation. Oxalate decarboxylase degraded oxalic acid faster than oxalate oxidase in eight of the filtrates, while oxalate oxidase performed better in one filtrate. One of the filtrates inhibited both enzymes. The potential inhibitory effect of selected compounds on the enzymatic activity was tested. Oxalate decarboxylase was more sensitive than oxalate oxidase to hydrogen peroxide. Oxalate decarboxylase was not as sensitive to chlorate and chlorite as oxalate oxidase. Up to 4 mM chlorate ions, the highest concentration tested, had no inhibitory effect on oxalate decarboxylase. Analysis of the filtrates suggests that high concentrations of chlorate present in some of the filtrates were responsible for the higher sensitivity of oxalate oxidase in these filtrates. Oxalate decarboxylase was thus a better choice than oxalate oxidase for treatment of filtrates from chlorine dioxide bleaching.

  4. Quantitation of immunoadsorbed flavoprotein oxidases by luminol-mediated chemiluminescence.

    Science.gov (United States)

    Hinkkanen, A; Maly, F E; Decker, K

    1983-04-01

    The detection of the flavoenzymes 6-hydroxy-L-nicotine oxidase and 6-hydroxy-D-nicotine oxidase at the sub-femtomol level was achieved by coupling the reaction of the immunoadsorbed proteins to the peroxidase-catalysed oxidation of luminol. The H2O2-producing oxidases retained their full activity when bound to the respective immobilized antibodies. This fact allowed the concentration of the enzymes from very dilute solutions and the quantitative assay of their activities in the microU range. Due to strict stereoselectivity and the absence of immunological cross-reactivity, the two flavoproteins could be determined in the same solution. This method was used to measure the 6-hydroxy-D-nicotine oxidase and 6-hydroxy-L-nicotine oxidase activities in Escherichia coli RR1 and different Arthrobacter strains cultured under non-inducing conditions. The same activity ratio of 6-hydroxy-L-nicotine oxidase/6-hydroxy-D-nicotine oxidase as in D L-nicotine-induced cells of A. oxidans was observed in non-induced wild type and in riboflavin-requiring (rf-) mutant cells of this aerob.

  5. Platelet monoamine oxidase: specific activity and turnover number in headache

    International Nuclear Information System (INIS)

    Summers, K.M.; Brown, G.K.; Craig, I.W.; Peatfield, R.; Rose, F.C.

    1982-01-01

    Monoamine oxidase turnover numbers (molecules of substrate converted to product per minute per active site) have been calculated for the human platelet enzyme using [ 3 H]pargyline. Headache patients with high and low monoamine oxidase specific activities relative to controls were found to have turnover numbers very close to those for controls. This finding suggests that their specific activities vary because of differences in the concentration of active monoamine oxidase molecules, rather than differences in the ability of those enzyme molecules to catalyse the deamination reaction. (Auth.)

  6. Effect of a heme oxygenase-1 inducer on NADPH oxidase ...

    African Journals Online (AJOL)

    Effect of a heme oxygenase-1 inducer on NADPH oxidase expression in ... and immunohistochemistry of hepatic NOX1 and NOX4 were investigated in week 4. ... (HO-1 inhibitor) administration caused upregulation of NOX gene expression ...

  7. Effects of glucose oxidase on the growth performance, serum ...

    African Journals Online (AJOL)

    Martina

    2016-02-06

    Feb 6, 2016 ... The experiment was conducted to investigate the effects of diets supplemented with ... Keywords: Glucose oxidase, intestinal health, performance, swine ..... This work was supported by the National High-Tech Research and ...

  8. Structure and activity of Aspergillus nidulans copper amine oxidase

    DEFF Research Database (Denmark)

    McGrath, Aaron P; Mithieux, Suzanne M; Collyer, Charles A

    2011-01-01

    Aspergillus nidulans amine oxidase (ANAO) has the unusual ability among the family of copper and trihydroxyphenylalanine quinone-containing amine oxidases of being able to oxidize the amine side chains of lysine residues in large peptides and proteins. We show here that in common with the related...... enzyme from the yeast Pichia pastoris, ANAO can promote the cross-linking of tropoelastin and oxidize the lysine residues in α-casein proteins and tropoelastin. The crystal structure of ANAO, the first for a fungal enzyme in this family, has been determined to a resolution of 2.4 Å. The enzyme is a dimer...... with the archetypal fold of a copper-containing amine oxidase. The active site is the most open of any of those of the structurally characterized enzymes in the family and provides a ready explanation for its lysine oxidase-like activity....

  9. Gene cloning and characterization of NADH oxidase from ...

    African Journals Online (AJOL)

    use

    2011-12-07

    Dec 7, 2011 ... potent inhibitors of NADH oxidases, silver nitrate and potassium cyanide did not show any significant ... anaerobes, a class of organisms that have not been ... DNA and amino acid sequence analyses were performed using.

  10. Production of rabbit antibodies against purified Glucose oxidase

    OpenAIRE

    Zia,Muhammad Anjum; Ain,Qurat-ul; Iftikhar,Tehreema; Abbas,Rao Zahid; Rahman,Khalil-ur

    2012-01-01

    Glucose oxidase is an active oxygen species generating enzyme produced from Aspergillus niger grown in submerged fermentation. Disintegration of the mycelium resulted in high glucose oxidase activity that was subjected to ammonium sulfate precipitation at 60-85% saturation rates that resulted to 6.14 U mg -1 specific activity. Purification of enzyme by anion exchange column (DEAE-Cellulose) resulted into 22.53 U mg-1 specific activity and 10.27 fold purification. This was applied to sephadex ...

  11. Confirmation of a blocked amino terminus of sulfhydryl oxidase

    International Nuclear Information System (INIS)

    Janolino, V.G.; Morrison-Rowe, S.J.; Swaisgood, H.E.

    1990-01-01

    The isolation of sulfhydryl oxidase from bovine milk in a suitably pure form for sequencing was carried out by transient covalent affinity chromatography of diafiltered whey using cysteinylsuccinamidopropyl-glass as matrix. The glutathione-eluted proteins were separated by SDS-PAGE. By radiolabeling the affinity chromatography-purified enzyme with [ 14 C]iodoacetate before subjecting to SDS-PAGE, the sulfhydryl oxidase band was identified, because sulfhydryl oxidase is known to be inactivated by alkylation of one sulfhydryl group per mole. The results confirmed that sulfhydryl oxidase corresponds to the 85 (± 5)-kDa band observed on SDS-PAGE. The protein band corresponding to radiolabeled sulfhydryl oxidase was recovered from SDS-PAGE gels by electrophoretic elution and by electroblotting on polyvinylidene difluoride membrane and subjected to gas phase sequencing. Precautions were taken during electrophoretic elution to prevent reactions that result in N-terminal blocking. Both methods of protein recovery yielded negative results when subjected to sequence analysis indicating that the N-terminus of sulfhydryl oxidase is blocked

  12. MONOAMINE OXIDASE: RADIOTRACER DEVELOPMENT AND HUMAN STUDIES.

    Energy Technology Data Exchange (ETDEWEB)

    FOWLER,J.S.; LOGAN,J.; VOLKOW,N.D.; WANG,G.J.; MACGREGOR,R.R.; DING,Y.S.

    2000-09-28

    PET is uniquely capable of providing information on biochemical transformations in the living human body. Although most of the studies of monoamine oxidase (MAO) have focused on measurements in the brain, the role of peripheral MAO as a phase 1 enzyme for the metabolism of drugs and xenobiotics is gaining attention (Strolin Benedetti and Tipton, 1998; Castagnoli et al., 1997.). MAO is well suited for this role because its concentration in organs such as kidneys, liver and digestive organs is high sometimes exceeding that in the brain. Knowledge of the distribution of the MAO subtypes within different organs and different cells is important in determining which substrates (and which drugs and xenobiotics) have access to which MAO subtypes. The highly variable subtype distribution with different species makes human studies even more important. In addition, the deleterious side effects of combining MAO inhibitors with other drugs and with foodstuffs makes it important to know the MAO inhibitory potency of different drugs both in the brain and in peripheral organs (Ulus et al., 2000). Clearly PET can play a role in answering these questions, in drug research and development and in discovering some of the factors which contribute to the highly variable MAO levels in different individuals.

  13. Covalently bound phosphate residues in bovine milk xanthine oxidase and in glucose oxidase from Aspergillus niger: A reevaluation

    Energy Technology Data Exchange (ETDEWEB)

    Johnson, J.L.; Rajagopalan, K.V. (Duke Univ. Medical Center, Durham, NC (USA)); London, R.E. (National Institute of Environmental Health Science, Research Triangle Park, NC (USA))

    1989-09-01

    The reported presence of covalently bound phosphate residues in flavoproteins has significant implications with regard to the catalytic mechanisms and structural stability of the specific enzymes themselves and in terms of general cellular metabolic regulation. These considerations have led to a reevaluation of the presence of covalently bound phosphorus in the flavoproteins xanthine oxidase and glucose oxidase. Milk xanthine oxidase purified by a procedure that includes anion-exchange chromatography is shown to contain three phosphate residues. All three are noncovalently associated with the protein, two with the FAD cofactor, and one with the molybdenum cofactor. Results of chemical analysis and {sup 31}P NMR spectroscopy indicate that enzyme purified by this method contains no phosphoserine residues. Xanthine oxidase preparations purified by chromatography on calcium phosphate gel in place of DEAE-Sephadex yielded higher phosphate-to-protein ratios, which could be reduced to the expected values by additional purification on a folate affinity column. Highly active, highly purified preparations of glucose oxidase are shown to contain only the two phosphate residues of the FAD cofactor. The covalently bound bridging phosphate reported by others may arise in aged or degraded preparations of the enzyme but appears not to be a constituent of functional glucose oxidase. These results suggest that the presence of covalent phosphate residues in other flavoproteins should be rigorously reevaluated as well.

  14. Covalently bound phosphate residues in bovine milk xanthine oxidase and in glucose oxidase from Aspergillus niger: A reevaluation

    International Nuclear Information System (INIS)

    Johnson, J.L.; Rajagopalan, K.V.; London, R.E.

    1989-01-01

    The reported presence of covalently bound phosphate residues in flavoproteins has significant implications with regard to the catalytic mechanisms and structural stability of the specific enzymes themselves and in terms of general cellular metabolic regulation. These considerations have led to a reevaluation of the presence of covalently bound phosphorus in the flavoproteins xanthine oxidase and glucose oxidase. Milk xanthine oxidase purified by a procedure that includes anion-exchange chromatography is shown to contain three phosphate residues. All three are noncovalently associated with the protein, two with the FAD cofactor, and one with the molybdenum cofactor. Results of chemical analysis and 31 P NMR spectroscopy indicate that enzyme purified by this method contains no phosphoserine residues. Xanthine oxidase preparations purified by chromatography on calcium phosphate gel in place of DEAE-Sephadex yielded higher phosphate-to-protein ratios, which could be reduced to the expected values by additional purification on a folate affinity column. Highly active, highly purified preparations of glucose oxidase are shown to contain only the two phosphate residues of the FAD cofactor. The covalently bound bridging phosphate reported by others may arise in aged or degraded preparations of the enzyme but appears not to be a constituent of functional glucose oxidase. These results suggest that the presence of covalent phosphate residues in other flavoproteins should be rigorously reevaluated as well

  15. Cytochemical localization of catalase and several hydrogen peroxide-producing oxidases in the nucleoids and matrix of rat liver peroxisomes

    NARCIS (Netherlands)

    Veenhuis, M.; Wendelaar Bonga, S.E.

    1979-01-01

    The distribution of catalase, amino acid oxidase, α-hydroxy acid oxidase, urate oxidase and alcohol oxidase was studied cytochemically in rat hepatocytes. The presence of catalase was demonstrated with the conventional diaminobenzidine technique. Oxidase activities were visualized with methods based

  16. Serum diamine oxidase activity in patients with histamine intolerance.

    Science.gov (United States)

    Manzotti, G; Breda, D; Di Gioacchino, M; Burastero, S E

    2016-03-01

    Intolerance to various foods, excluding bona fide coeliac disease and lactose intolerance, represents a growing cause of patient visits to allergy clinics.Histamine intolerance is a long-known, multifaceted clinical condition triggered by histamine-rich foods and alcohol and/or by drugs that liberate histamine or block diamine oxidase (DAO), the main enzyme involved in the metabolism of ingested histamine. Histamine limitation diets impose complex, non-standardized restrictions that may severely impact the quality of life of patients. We retrospectively evaluated 14 patients who visited allergy outpatient facilities in northern Italy with a negative diagnosis for IgE-mediated food hypersensitivity, coeliac disease, conditions related to gastric hypersecretion, and systemic nickel hypersensitivity, and who previously underwent a histamine limitation diet with benefits for their main symptoms. Serum diamine oxidase levels and the clinical response to diamine oxidase supplementation were investigated. We found that 10 out of 14 patients had serum DAO activityintolerance. Moreover, 13 out of 14 patients subjectively reported a benefit in at least one of the disturbances related to food intolerances following diamine oxidase supplementation. The mean value (±SD) of diamine oxidase activity in the cohort of patients with histamine intolerance symptoms was 7.04±6.90 U/mL compared to 39.50±18.16 U/mL in 34 healthy controls (P=0.0031). In patients with symptoms triggered by histamine-rich food, measuring the serum diamine oxidase activity can help identify subjects who can benefit from a histamine limitation diet and/or diamine oxidase supplementation.Properly designed, controlled studies investigating histamine intolerance that include histamine provocation are indispensable for providing insights into the area of food intolerances, which are currently primarily managed with non-scientific approaches in Italy. © The Author(s) 2015.

  17. Monoamine Oxidase B Inhibitors in Parkinson's Disease.

    Science.gov (United States)

    Dezsi, Livia; Vecsei, Laszlo

    2017-01-01

    Parkinson's disease (PD) is a neurodegenerative disorder with a prevalence increasing with age. Oxidative stress and glutamate toxicity are involved in its pathomechanism. There are still many unmet needs of PD patients, including the alleviation of motor fluctuations and dyskinesias, and the development of therapies with neuroprotective potential. To give an overview of the pharmacological properties, the efficacy and safety of the monoamine oxidase B (MAO-B) inhibitors in the treatment of PD, with special focus on the results of randomized clinical trials. A literature search was conducted in PubMed for 'PD treatment', 'MAO-B inhibitors', 'selegiline', 'rasagiline', 'safinamide' and 'clinical trials' with 'MAO-B inhibitors' in 'Parkinson' disease'. MAO-B inhibitors have a favorable pharmacokinetic profile, improve the dopamine deficient state and may have neuroprotective properties. Safinamide exhibits an anti-glutamatergic effect as well. When applied as monotherapy, MAO-B inhibitors provide a modest, but significant improvement of motor function and delay the need for levodopa. Rasagiline and safinamide were proven safe and effective when added to a dopamine agonist in early PD. As add-on to levodopa, MAO-B inhibitors significantly reduced off-time and were comparable in efficacy to COMT inhibitors. Improvements were achieved as regards certain non-motor symptoms as well. Due to the efficacy shown in clinical trials and their favorable side-effect profile, MAO-B inhibitors are valuable drugs in the treatment of PD. They are recommended as monotherapy in the early stages of the disease and as add-on therapy to levodopa in advanced PD. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  18. Forage Polyphenol Oxidase and Ruminant Livestock Nutrition

    Directory of Open Access Journals (Sweden)

    Michael Richard F. Lee

    2014-12-01

    Full Text Available Polyphenol oxidase (PPO is associated with the detrimental effect of browning fruit and vegetables, however interest within PPO containing forage crops has grown since the brownng reaction was associated with reduced nitrogen (N losses in silo and the rumen. The reduction in protein breakdown in silo of red clover (high PPO forage increased the quality of protein, improving N-use efficiency (NUE when fed to ruminants. A further benefit of red clover silage feeding is a significant reduction in lipolysis in silo and an increase in the deposition of beneficial C18 polyunsaturated fatty acid (PUFA in animal products, which has also been linked to PPO activity. PPOs protection of plant protein and glycerol based-PUFA in silo is related to the deactivation of plant proteases and lipases. This deactivation occurs through PPO catalysing the conversion of diphenols to quinones which bind with cellular nucleophiles such as protein reforming a protein-bound phenol (PBP. If the protein is an enzyme the complexing denatures the enzyme. However, PPO is inactive in the anaerobic rumen and therefore any subsequent protection of plant protein and glycerol based-PUFA in the rumen must be as a result of events that occurred to the forage pre-ingestion. Reduced activity of plant proteases and lipases would have little effect on NUE and glycerol based-PUFA in the rumen due to the greater concentration of rumen microbial proteases and lipases. The mechanism for PPOs protection of plant protein in the rumen is a consequence of complexing plant protein, rather than protease deactivation per se. These complexed proteins reduce protein digestibility in the rumen and subsequently increase un-degraded dietary protein flow to the small intestine. The mechanism for protecting glycerol-based PUFA has yet to be fully elucidated but may be associated with entrapment within PBP reducing access to microbial lipases or differences in rumen digestion kinetics of red clover.

  19. Monoamine oxidase and agitation in psychiatric patients.

    Science.gov (United States)

    Nikolac Perkovic, Matea; Svob Strac, Dubravka; Nedic Erjavec, Gordana; Uzun, Suzana; Podobnik, Josip; Kozumplik, Oliver; Vlatkovic, Suzana; Pivac, Nela

    2016-08-01

    Subjects with schizophrenia or conduct disorder display a lifelong pattern of antisocial, aggressive and violent behavior and agitation. Monoamine oxidase (MAO) is an enzyme involved in the degradation of various monoamine neurotransmitters and neuromodulators and therefore has a role in various psychiatric and neurodegenerative disorders and pathological behaviors. Platelet MAO-B activity has been associated with psychopathy- and aggression-related personality traits, while variants of the MAOA and MAOB genes have been associated with diverse clinical phenotypes, including aggressiveness, antisocial problems and violent delinquency. The aim of the study was to evaluate the association of platelet MAO-B activity, MAOB rs1799836 polymorphism and MAOA uVNTR polymorphism with severe agitation in 363 subjects with schizophrenia and conduct disorder. The results demonstrated significant association of severe agitation and smoking, but not diagnosis or age, with platelet MAO-B activity. Higher platelet MAO-B activity was found in subjects with severe agitation compared to non-agitated subjects. Platelet MAO-B activity was not associated with MAOB rs1799836 polymorphism. These results suggested the association between increased platelet MAO-B activity and severe agitation. No significant association was found between severe agitation and MAOA uVNTR or MAOB rs1799836 polymorphism, revealing that these individual polymorphisms in MAO genes are not related to severe agitation in subjects with schizophrenia and conduct disorder. As our study included 363 homogenous Caucasian male subjects, our data showing this negative genetic association will be a useful addition to future meta-analyses. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. [Effect of Kaixinsan on monoamine oxidase activity].

    Science.gov (United States)

    Wang, Shi; Dong, Xian-Zhe; Tan, Xiao; Wang, Yu-Ning; Liu, Ping

    2016-05-01

    To observe the effect of antidepressant medicine prescription, Kaixinsan (KXS) on monoamine oxidase (MAO) activity, and explore the mechanism of KXS in elevating the levels of monoamine neurotransmitter from the perspective of metabolism, in vitro enzyme reaction system and C6 neuroglial cells, the effect of KXS at different concentrations on MAO-A and MAO-B activity was observed. In animal studies, the effect of KXS at different concentrations on MAO-A and MAO-B activities of brain mitochondrialin normal rats and solitary chronic unpredictable moderate stress (CMS) model rats after intragastric administration for 1, 2, 3 weeks. Results showed that 10 g•L⁻¹ KXS could significantly reduce the activity of MAO-A and MAO-B in enzyme reaction system; and in C6 cells, KXS within 0.625-10 g•L⁻¹ concentration range had no significant effect on the activity of MAO-A, but had obvious inhibitory effect on the activity of MAO-B in a dose dependent manner. KXS had no significant effect on the activity of MAO-A and MAO-B in brains of normal rats after action for 1, 2, 3 weeks. After 2 and 3 weeks treatment with 338 mg•kg⁻¹ dose KXS, MAO-A activity in the brain of CMS rats was decreased as compared with the model group (PMAO-B activity after 1, 2, 3 weeks of treatment. The results indicated that KXS had certain effect on in vitro MAO-A and MAO-B activity, had no effect on brain MAO-A and MAO-B activity in vivo in normal rats, and had certain inhibitory effect on MAO-A activity in brains of CMS rats. Copyright© by the Chinese Pharmaceutical Association.

  1. Lysyl oxidase and adipose tissue dysfunction.

    Science.gov (United States)

    Pastel, Emilie; Price, Emily; Sjöholm, Kajsa; McCulloch, Laura J; Rittig, Nikolaj; Liversedge, Neil; Knight, Bridget; Møller, Niels; Svensson, Per-Arne; Kos, Katarina

    2018-01-01

    Lysyl oxidase (LOX) is an enzyme crucial for collagen fibre crosslinking and thus for fibrosis development. Fibrosis is characterised by a surplus of collagen fibre accumulation and is amongst others also a feature of obesity-associated dysfunctional adipose tissue (AT) which has been linked with type 2 diabetes. We hypothesised that in type 2 diabetes and obesity LOX expression and activity will be increased as a consequence of worsening AT dysfunction. This study aimed to provide a comprehensive characterisation of LOX in human AT. LOX mRNA expression was analysed in omental and abdominal subcutaneous AT obtained during elective surgery from subjects with a wide range of BMI, with and without diabetes. In addition, LOX expression was studied in subcutaneous AT before and 9.5months after bariatric surgery. To study the mechanism of LOX changes, its expression and activity were assessed after either hypoxia, recombinant human leptin or glucose treatment of AT explants. In addition, LOX response to acute inflammation was tested after stimulation by a single injection of lipopolysaccharide versus saline solution (control) in healthy men, in vivo. Quantity of mRNA was measured by RT-qPCR. LOX expression was higher in obesity and correlated with BMI whilst, in vitro, leptin at high concentrations, as a potential feedback mechanism, suppressed its expression. Neither diabetes status, nor hyperglycaemia affected LOX. Hypoxia and lipopolysaccharide-induced acute inflammation increased LOX AT expression, latter was independent of macrophage infiltration. Whilst LOX may not be affected by obesity-associated complications such as diabetes, our results confirm that LOX is increased by hypoxia and inflammation as underlying mechanism for its upregulation in adipose tissue with obesity. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Production of rabbit antibodies against purified Glucose oxidase

    Directory of Open Access Journals (Sweden)

    Muhammad Anjum Zia

    2012-02-01

    Full Text Available Glucose oxidase is an active oxygen species generating enzyme produced from Aspergillus niger grown in submerged fermentation. Disintegration of the mycelium resulted in high glucose oxidase activity that was subjected to ammonium sulfate precipitation at 60-85% saturation rates that resulted to 6.14 U mg -1 specific activity. Purification of enzyme by anion exchange column (DEAE-Cellulose resulted into 22.53 U mg-1 specific activity and 10.27 fold purification. This was applied to sephadex G-200 column for gel filtration chromatography. It was observed that enzyme achieved 59.37 U mg-1of specific activity with 27.08 fold purity and 64.36% recovery. Purified glucose oxidase was injected into rabbits through intravenous route, to raise the glucose oxidase antibodies. After 30 days incubation period, the rabbits were slaughtered and serum was separated from blood. The antibodies were isolated by ammonium sulfate precipitation and confirmed by agar gel precipitation test. This could be a convenient and low cost alternate assay for the estimation of glucose oxidase in biological fluids. Moreover, such antibodies against the said enzyme could be used in various therapeutic and diagnostic applications.

  3. Calcium transport in vesicles energized by cytochrome oxidase

    Energy Technology Data Exchange (ETDEWEB)

    Rosier, Randy N. [Univ. of Rochester, NY (United States)

    1979-01-01

    Experiments on the reconstitution of cytochrome oxidase into phospholipid vesicles were carried out using techniques of selectivity energizing the suspensions with ascorbate and cytochrome c or ascorbate, PMS, and internally trapped cytochrome c. It was found that the K+ selective ionophore valinomycin stimulated the rate of respiration of cytochrome oxidase vesicles regardless of the direction of the K+ flux across the vesicle membranes. The stimulation occurred in the presence of protonophoric uncouplers and in the complete absence of potassium or in detergent-lysed suspensions. Gramicidin had similar effects and it was determined that the ionophores acted by specific interaction with cytochrome oxidase rather than by the previously assumed collapse of membrane potentials. When hydrophobic proteins and appropriate coupling factors were incorporated into the cytochrome oxidase, vesicles phosphorylation of ADP could be coupled to the oxidation reaction of cytochrome oxidase. Relatively low P:O, representing poor coupling of the system, were problematical and precluded measurements of protonmotive force. However the system was used to study ion translocation.

  4. Immobilization of xanthine oxidase on a polyaniline silicone support.

    Science.gov (United States)

    Nadruz, W; Marques, E T; Azevedo, W M; Lima-Filho, J L; Carvalho, L B

    1996-03-01

    A polyaniline silicone support to immobilize xanthine oxidase is proposed as a reactor coil to monitor the action of xanthine oxidase on hypoxanthine, xanthine and 6-mercaptopurine. A purified xanthine oxidase immobilized on this support lost 80% of the initial activity after 12 min of use. Co-immobilization of superoxide dismutase and catalase increased the stability of immobilized xanthine oxidase so that the derivative maintained 79% of its initial activity after 4.6 h of continuous use in which 1.5 mumol purine bases were converted by the immobilized enzyme system. There is no evidence of either polyaniline or protein leaching from the coil during 3 h of continuous use. When solutions (10 ml) of hypoxanthine, xanthine and 6-mercaptopurine were circulated individually through the xanthine oxidase-superoxide dismutase-catalase-polyaniline coil (1 mm internal diameter and 3 m in length, 3 ml internal volume) activities of 8.12, 11.17 and 1.09 nmol min-1 coil-1, respectively, were obtained. The advantages of the reactor configuration and the redox properties of the polymer, particularly with respect to immobilized oxidoreductases, make this methodology attractive for similar enzyme systems. This immobilized enzyme system using polyaniline-silicone as support converted 6-mercaptopurine to 6-thiouric acid with equal efficiency as resins based on polyacrylamide and polyamide 11.

  5. Fluorescent Probes for Analysis and Imaging of Monoamine Oxidase Activity

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Dokyoung; Jun, Yong Woong; Ahn, Kyo Han [POSTECH, Pohang (Korea, Republic of)

    2014-05-15

    Monoamine oxidases catalyze the oxidative deamination of dietary amines and amine neurotransmitters, and assist in maintaining the homeostasis of the amine neurotransmitters in the brain. Dysfunctions of these enzymes can cause neurological and behavioral disorders including Parkinson's and Alzheimer's diseases. To understand their physiological roles, efficient assay methods for monoamine oxidases are essential. Reviewed in this Perspective are the recent progress in the development of fluorescent probes for monoamine oxidases and their applications to enzyme assays in cells and tissues. It is evident that still there is strong need for a fluorescent probe with desirable substrate selectivity and photophysical properties to challenge the much unsolved issues associated with the enzymes and the diseases.

  6. Characterization of wheat germin (oxalate oxidase) expressed by Pichia pastoris

    International Nuclear Information System (INIS)

    Pan, Heng-Yen; Whittaker, Mei M.; Bouveret, Romaric; Berna, Anne; Bernier, Francois; Whittaker, James W.

    2007-01-01

    High-level secretory expression of wheat (Triticum aestivum) germin/oxalate oxidase was achieved in Pichia pastoris fermentation cultures as an α-mating factor signal peptide fusion, based on the native wheat cDNA coding sequence. The oxalate oxidase activity of the recombinant enzyme is substantially increased (7-fold) by treatment with sodium periodate, followed by ascorbate reduction. Using these methods, approximately 1 g (4 x 10 4 U) of purified, activated enzyme was obtained following eight days of induction of a high density Pichia fermentation culture, demonstrating suitability for large-scale production of oxalate oxidase for biotechnological applications. Characterization of the recombinant protein shows that it is glycosylated, with N-linked glycan attached at Asn47. For potential biomedical applications, a nonglycosylated (S49A) variant was also prepared which retains essentially full enzyme activity, but exhibits altered protein-protein interactions

  7. The use of galactose oxidase in lipid labeling

    International Nuclear Information System (INIS)

    Radin, N.S.; Evangelatos, G.P.

    1981-01-01

    Galactose oxidase can be used to oxidize the terminal carbon atom of lipids containing galactose or N-acetylgalactosamine, and the resultant aldehyde group can be reduced back to the original carbinol with radioactive borohydride. The efficiency of the first reaction has been investigated systematically by using [6- 3 H]galactosyl ceramide as substrate and measuring the amount of radioactive water formed. This enabled us to establish that the addition of catalase and peroxidase greatly speeded the oxidation, that phosphate and PIPES buffers were the best among those tested, that the reaction continued for 24 hr without a second addition of galactose oxidase, and that the optimum concentration of organic solvent (tetrahydrofuran) was 50%. The suggestion if made that a similar set of variables be studied for each lipid or nonlipid by the same basic technique: labeling by the oxidase/borohydride method and use of the resultant compound as substrate

  8. Increased xanthine oxidase during labour--implications for oxidative stress.

    Science.gov (United States)

    Many, A; Roberts, J M

    1997-11-01

    Xanthine dehydrogenase/oxidase (XDH/XO) produces uric acid. When in the oxidase form, this production is coupled with the generation of free radicals. Hypoxia-reperfusion enhances conversion of XDH to XO. Since the placenta is exposed to short periods of hypoxia reperfusion during labour, 17 placentae of pregnancy terminated by elective caesarean section and five placentae of pregnancies terminated by caesarean section during labour were examined for XDH/XO activity. It was found that XO activity was higher in the placentae of labouring women (P = 0.003), which suggests that labour enhances conversion of XDH to XO, facilitating free radical production.

  9. Two X-linked chronic granulomatous disease patients with unusual NADPH oxidase properties

    NARCIS (Netherlands)

    Wolach, Baruch; Broides, Arnon; Zeeli, Tal; Gavrieli, Ronit; de Boer, Martin; van Leeuwen, Karin; Levy, Jacov; Roos, Dirk

    2011-01-01

    Chronic granulomatous disease (CGD) is an immune deficiency syndrome caused by defects in the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, the enzyme that generates reactive oxygen species (ROS) in phagocytizing leukocytes. This study evaluates the NADPH oxidase capacity in two

  10. Peroxisomal Polyamine Oxidase and NADPH-Oxidase cross-talk for ROS homeostasis which affects respiration rate in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Efthimios A. Andronis

    2014-04-01

    Full Text Available Homeostasis of reactive oxygen species (ROS in the intracellular compartments is of critical importance as ROS have been linked with nearly all cellular processes and more importantly with diseases and aging. PAs are nitrogenous molecules with an evolutionary conserved role in the regulation of metabolic and energetic status of cells. Recent evidence also suggests that polyamines (PA are major regulators of ROS homeostasis. In Arabidopsis the backconversion of the PAs spermidine (Spd and spermine (Spm to putrescine (Put and Spd, respectively is catalyzed by two peroxisomal PA oxidases (AtPAO. However, the physiological role of this pathway remains largely elusive. Here we explore the role of peroxisomal PA backconversion and in particular that catalyzed by the highly expressed AtPAO3 in the regulation of ROS homeostasis and mitochondrial respiratory burst. Exogenous PAs exert an NADPH-oxidase dependent stimulation of oxygen consumption, with Spd exerting the strongest effect. This increase is attenuated by treatment with the NADPH-oxidase blocker diphenyleneiodonium iodide (DPI. Loss-of-function of AtPAO3 gene results to increased NADPH-oxidase-dependent production of superoxide anions (O2.-, but not H2O2, which activate the mitochondrial alternative oxidase pathway (AOX. On the contrary, overexpression of AtPAO3 results to an increased but balanced production of both H2O2 and O2.-. These results suggest that the ratio of O2.-/H2O2 regulates respiratory chain in mitochondria, with PA-dependent production of O2.- by NADPH-oxidase tilting the balance of electron transfer chain in favor of the AOX pathway. In addition, AtPAO3 seems to be an important component in the regulating module of ROS homeostasis, while a conserved role for PA backconversion and ROS across kingdoms is discussed.

  11. Cytochrome cbb3 of Thioalkalivibrio is a Na+-pumping cytochrome oxidase

    NARCIS (Netherlands)

    Muntyan, M.S.; Cherepanov, D.A.; Malinen, A.M.; Bloch, D.A.; Sorokin, D.Y.; Severina, I.I.; Ivashina, T.V.; Lahti, R.; Muyzer, G.; Skulachev, V.P.

    2015-01-01

    Cytochrome c oxidases (Coxs) are the basic energy transducers in the respiratory chain of the majority of aerobic organisms. Coxs studied to date are redox-driven proton-pumping enzymes belonging to one of three subfamilies: A-, B-, and C-type oxidases. The C-type oxidases (cbb3 cytochromes), which

  12. Enhanced production of glucose oxidase from UVmutant of ...

    African Journals Online (AJOL)

    UV rays were used as mutagen in wild type strain of Aspergillus niger for enhanced production of glucose oxidase. After mutangenization and selection, mutant A. niger strains, resistant to 2-deoxy-Dglucose were obtained. The mutants showed 1.57 and 1.98 fold increase in activities of extra and intra cellular glucose ...

  13. Localization of Glucose Oxidase and Catalase Activities in Aspergillus niger

    NARCIS (Netherlands)

    Witteveen, Cor F.B.; Veenhuis, Marten; Visser, Jaap

    The subcellular localization of glucose oxidase (EC 1.1.3.4) in Aspergillus niger N400 (CBS 120.49) was investigated by (immuno)cytochemical methods. By these methods, the bulk of the enzyme was found to be localized in the cell wall. In addition, four different catalases (EC 1.11.1.6) were

  14. The glucose oxidase-peroxidase assay for glucose

    Science.gov (United States)

    The glucose oxidase-peroxidase assay for glucose has served as a very specific, sensitive, and repeatable assay for detection of glucose in biological samples. It has been used successfully for analysis of glucose in samples from blood and urine, to analysis of glucose released from starch or glycog...

  15. Nanoporous gold assembly of glucose oxidase for electrochemical biosensing

    DEFF Research Database (Denmark)

    Xiao, Xinxin; Ulstrup, Jens; Li, Hui

    2014-01-01

    Nanoporous gold (NPG) is composed of three-dimensional (3D) bicontinuous nanostructures with large surface area. Nano-channels inside NPG provide an ideal local environment for immobilization of enzyme molecules with expected stabilization of the protein molecules. In this work, glucose oxidase (...

  16. Gene cloning and characterization of NADH oxidase from ...

    African Journals Online (AJOL)

    The genome search of Thermococcus kodakarensis revealed three open reading frames, Tk0304, Tk1299 and Tk1392 annotated as nicotinamide adenine dinucleotide (NADH) oxidases. This study deals with cloning, and characterization of Tk0304. The gene, composed of 1320 nucleotides, encodes a protein of 439 ...

  17. Optimization of cholesterol oxidase production by Brevibacterium sp ...

    African Journals Online (AJOL)

    An ultrasound-assisted emulsification as a pretreatment for cholesterol oxidase production by submerge fermentation using Brevibacterium sp. in a batch system was studied. Medium improvement for the production employing response surface methodology (RSM) was optimized in this paper. The concentration of ...

  18. in Escherichia coli with native cholesterol oxidase expressed

    African Journals Online (AJOL)

    The structure and bio-activity of an endogenous cholesterol oxidase from Brevibacterium sp. was compared to the same enzyme exogenously expressed in Escherichia coli BL21 (DE3) with and without N- or C-terminal his-tags. The different proteins were purified with affinity and subtractive protocols. The specific activity of ...

  19. Cytokinin oxidase or dehydrogenase? Mechanism of cytokinin degradation in cereals

    DEFF Research Database (Denmark)

    Galuszka, P.; Frebort, I.; Sebela, M.

    2001-01-01

    An enzyme degrading cytokinins with isoprenoid side chain, previously named cytokinin oxidase, was purified to near homogeneity from wheat and barley grains. New techniques were developed for the enzyme activity assay and staining on native electrophoretic gels to identify the protein. The purifi...

  20. Effect of heat treatment on polyphenol oxidase and peroxidase ...

    African Journals Online (AJOL)

    Effect of heat treatment (55°C/20 min) on polyphenol oxidase (PPO) and peroxidase (POD) activities and total phenolic compounds was investigated in Algerian dates (Deglet Nour variety) at Tamar (fully ripe) stage and in dates stored for 5 months at ambient temperature and in cold storage (10°C). Results obtained ...

  1. Lysyl oxidases regulate fibrillar collagen remodelling in idiopathic pulmonary fibrosis

    NARCIS (Netherlands)

    Tjin, Gavin; White, Eric S; Faiz, Alen; Sicard, Delphine; Tschumperlin, Daniel J; Mahar, Annabelle; Kable, Eleanor P W; Burgess, Janette K

    2017-01-01

    Idiopathic pulmonary fibrosis (IPF) is a progressive scarring disease of the lung with feweffective therapeutic options. Structural remodelling of the extracellular matrix [i.e. collagen cross-linkingmediated by the lysyl oxidase (LO) family of enzymes (LOX, LOXL1-4)] might contribute to disease

  2. Novel thidiazuron-derived inhibitors of cytokinin oxidase/dehydrogenase

    Czech Academy of Sciences Publication Activity Database

    Nisler, Jaroslav; Kopečný, D.; Končitíková, R.; Zatloukal, Marek; Bazgier, Václav; Berka, K.; Zalabák, D.; Briozzo, P.; Strnad, Miroslav; Spíchal, Lukáš

    2016-01-01

    Roč. 92, 1-2 (2016), s. 235-248 ISSN 0167-4412 R&D Projects: GA MŠk(CZ) LO1204; GA ČR GA15-22322S Institutional support: RVO:61389030 Keywords : Cytokinin oxidase/dehydrogenase * Crystal structure * Molecular docking Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.356, year: 2016

  3. DNA damage and decrease of cellular oxidase activity in piglet ...

    African Journals Online (AJOL)

    DNA damage and decrease of cellular oxidase activity in piglet sertoli cells exposed to gossypol. Ming Zhang, Hui Yuan, Zuping He, Liyun Yuan, Jine Yi, Sijun Deng, Li Zhu, Chengzhi Guo, Yin Lu, Jing Wu, Lixin Wen, Qiang Wei, Liqun Xue ...

  4. Inactivation of 1-aminocyclopropane-1-carboxylate oxidase involves oxidative modifications.

    Science.gov (United States)

    Barlow, J N; Zhang, Z; John, P; Baldwin, J E; Schofield, C J

    1997-03-25

    1-Aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the final step in the biosynthesis of the plant signaling molecule ethylene. It is a member of the ferrous iron dependent family of oxidases and dioxygenases and is unusual in that it displays a very short half-life under catalytic conditions, typically less than 20 min, and a requirement for CO2 as an activator. The rates of inactivation of purified, recombinant ACC oxidase from tomato under various combinations of substrates and cofactors were measured. Inactivation was relatively slow in the presence of buffer alone (t1/2 > 1 h), but fast in the presence of ferrous iron and ascorbate (t1/2 approximately 10 min). The rate of iron/ascorbate-mediated inactivation was increased by the addition of ACC, unaffected by the addition of CO2 at saturation (supplied as bicarbonate) but decreased by the addition of catalase or ACC + CO2 at saturation (supplied as bicarbonate). Iron/ascorbate-mediated inactivation was accompanied by partial proteolysis as observed by SDS-PAGE analysis. The fragmentation pattern was altered when ACC was also included, suggesting that ACC can bind to ACC oxidase in the absence of bicarbonate. N-terminal sequencing of fragments resulted in identification of an internal cleavage site which we propose is proximate to active-site bound iron. Thus, ACC oxidase inactivates via relatively slow partial unfolding of the catalytically active conformation, oxidative damage mediated via hydrogen peroxide which is catalase protectable and oxidative damage to the active site which results in partial proteolysis and is not catalase protectable.

  5. Expression Studies of Gibberellin Oxidases in Developing Pumpkin Seeds1

    Science.gov (United States)

    Frisse, Andrea; Pimenta, Maria João; Lange, Theo

    2003-01-01

    Two cDNA clones, 3-ox and 2-ox, have been isolated from developing pumpkin (Cucurbita maxima) embryos that show significant amino acid homology to gibberellin (GA) 3-oxidases and 2-oxidases, respectively. Recombinant fusion protein of clone 3-ox converted GA12-aldehyde, GA12, GA15, GA24, GA25, and GA9 to GA14-aldehyde, GA14, GA37, GA36, GA13, and GA4, respectively. Recombinant 2-ox protein oxidized GA9, GA4, and GA1 to GA51, GA34, and GA8, respectively. Previously cloned GA 7-oxidase revealed additional 3β-hydroxylation activity of GA12. Transcripts of this gene were identified in endosperm and embryo of the developing seed by quantitative reverse transcriptase-polymerase chain reaction and localized in protoderm, root apical meristem, and quiescent center by in situ hybridization. mRNA of the previously cloned GA 20-oxidase from pumpkin seeds was localized in endosperm and in tissues of protoderm, ground meristem, and cotyledons of the embryo. However, transcripts of the recently cloned GA 20-oxidase from pumpkin seedlings were found all over the embryo, and in tissues of the inner seed coat at the micropylar end. Previously cloned GA 2β,3β-hydroxylase mRNA molecules were specifically identified in endosperm tissue. Finally, mRNA molecules of the 3-ox and 2-ox genes were found in the embryo only. 3-ox transcripts were localized in tissues of cotyledons, protoderm, and inner cell layers of the root apical meristem, and 2-ox transcripts were found in all tissues of the embryo except the root tips. These results indicate tissue-specific GA-biosynthetic pathways operating within the developing seed. PMID:12644672

  6. Cyanobacterial lactate oxidases serve as essential partners in N2-fixation and evolved into photorespiratory glycolate oxidases in plants.

    NARCIS (Netherlands)

    Hackenberg, C.; Kern, R.; Hüge, J; Stal, L.J.; Tsuji, Y.; Kopka, J.; Shiraiwa, Y.; Bauwe, H.; Hagemann, M.

    2011-01-01

    Glycolate oxidase (GOX) is an essential enzyme involved in photorespiratory metabolism in plants. In cyanobacteria and green algae, the corresponding reaction is catalyzed by glycolate dehydrogenases (GlcD). The genomes of N2-fixing cyanobacteria, such as Nostoc PCC 7120 and green algae, appear to

  7. Cyanobacterial lactate oxidases serve as essential partners of N2-fixation and evolved to photorespiratory glycolate oxidases in plants

    NARCIS (Netherlands)

    Hackenberg, C.; Kern, R.; Hüge, J.; Stal, L.J.; Tsuji, Y.; Kopka, J.; Shiraiwa, Y.; Bauwe, H.; Hagemann, M.

    2011-01-01

    Glycolate oxidase (GOX) is an essential enzyme involved in photorespiratory metabolism in plants. In cyanobacteria and green algae, the corresponding reaction is catalyzed by glycolate dehydrogenases (GlcD). The genomes of N2-fixing cyanobacteria, such as Nostoc PCC 7120 and green algae, appear to

  8. The structure of SSO2064, the first representative of Pfam family PF01796, reveals a novel two-domain zinc-ribbon OB-fold architecture with a potential acyl-CoA-binding role

    International Nuclear Information System (INIS)

    Krishna, S. Sri; Aravind, L.; Bakolitsa, Constantina; Caruthers, Jonathan; Carlton, Dennis; Miller, Mitchell D.; Abdubek, Polat; Astakhova, Tamara; Axelrod, Herbert L; Chiu, Hsiu-Ju; Clayton, Thomas; Deller, Marc C.; Duan, Lian; Feuerhelm, Julie; Grant, Joanna C.; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K.; Klock, Heath E.; Knuth, Mark W.; Kumar, Abhinav; Marciano, David; McMullan, Daniel; Morse, Andrew T.; Nigoghossian, Edward; Okach, Linda; Reyes, Ron; Rife, Christopher L.; Bedem, Henry van den; Weekes, Dana; Xu, Qingping; Hodgson, Keith O.; Wooley, John; Elsliger, Marc-André; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.

    2010-01-01

    The crystal structure of SSO2064, the first structural representative of Pfam family PF01796 (DUF35), reveals a two-domain architecture comprising an N-terminal zinc-ribbon domain and a C-terminal OB-fold domain. Analysis of the domain architecture, operon organization and bacterial orthologs combined with the structural features of SSO2064 suggests a role involving acyl-CoA binding for this family of proteins. SSO2064 is the first structural representative of PF01796 (DUF35), a large prokaryotic family with a wide phylogenetic distribution. The structure reveals a novel two-domain architecture comprising an N-terminal, rubredoxin-like, zinc ribbon and a C-terminal, oligonucleotide/oligosaccharide-binding (OB) fold domain. Additional N-terminal helical segments may be involved in protein–protein interactions. Domain architectures, genomic context analysis and functional evidence from certain bacterial representatives of this family suggest that these proteins form a novel fatty-acid-binding component that is involved in the biosynthesis of lipids and polyketide antibiotics and that they possibly function as acyl-CoA-binding proteins. This structure has led to a re-evaluation of the DUF35 family, which has now been split into two entries in the latest Pfam release (v.24.0)

  9. Lactate Biosensor Based on Cellulose Acetate Membrane Bound Lactate Oxidase

    Directory of Open Access Journals (Sweden)

    Suman

    2007-05-01

    Full Text Available Lactate biosensor was fabricated by immobilizing lactate oxidase in cellulose acetate membrane and by mounting over the sensing part of Pt electrode (working and connected to Ag/AgCl electrode (reference along with auxillary electrode through potentiostat. The enzyme electrode was anodically polarized at +400 mV to generate electrons from H2O2, which was formed from oxidation of serum lactate by immobilized lactate oxidase. The minimum detection limit of the electrode was 0.1mmoles/L and sensitivity of the sensor was 0.008 mA/mM/L lactate. Assay coefficients of variation were < 2% .A good correlation (r=0.99 was found between lactate values obtained by colorimetric method and lactate biosensor. The self-life of the biosensor was 18 days at 4ºC and enzyme electrode can be re-used 150 times without any significant loss in enzyme activity.

  10. Dual inhibitors of cholinesterases and monoamine oxidases for Alzheimer's disease.

    Science.gov (United States)

    Knez, Damijan; Sova, Matej; Košak, Urban; Gobec, Stanislav

    2017-05-01

    Accumulating evidence indicates a solid relationship between several enzymes and Alzheimer's disease. Cholinesterases and monoamine oxidases are closely associated with the disease symptomatology and progression and have been tackled simultaneously using several multifunctional ligands. This design strategy offers great chances to alter the course of Alzheimer's disease, in addition to alleviation of the symptoms. More than 15 years of research has led to the identification of various dual cholinesterase/monoamine oxidase inhibitors, while some showing positive outcomes in clinical trials, thus giving rise to additional research efforts in the field. The aim of this review is to provide an update on the novel dual inhibitors identified recently and to shed light on their therapeutic potential.

  11. Involvement of NADH Oxidase in Biofilm Formation in Streptococcus sanguinis.

    Directory of Open Access Journals (Sweden)

    Xiuchun Ge

    Full Text Available Biofilms play important roles in microbial communities and are related to infectious diseases. Here, we report direct evidence that a bacterial nox gene encoding NADH oxidase is involved in biofilm formation. A dramatic reduction in biofilm formation was observed in a Streptococcus sanguinis nox mutant under anaerobic conditions without any decrease in growth. The membrane fluidity of the mutant bacterial cells was found to be decreased and the fatty acid composition altered, with increased palmitic acid and decreased stearic acid and vaccenic acid. Extracellular DNA of the mutant was reduced in abundance and bacterial competence was suppressed. Gene expression analysis in the mutant identified two genes with altered expression, gtfP and Idh, which were found to be related to biofilm formation through examination of their deletion mutants. NADH oxidase-related metabolic pathways were analyzed, further clarifying the function of this enzyme in biofilm formation.

  12. Variations in epidermal cytochrome oxidase activity after local irradiation

    International Nuclear Information System (INIS)

    Itoiz, M.E.; Rey, B.M. de; Cabrini, R.L.

    1982-01-01

    Cytochrome oxidase activity was evaluated histochemically as an index of mitochondrial damage after local irradiation with X-rays. It was determined by microphotometry on the tail skin of newly born Wistar rats four days after irradiation with doses ranging from 2 to 16krad. The enzyme activity of the whole epidermis increased after irradiation, the increases being related to the increase in thickness of the epithelium which was observed as a response to irradiation injury. Within the dose range tested, the enzyme concentration (expressed per unit volume of tissue) decreased in relation to the dose applied. At the electron microscopy level, the cytochemical demonstration of cytochrome oxidase revealed an irregular reaction over the cristae, intramitochondrial vacuolization and partial homogenization of the matrix. Positive membrane fragments were seen around lipid droplets. This reaction confirms the mitochondrial origin of these previously observed radiation-induced vacuoles. (author)

  13. Glucose Oxidase Catalyzed Self-Assembly of Bioelectroactive Gold Nanostructures

    Science.gov (United States)

    2010-01-01

    polymer matrix), however, electrons generated at the FAD/FADH2 active site of glucose oxidase (GOx) must tunnel ca. 15 through the protein shell...described as a surface bound thiolate [33]. Recently, the presence of free thiol groups has been proposed as a mechanism for gold reduction in pure enzymes...simultaneously [38]. The oxidative polymerization of the amines proceeds simulta- neously with the formation of gold nanoparticles such that the polymerized amine

  14. Graphene-glucose oxidase bioanodes for enzymatic biofuel cells

    DEFF Research Database (Denmark)

    Tang, Jing; Werchmeister, Rebecka Maria Larsen; Engelbrekt, Christian

    2017-01-01

    as supporting material, polyethyleneimine (PEI) as linker and glucose oxidase (GOD) as the chosen enzyme. GOD can catalyze oxidation of glucose to gluconolactone, but needs a mediator to assist electron transfer between the enzyme and electrodes. The redox molecule ferrocene carboxylic acid (Fc...... and systematically investigated. The assembled EBFCs show good reproducibility. EBFCs provide maximum output power density 2.47 μW cm-2 at 35 ℃, indicating the optimized activity of EBFCs fed with glucose....

  15. Multiple controls affect arsenite oxidase gene expression in Herminiimonas arsenicoxydans

    Directory of Open Access Journals (Sweden)

    Coppée Jean-Yves

    2010-02-01

    Full Text Available Abstract Background Both the speciation and toxicity of arsenic are affected by bacterial transformations, i.e. oxidation, reduction or methylation. These transformations have a major impact on environmental contamination and more particularly on arsenic contamination of drinking water. Herminiimonas arsenicoxydans has been isolated from an arsenic- contaminated environment and has developed various mechanisms for coping with arsenic, including the oxidation of As(III to As(V as a detoxification mechanism. Results In the present study, a differential transcriptome analysis was used to identify genes, including arsenite oxidase encoding genes, involved in the response of H. arsenicoxydans to As(III. To get insight into the molecular mechanisms of this enzyme activity, a Tn5 transposon mutagenesis was performed. Transposon insertions resulting in a lack of arsenite oxidase activity disrupted aoxR and aoxS genes, showing that the aox operon transcription is regulated by the AoxRS two-component system. Remarkably, transposon insertions were also identified in rpoN coding for the alternative N sigma factor (σ54 of RNA polymerase and in dnaJ coding for the Hsp70 co-chaperone. Western blotting with anti-AoxB antibodies and quantitative RT-PCR experiments allowed us to demonstrate that the rpoN and dnaJ gene products are involved in the control of arsenite oxidase gene expression. Finally, the transcriptional start site of the aoxAB operon was determined using rapid amplification of cDNA ends (RACE and a putative -12/-24 σ54-dependent promoter motif was identified upstream of aoxAB coding sequences. Conclusion These results reveal the existence of novel molecular regulatory processes governing arsenite oxidase expression in H. arsenicoxydans. These data are summarized in a model that functionally integrates arsenite oxidation in the adaptive response to As(III in this microorganism.

  16. Biochemical Conservation and Evolution of Germacrene A Oxidase in Asteraceae*

    Science.gov (United States)

    Nguyen, Don Trinh; Göpfert, Jens Christian; Ikezawa, Nobuhiro; MacNevin, Gillian; Kathiresan, Meena; Conrad, Jürgen; Spring, Otmar; Ro, Dae-Kyun

    2010-01-01

    Sesquiterpene lactones are characteristic natural products in Asteraceae, which constitutes ∼8% of all plant species. Despite their physiological and pharmaceutical importance, the biochemistry and evolution of sesquiterpene lactones remain unexplored. Here we show that germacrene A oxidase (GAO), evolutionarily conserved in all major subfamilies of Asteraceae, catalyzes three consecutive oxidations of germacrene A to yield germacrene A acid. Furthermore, it is also capable of oxidizing non-natural substrate amorphadiene. Co-expression of lettuce GAO with germacrene synthase in engineered yeast synthesized aberrant products, costic acids and ilicic acid, in an acidic condition. However, cultivation in a neutral condition allowed the de novo synthesis of a single novel compound that was identified as germacrene A acid by gas and liquid chromatography and NMR analyses. To trace the evolutionary lineage of GAO in Asteraceae, homologous genes were further isolated from the representative species of three major subfamilies of Asteraceae (sunflower, chicory, and costus from Asteroideae, Cichorioideae, and Carduoideae, respectively) and also from the phylogenetically basal species, Barnadesia spinosa, from Barnadesioideae. The recombinant GAOs from these genes clearly showed germacrene A oxidase activities, suggesting that GAO activity is widely conserved in Asteraceae including the basal lineage. All GAOs could catalyze the three-step oxidation of non-natural substrate amorphadiene to artemisinic acid, whereas amorphadiene oxidase diverged from GAO displayed negligible activity for germacrene A oxidation. The observed amorphadiene oxidase activity in GAOs suggests that the catalytic plasticity is embedded in ancestral GAO enzymes that may contribute to the chemical and catalytic diversity in nature. PMID:20351109

  17. Biocompatibility selenium nanoparticles with an intrinsic oxidase-like activity

    Science.gov (United States)

    Guo, Leilei; Huang, Kaixun; Liu, Hongmei

    2016-03-01

    Selenium nanoparticles (SeNPs) are considered to be the new selenium supplement forms with high biological activity and low toxicity; however, the molecular mechanism by which SeNPs exert the biological function is unclear. Here, we reported that biocompatibility SeNPs possessed intrinsic oxidase-like activity. Using Na2SeO3 as a precursor and glutathione as a reductant, biocompatibility SeNPs were synthesized by the wet chemical reduction method in the presence of bovine serum albumin (BSA). The results of structure characterization revealed that synthesized SeNPs were amorphous red elementary selenium with spherical morphology, and ranged in size from 25 to 70 nm size with a narrow distribution (41.4 ± 6.7 nm). The oxidase-like activity of the as-synthesized SeNPs was tested with 3,3',5,5'-tetramethylbenzidine (TMB) as a substrate. The results indicated that SeNPs could catalyze the oxidization of TMB by dissolved oxygen. These SeNPs showed an optimum catalytic activity at pH 4 and 30 °C, and the oxidase-like activity was higher as the concentration of SeNPs increased and the size of SeNPs decreased. The Michaelis constant ( K m) values and maximal reaction velocity ( V max) of the SeNPs for TMB oxidation were 0.0083 mol/L and 3.042 μmol/L min, respectively.

  18. Direct comparison of gluco-oligosaccharide oxidase variants and glucose oxidase: substrate range and H2O2 stability.

    Science.gov (United States)

    Vuong, Thu V; Foumani, Maryam; MacCormick, Benjamin; Kwan, Rachel; Master, Emma R

    2016-11-21

    Glucose oxidase (GO) activity is generally restricted to glucose and is susceptible to inactivation by H 2 O 2 . By comparison, the Y300A variant of gluco-oligosaccharide oxidase (GOOX) from Sarocladium strictum showed broader substrate range and higher H 2 O 2 stability. Specifically, Y300A exhibited up to 40 times higher activity on all tested sugars except glucose, compared to GO. Moreover, fusion of the Y300A variant to a family 22 carbohydrate binding module from Clostridium thermocellum (CtCBM22A) nearly doubled its catalytic efficiency on glucose, while retaining significant activity on oligosaccharides. In the presence of 200 mM of H 2 O 2 , the recombinant CtCBM22A_Y300A retained 80% of activity on glucose and 100% of activity on cellobiose, the preferred substrate for this enzyme. By contrast, a commercial glucose oxidase reported to contain ≤0.1 units catalase/ mg protein, retained 60% activity on glucose under the same conditions. GOOX variants appear to undergo a different mechanism of inactivation, as a loss of histidine instead of methionine was observed after H 2 O 2 incubation. The addition of CtCBM22A also promoted functional binding of the fusion enzyme to xylan, facilitating its simultaneous purification and immobilization using edible oat spelt xylan, which might benefit the usage of this enzyme preparation in food and baking applications.

  19. Crystallization of the two-domain N-terminal fragment of the archaeal ribosomal protein L10(P0) in complex with a specific fragment of 23S rRNA

    Science.gov (United States)

    Kravchenko, O. V.; Mitroshin, I. V.; Gabdulkhakov, A. G.; Nikonov, S. V.; Garber, M. B.

    2011-07-01

    Lateral L12-stalk (P1-stalk in Archaea, P1/P2-stalk in eukaryotes) is an obligatory morphological element of large ribosomal subunits in all organisms studied. This stalk is composed of the complex of ribosomal proteins L10(P0) and L12(P1) and interacts with 23S rRNA through the protein L10(P0). L12(P1)-stalk is involved in the formation of GTPase center of the ribosome and plays an important role in the ribosome interaction with translation factors. High mobility of this stalk puts obstacles in determination of its structure within the intact ribosome. Crystals of a two-domain N-terminal fragment of ribosomal protein L10(P0) from the archaeon Methanococcus jannaschii in complex with a specific fragment of rRNA from the same organism have been obtained. The crystals diffract X-rays at 3.2 Å resolution.

  20. Crystallization of the two-domain N-terminal fragment of the archaeal ribosomal protein L10(P0) in complex with a specific fragment of 23S rRNA

    Energy Technology Data Exchange (ETDEWEB)

    Kravchenko, O. V.; Mitroshin, I. V.; Gabdulkhakov, A. G.; Nikonov, S. V.; Garber, M. B., E-mail: garber@vega.protres.ru [Institute of Protein Research RAS (Russian Federation)

    2011-07-15

    Lateral L12-stalk (P1-stalk in Archaea, P1/P2-stalk in eukaryotes) is an obligatory morphological element of large ribosomal subunits in all organisms studied. This stalk is composed of the complex of ribosomal proteins L10(P0) and L12(P1) and interacts with 23S rRNA through the protein L10(P0). L12(P1)-stalk is involved in the formation of GTPase center of the ribosome and plays an important role in the ribosome interaction with translation factors. High mobility of this stalk puts obstacles in determination of its structure within the intact ribosome. Crystals of a two-domain N-terminal fragment of ribosomal protein L10(P0) from the archaeon Methanococcus jannaschii in complex with a specific fragment of rRNA from the same organism have been obtained. The crystals diffract X-rays at 3.2 Angstrom-Sign resolution.

  1. Protein structural development of threadfin bream ( Nemipterus spp.) surimi gels induced by glucose oxidase.

    Science.gov (United States)

    Wang, Lei; Fan, Daming; Fu, Lulu; Jiao, Xidong; Huang, Jianlian; Zhao, Jianxin; Yan, Bowen; Zhou, Wenguo; Zhang, Wenhai; Ye, Weijian; Zhang, Hao

    2018-01-01

    This study investigated the effect of glucose oxidase on the gel properties of threadfin bream surimi. The gel strength of surimi increased with the addition of 0.5‰ glucose oxidase after two-step heating. Based on the results of the chemical interactions, the hydrophobic interaction and disulfide bond of glucose oxidase-treated surimi samples increased compared with the control samples at the gelation temperature and gel modori temperature. The surface hydrophobicity of samples with glucose oxidase and glucose increased significantly ( p glucose oxidase induced more α-helixes to turn into a more elongated random and flocculent structure. Glucose oxidase changes the secondary structure of the surimi protein, making more proteins depolarize and stretch and causing actomyosin to accumulate to each other, resulting in the formation of surimi gel.

  2. Amine oxidases as important agents of pathological processes of rhabdomyolysis in rats.

    Science.gov (United States)

    Gudkova, O O; Latyshko, N V; Shandrenko, S G

    2016-01-01

    In this study we have tested an idea on the important role of amine oxidases (semicarbazide-sensitive amine oxidase, diamine oxidase, polyamine oxidase) as an additional source of oxidative/carbonyl stress under glycerol-induced rhabdomyolysis, since the enhanced formation of reactive oxygen species and reactive carbonyl species in a variety of tissues is linked to various diseases. In our experiments we used the sensitive fluorescent method devised for estimation of amine oxidases activity in the rat kidney and thymus as targeted organs under rhabdomyolysis. We have found in vivo the multiple rises in activity of semicarbazide-sensitive amine oxidase, diamine oxidase, polyamine oxidase (2-4.5 times) in the corresponding cell fractions, whole cells or their lysates at the 3-6th day after glycerol injection. Aberrant antioxidant activities depended on rhabdomyolysis stage and had organ specificity. Additional treatment of animals with metal chelator ‘Unithiol’ adjusted only the activity of antioxidant enzymes but not amine oxidases in both organs. Furthermore the in vitro experiment showed that Fenton reaction (hydrogen peroxide in the presence of iron) products alone had no effect on semicarbazide-sensitive amine oxidase activity in rat liver cell fraction whereas supplementation with methylglyoxal resulted in its significant 2.5-fold enhancement. Combined action of the both agents had additive effect on semicarbazide-sensitive amine oxidase activity. We can assume that biogenic amine and polyamine catabolism by amine oxidases is upregulated by oxidative and carbonyl stress factors directly under rhabdomyolysis progression, and the increase in catabolic products concentration contributes to tissue damage in glycerol-induced acute renal failure and apoptosis stimulation in thymus.

  3. Up-Streaming Process for Glucose Oxidase by Thermophilic Penicillium sp. in Shake Flask

    OpenAIRE

    Muhammad Mohsin JAVED; Aroosh SHABIR; Sana ZAHOOR; Ikram UL-HAQ

    2012-01-01

    The present study is concerned with the production of glucose oxidase (GOD) from thermophilic Penicillium sp. in 250 mL shake flask. Fourteen different strains of thermophilic Penicillium sp. were isolated from the soil and were screened for glucose oxidase production. IIBP-13 strain gave maximum extra-cellular glucose oxidase production as compared to other isolates. Effect of submerged fermentation in shaking and static conditions, different carbon sources and incubation period on the produ...

  4. Molecular evolution of the polyamine oxidase gene family in Metazoa

    Directory of Open Access Journals (Sweden)

    Polticelli Fabio

    2012-06-01

    Full Text Available Abstract Background Polyamine oxidase enzymes catalyze the oxidation of polyamines and acetylpolyamines. Since polyamines are basic regulators of cell growth and proliferation, their homeostasis is crucial for cell life. Members of the polyamine oxidase gene family have been identified in a wide variety of animals, including vertebrates, arthropodes, nematodes, placozoa, as well as in plants and fungi. Polyamine oxidases (PAOs from yeast can oxidize spermine, N1-acetylspermine, and N1-acetylspermidine, however, in vertebrates two different enzymes, namely spermine oxidase (SMO and acetylpolyamine oxidase (APAO, specifically catalyze the oxidation of spermine, and N1-acetylspermine/N1-acetylspermidine, respectively. Little is known about the molecular evolutionary history of these enzymes. However, since the yeast PAO is able to catalyze the oxidation of both acetylated and non acetylated polyamines, and in vertebrates these functions are addressed by two specialized polyamine oxidase subfamilies (APAO and SMO, it can be hypothesized an ancestral reference for the former enzyme from which the latter would have been derived. Results We analysed 36 SMO, 26 APAO, and 14 PAO homologue protein sequences from 54 taxa including various vertebrates and invertebrates. The analysis of the full-length sequences and the principal domains of vertebrate and invertebrate PAOs yielded consensus primary protein sequences for vertebrate SMOs and APAOs, and invertebrate PAOs. This analysis, coupled to molecular modeling techniques, also unveiled sequence regions that confer specific structural and functional properties, including substrate specificity, by the different PAO subfamilies. Molecular phylogenetic trees revealed a basal position of all the invertebrates PAO enzymes relative to vertebrate SMOs and APAOs. PAOs from insects constitute a monophyletic clade. Two PAO variants sampled in the amphioxus are basal to the dichotomy between two well supported

  5. Regulation of the NADPH Oxidase RBOHD During Plant Immunity.

    Science.gov (United States)

    Kadota, Yasuhiro; Shirasu, Ken; Zipfel, Cyril

    2015-08-01

    Pathogen recognition induces the production of reactive oxygen species (ROS) by NADPH oxidases in both plants and animals. ROS have direct antimicrobial properties, but also serve as signaling molecules to activate further immune outputs. However, ROS production has to be tightly controlled to avoid detrimental effects on host cells, but yet must be produced in the right amount, at the right place and at the right time upon pathogen perception. Plant NADPH oxidases belong to the respiratory burst oxidase homolog (RBOH) family, which contains 10 members in the model plant Arabidopsis thaliana. The perception of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) leads to a rapid, specific and strong production of ROS, which is dependent on RBOHD. RBOHD is mainly controlled by Ca(2+) via direct binding to EF-hand motifs and phosphorylation by Ca(2+)-dependent protein kinases. Recent studies have, however, revealed a critical role for a Ca(2+)-independent regulation of RBOHD. The plasma membrane-associated cytoplasmic kinase BIK1 (BOTRYTIS-INDUCED KINASE1), which is a direct substrate of the PRR complex, directly interacts with and phosphorylates RBOHD upon PAMP perception. Impairment of these phosphorylation events completely abolishes the function of RBOHD in immunity. These results suggest that RBOHD activity is tightly controlled by multilayered regulations. In this review, we summarize recent advances in our understanding of the regulatory mechanisms controlling RBOHD activation. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  6. Biocompatibility selenium nanoparticles with an intrinsic oxidase-like activity

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Leilei; Huang, Kaixun; Liu, Hongmei, E-mail: hmliu2004@126.com [Huazhong University of Science and Technology, School of Chemistry and Chemical Engineering (China)

    2016-03-15

    Selenium nanoparticles (SeNPs) are considered to be the new selenium supplement forms with high biological activity and low toxicity; however, the molecular mechanism by which SeNPs exert the biological function is unclear. Here, we reported that biocompatibility SeNPs possessed intrinsic oxidase-like activity. Using Na{sub 2}SeO{sub 3} as a precursor and glutathione as a reductant, biocompatibility SeNPs were synthesized by the wet chemical reduction method in the presence of bovine serum albumin (BSA). The results of structure characterization revealed that synthesized SeNPs were amorphous red elementary selenium with spherical morphology, and ranged in size from 25 to 70 nm size with a narrow distribution (41.4 ± 6.7 nm). The oxidase-like activity of the as-synthesized SeNPs was tested with 3,3′,5,5′-tetramethylbenzidine (TMB) as a substrate. The results indicated that SeNPs could catalyze the oxidization of TMB by dissolved oxygen. These SeNPs showed an optimum catalytic activity at pH 4 and 30 °C, and the oxidase-like activity was higher as the concentration of SeNPs increased and the size of SeNPs decreased. The Michaelis constant (K{sub m}) values and maximal reaction velocity (V{sub max}) of the SeNPs for TMB oxidation were 0.0083 mol/L and 3.042 μmol/L min, respectively.

  7. Biocompatibility selenium nanoparticles with an intrinsic oxidase-like activity

    International Nuclear Information System (INIS)

    Guo, Leilei; Huang, Kaixun; Liu, Hongmei

    2016-01-01

    Selenium nanoparticles (SeNPs) are considered to be the new selenium supplement forms with high biological activity and low toxicity; however, the molecular mechanism by which SeNPs exert the biological function is unclear. Here, we reported that biocompatibility SeNPs possessed intrinsic oxidase-like activity. Using Na 2 SeO 3 as a precursor and glutathione as a reductant, biocompatibility SeNPs were synthesized by the wet chemical reduction method in the presence of bovine serum albumin (BSA). The results of structure characterization revealed that synthesized SeNPs were amorphous red elementary selenium with spherical morphology, and ranged in size from 25 to 70 nm size with a narrow distribution (41.4 ± 6.7 nm). The oxidase-like activity of the as-synthesized SeNPs was tested with 3,3′,5,5′-tetramethylbenzidine (TMB) as a substrate. The results indicated that SeNPs could catalyze the oxidization of TMB by dissolved oxygen. These SeNPs showed an optimum catalytic activity at pH 4 and 30 °C, and the oxidase-like activity was higher as the concentration of SeNPs increased and the size of SeNPs decreased. The Michaelis constant (K m ) values and maximal reaction velocity (V max ) of the SeNPs for TMB oxidation were 0.0083 mol/L and 3.042 μmol/L min, respectively.

  8. Electroconvulsive therapy in patients taking monoamine oxidase inhibitors.

    Science.gov (United States)

    Dolenc, Tamara J; Habl, Samar S; Barnes, Roxann D; Rasmussen, Keith G

    2004-12-01

    Concerns have been expressed regarding the use of general anesthesia for electroconvulsive therapy (ECT) in patients taking monoamine oxidase inhibitors (MAOIs). We review the published literature and present 4 new cases and conclude that there is no evidence of a dangerous interaction between ECT and MAOI use. In general, a cautious approach would be to discontinue MAOIs before ECT if the medication has not been helpful; however, there is no need for a washout interval before starting ECT. Furthermore, if there is otherwise a reason for continuing the MAOI, it can be continued during index ECT or initiated during maintenance ECT.

  9. Lysyl Oxidase, a Targetable Secreted Molecule Involved in Cancer Metastasis

    DEFF Research Database (Denmark)

    Cox, Thomas Robert; Gartland, Alison; Erler, Janine T

    2016-01-01

    to improve the tools available in our arsenal against this disease, both in terms of treatment, but also prevention. Recently, we showed that hypoxic induction of the extracellular matrix modifying enzyme lysyl oxidase (LOX) correlates with metastatic dissemination to the bone in estrogen receptor negative...... of this enzyme as a therapeutic target for metastatic breast cancer. Our work is the latest in an emerging body of work supporting the targeting of LOX and calls for greater efforts in developing therapeutics against this extracellular secreted factor in the prevention of cancer progression across multiple solid...

  10. Putting together a plasma membrane NADH oxidase: a tale of three laboratories.

    Science.gov (United States)

    Löw, Hans; Crane, Frederick L; Morré, D James

    2012-11-01

    The observation that high cellular concentrations of NADH were associated with low adenylate cyclase activity led to a search for the mechanism of the effect. Since cyclase is in the plasma membrane, we considered the membrane might have a site for NADH action, and that NADH might be oxidized at that site. A test for NADH oxidase showed very low activity, which could be increased by adding growth factors. The plasma membrane oxidase was not inhibited by inhibitors of mitochondrial NADH oxidase such as cyanide, rotenone or antimycin. Stimulation of the plasma membrane oxidase by iso-proterenol or triiodothyronine was different from lack of stimulation in endoplasmic reticulum. After 25 years of research, three components of a trans membrane NADH oxidase have been discovered. Flavoprotein NADH coenzyme Q reductases (NADH cytochrome b reductase) on the inside, coenzyme Q in the middle, and a coenzyme Q oxidase on the outside as a terminal oxidase. The external oxidase segment is a copper protein with unique properties in timekeeping, protein disulfide isomerase and endogenous NADH oxidase activity, which affords a mechanism for control of cell growth by the overall NADH oxidase and the remarkable inhibition of oxidase activity and growth of cancer cells by a wide range of anti-tumor drugs. A second trans plasma membrane electron transport system has been found in voltage dependent anion channel (VDAC), which has NADH ferricyanide reductase activity. This activity must be considered in relation to ferricyanide stimulation of growth and increased VDAC antibodies in patients with autism. Copyright © 2012 Elsevier Ltd. All rights reserved.

  11. Analysis of a two-domain binding site for the urokinase-type plasminogen activator-plasminogen activator inhibitor-1 complex in low-density-lipoprotein-receptor-related protein.

    Science.gov (United States)

    Andersen, O M; Petersen, H H; Jacobsen, C; Moestrup, S K; Etzerodt, M; Andreasen, P A; Thøgersen, H C

    2001-07-01

    The low-density-lipoprotein-receptor (LDLR)-related protein (LRP) is composed of several classes of domains, including complement-type repeats (CR), which occur in clusters that contain binding sites for a multitude of different ligands. Each approximately 40-residue CR domain contains three conserved disulphide linkages and an octahedral Ca(2+) cage. LRP is a scavenging receptor for ligands from extracellular fluids, e.g. alpha(2)-macroglobulin (alpha(2)M)-proteinase complexes, lipoprotein-containing particles and serine proteinase-inhibitor complexes, like the complex between urokinase-type plasminogen activator (uPA) and the plasminogen activator inhibitor-1 (PAI-1). In the present study we analysed the interaction of the uPA-PAI-1 complex with an ensemble of fragments representing a complete overlapping set of two-domain fragments accounting for the ligand-binding cluster II (CR3-CR10) of LRP. By ligand blotting, solid-state competition analysis and surface-plasmon-resonance analysis, we demonstrate binding to multiple CR domains, but show a preferential interaction between the uPA-PAI-1 complex and a two-domain fragment comprising CR domains 5 and 6 of LRP. We demonstrate that surface-exposed aspartic acid and tryptophan residues at identical positions in the two homologous domains, CR5 and CR6 (Asp(958,CR5), Asp(999,CR6), Trp(953,CR5) and Trp(994,CR6)), are critical for the binding of the complex as well as for the binding of the receptor-associated protein (RAP) - the folding chaperone/escort protein required for transport of LRP to the cell surface. Accordingly, the present work provides (1) an identification of a preferred binding site within LRP CR cluster II; (2) evidence that the uPA-PAI-1 binding site involves residues from two adjacent protein domains; and (3) direct evidence identifying specific residues as important for the binding of uPA-PAI-1 as well as for the binding of RAP.

  12. Plasma amine oxidase activities in Norrie disease patients with an X-chromosomal deletion affecting monoamine oxidase.

    Science.gov (United States)

    Murphy, D L; Sims, K B; Karoum, F; Garrick, N A; de la Chapelle, A; Sankila, E M; Norio, R; Breakefield, X O

    1991-01-01

    Two individuals with an X-chromosomal deletion were recently found to lack the genes encoding monoamine oxidase type A (MAO-A) and MAO-B. This abnormality was associated with almost total (90%) reductions in the oxidatively deaminated urinary metabolites of the MAO-A substrate, norepinephrine, and with marked (100-fold) increases in an MAO-B substrate, phenylethylamine, confirming systemic functional consequences of the genetic enzyme deficiency. However, urinary concentrations of the deaminated metabolites of dopamine and serotonin (5-HT) were essentially normal. To investigate other deaminating systems besides MAO-A and MAO-B that might produce these metabolites of dopamine and 5-HT, we examined plasma amine oxidase (AO) activity in these two patients and two additional patients with the same X-chromosomal deletion. Normal plasma AO activity was found in all four Norrie disease-deletion patients, in four patients with classic Norrie disease without a chromosomal deletion, and in family members of patients from both groups. Marked plasma amine metabolite abnormalities and essentially absent platelet MAO-B activity were found in all four Norrie disease-deletion patients, but in none of the other subjects in the two comparison groups. These results indicate that plasma AO is encoded by gene(s) independent of those for MAO-A and MAO-B, and raise the possibility that plasma AO, and perhaps the closely related tissue AO, benzylamine oxidase, as well as other atypical AOs or MAOs encoded independently from MAO-A and MAO-B may contribute to the oxidative deamination of dopamine and 5-HT in humans.

  13. Hofmeister effects on the glucose oxidase hydrogel-modified electrode

    International Nuclear Information System (INIS)

    Suzuki, Aimi; Tsujimura, Seiya

    2016-01-01

    We describe the consistent effect of salts in the electrolyte solution on glucose oxidation current production in the redox hydrogel-modified electrode containing glucose oxidase as an electrocatalyst and Os complex mediator. The ions affect not only on the electron transfer between the enzyme and the Os complex, but also on the hydrogel structure. This study found that the degree of the effect can be characterized by Hofmeister series. The relative decrease in oxidization current is the lowest in the middle of the Hofmeister series, and increases monotonically on either side. An increase of ionic strength inhibits the electron transfer from the active site of glucose oxidase to Os complex. In addition to this, the kosmotropic anions, which are strongly hydrated, caused hydrogel deswelling (shrinking). The more chaotropic an ion is, the more it adsorbs to uncharged parts of polymer/enzyme with dispersion force, and the swelling of the hydrogel decreases the catalytic current. This study impacts the design of hydrogel electrode and selection of electrolyte ions for bioelectronic applications.

  14. Cytochrome oxidase assembly does not require catalytically active cytochrome C.

    Science.gov (United States)

    Barrientos, Antoni; Pierre, Danielle; Lee, Johnson; Tzagoloff, Alexander

    2003-03-14

    Cytochrome c oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain, catalyzes the transfer of electrons from reduced cytochrome c to molecular oxygen. COX assembly requires the coming together of nuclear- and mitochondrial-encoded subunits and the assistance of a large number of nuclear gene products acting at different stages of maturation of the enzyme. In Saccharomyces cerevisiae, expression of cytochrome c, encoded by CYC1 and CYC7, is required not only for electron transfer but also for COX assembly through a still unknown mechanism. We have attempted to distinguish between a functional and structural requirement of cytochrome c in COX assembly. A cyc1/cyc7 double null mutant strain was transformed with the cyc1-166 mutant gene (Schweingruber, M. E., Stewart, J. W., and Sherman, F. (1979) J. Biol. Chem. 254, 4132-4143) that expresses stable but catalytically inactive iso-1-cytochrome c. The COX content of the cyc1/cyc7 double mutant strain harboring non-functional iso-1-cytochrome c has been characterized spectrally, functionally, and immunochemically. The results of these studies demonstrate that cytochrome c plays a structural rather than functional role in assembly of cytochrome c oxidase. In addition to its requirement for COX assembly, cytochrome c also affects turnover of the enzyme. Mutants containing wild type apocytochrome c in mitochondria lack COX, suggesting that only the folded and mature protein is able to promote COX assembly.

  15. A broad distribution of the alternative oxidase in microsporidian parasites.

    Directory of Open Access Journals (Sweden)

    Bryony A P Williams

    2010-02-01

    Full Text Available Microsporidia are a group of obligate intracellular parasitic eukaryotes that were considered to be amitochondriate until the recent discovery of highly reduced mitochondrial organelles called mitosomes. Analysis of the complete genome of Encephalitozoon cuniculi revealed a highly reduced set of proteins in the organelle, mostly related to the assembly of iron-sulphur clusters. Oxidative phosphorylation and the Krebs cycle proteins were absent, in keeping with the notion that the microsporidia and their mitosomes are anaerobic, as is the case for other mitosome bearing eukaryotes, such as Giardia. Here we provide evidence opening the possibility that mitosomes in a number of microsporidian lineages are not completely anaerobic. Specifically, we have identified and characterized a gene encoding the alternative oxidase (AOX, a typically mitochondrial terminal oxidase in eukaryotes, in the genomes of several distantly related microsporidian species, even though this gene is absent from the complete genome of E. cuniculi. In order to confirm that these genes encode functional proteins, AOX genes from both A. locustae and T. hominis were over-expressed in E. coli and AOX activity measured spectrophotometrically using ubiquinol-1 (UQ-1 as substrate. Both A. locustae and T. hominis AOX proteins reduced UQ-1 in a cyanide and antimycin-resistant manner that was sensitive to ascofuranone, a potent inhibitor of the trypanosomal AOX. The physiological role of AOX microsporidia may be to reoxidise reducing equivalents produced by glycolysis, in a manner comparable to that observed in trypanosomes.

  16. Oxidase catalysis via aerobically generated hypervalent iodine intermediates

    Science.gov (United States)

    Maity, Asim; Hyun, Sung-Min; Powers, David C.

    2018-02-01

    The development of sustainable oxidation chemistry demands strategies to harness O2 as a terminal oxidant. Oxidase catalysis, in which O2 serves as a chemical oxidant without necessitating incorporation of oxygen into reaction products, would allow diverse substrate functionalization chemistry to be coupled to O2 reduction. Direct O2 utilization suffers from intrinsic challenges imposed by the triplet ground state of O2 and the disparate electron inventories of four-electron O2 reduction and two-electron substrate oxidation. Here, we generate hypervalent iodine reagents—a broadly useful class of selective two-electron oxidants—from O2. This is achieved by intercepting reactive intermediates of aldehyde autoxidation to aerobically generate hypervalent iodine reagents for a broad array of substrate oxidation reactions. The use of aryl iodides as mediators of aerobic oxidation underpins an oxidase catalysis platform that couples substrate oxidation directly to O2 reduction. We anticipate that aerobically generated hypervalent iodine reagents will expand the scope of aerobic oxidation chemistry in chemical synthesis.

  17. 2-acetylphenol analogs as potent reversible monoamine oxidase inhibitors

    Directory of Open Access Journals (Sweden)

    Legoabe LJ

    2015-07-01

    Full Text Available Lesetja J Legoabe,1 Anél Petzer,1 Jacobus P Petzer1,21Centre of Excellence for Pharmaceutical Sciences, 2Department of Pharmaceutical Chemistry, School of Pharmacy, North-West University, Potchefstroom, South AfricaAbstract: Based on a previous report that substituted 2-acetylphenols may be promising leads for the design of novel monoamine oxidase (MAO inhibitors, a series of C5-substituted 2-acetylphenol analogs (15 and related compounds (two were synthesized and evaluated as inhibitors of human MAO-A and MAO-B. Generally, the study compounds exhibited inhibitory activities against both MAO-A and MAO-B, with selectivity for the B isoform. Among the compounds evaluated, seven compounds exhibited IC50 values <0.01 µM for MAO-B inhibition, with the most selective compound being 17,000-fold selective for MAO-B over the MAO-A isoform. Analyses of the structure–activity relationships for MAO inhibition show that substitution on the C5 position of the 2-acetylphenol moiety is a requirement for MAO-B inhibition, and the benzyloxy substituent is particularly favorable in this regard. This study concludes that C5-substituted 2-acetylphenol analogs are potent and selective MAO-B inhibitors, appropriate for the design of therapies for neurodegenerative disorders such as Parkinson’s disease.Keywords: monoamine oxidase, MAO, inhibition, 2-acetylphenol, structure–activity relationship

  18. Biodegradation of phenolic compounds with oxidases from sorghum and non-defined mixed bacterium media

    International Nuclear Information System (INIS)

    Obame, C. E. L.; Savadogo, P. W.; Mamoudou, D. H.; Dembele, R. H.; Traore, A. S.

    2009-01-01

    The biodegradation of the phenolic compounds is performed using oxidative enzymes, e. g. polyphenol oxidases (PPOs) and peroxidases (POXs). These oxidases displaying a wide spectrum for the oxidation of phenolic compounds were isolated either from sorghum or mixed bacteria. Spectrophotometric methods were used to assess the monophenolase and diphenolase activities of PPOs as well as the hydrogen-dependant oxidation of POXs. (Author)

  19. Study on the preparation of immobilized glucose oxidase membrane and its application in clinic analysis

    International Nuclear Information System (INIS)

    Yu Ye; Cao Jin; Su Zongxian; Chen Zixiong

    1990-01-01

    The paper deals with the preparation of immobilized glucose oxidase membrane by using two steps irradiation (irradiation gratfting, irradiation entrapping). Some properties of membrane were discussed. The immobilized glucose oxidase membrane with oxygen electrode and oxygen analyser can be satisfied with the clinic analysis for the determination of serum glucose

  20. Switching an O2 sensitive glucose oxidase bioelectrode into an almost insensitive one by cofactor redesign.

    Science.gov (United States)

    Tremey, Emilie; Suraniti, Emmanuel; Courjean, Olivier; Gounel, Sébastien; Stines-Chaumeil, Claire; Louerat, Frédéric; Mano, Nicolas

    2014-06-04

    In the 5-8 mM glucose concentration range, of particular interest for diabetes management, glucose oxidase bioelectrodes are O2 dependent, which decrease their efficiencies. By replacing the natural cofactor of glucose oxidase, we succeeded in turning an O2 sensitive bioelectrode into an almost insensitive one.

  1. Hydroxychavicol: a potent xanthine oxidase inhibitor obtained from the leaves of betel, Piper betle.

    Science.gov (United States)

    Murata, Kazuya; Nakao, Kikuyo; Hirata, Noriko; Namba, Kensuke; Nomi, Takao; Kitamura, Yoshihisa; Moriyama, Kenzo; Shintani, Takahiro; Iinuma, Munekazu; Matsuda, Hideaki

    2009-07-01

    The screening of Piperaceous plants for xanthine oxidase inhibitory activity revealed that the extract of the leaves of Piper betle possesses potent activity. Activity-guided purification led us to obtain hydroxychavicol as an active principle. Hydroxychavicol is a more potent xanthine oxidase inhibitor than allopurinol, which is clinically used for the treatment of hyperuricemia.

  2. Analysis of cellulase and polyphenol oxidase production by southern pine beetle associated fungi

    Science.gov (United States)

    Abduvali Valiev; Zumrut B. Ogel; Dier D. Klepzig

    2009-01-01

    In this study, the production of extracellular enzymes by fungi associated with southern pine beetle was investigated for the first time. Cellulase and polyphenol oxidase production were analyzed for three beetle associated fungi. Only the mutualistic symbiont Entomocorticium sp. A was found to produce cellulases and polyphenol oxidase....

  3. Purification, characterization and decolorization of bilirubin oxidase from Myrothecium verrucaria 3.2190

    Science.gov (United States)

    Myrothecium verrucaria 3.2190 is a nonligninolytic fungus that produces bilirubin oxidase. Both Myrothecium verrucaria and the extracellular bilirubin oxidase were tested for their ability to decolorize indigo carmine. The biosorption and biodegradation of the dye were detected during the process of...

  4. Hot or not? Discovery and characterization of a thermostable alditol oxidase from Acidothermus cellulolyticus 11B

    NARCIS (Netherlands)

    Winter, Remko T.; Heuts, Dominic P. H. M.; Rijpkema, Egon M. A.; van Bloois, Edwin; Wijma, Hein J.; Fraaije, Marco W.

    We describe the discovery, isolation and characterization of a highly thermostable alditol oxidase from Acidothermus cellulolyticus 11B. This protein was identified by searching the genomes of known thermophiles for enzymes homologous to Streptomyces coelicolor A3(2) alditol oxidase (AldO). A gene

  5. Discovery, characterization, and kinetic analysis of an alditol oxidase from streptomyces coelicolor

    NARCIS (Netherlands)

    Heuts, Dominic P. H. M.; van Hellemond, Erik W.; Janssen, Dick B.; Fraaije, Marco W.

    2007-01-01

    A gene encoding an alditol oxidase was found in the genome of Streptomyces coelicolor A3(2). This newly identified oxidase, AldO, was expressed at extremely high levels in Escherichia coli when fused to maltose-binding protein. AldO is a soluble monomeric flavoprotein with subunits of 45.1 kDa, each

  6. Biodegradation of phenolic compounds with oxidases from sorghum and non-defined mixed bacterium media

    Energy Technology Data Exchange (ETDEWEB)

    Obame, C. E. L.; Savadogo, P. W.; Mamoudou, D. H.; Dembele, R. H.; Traore, A. S.

    2009-07-01

    The biodegradation of the phenolic compounds is performed using oxidative enzymes, e. g. polyphenol oxidases (PPOs) and peroxidases (POXs). These oxidases displaying a wide spectrum for the oxidation of phenolic compounds were isolated either from sorghum or mixed bacteria. Spectrophotometric methods were used to assess the monophenolase and diphenolase activities of PPOs as well as the hydrogen-dependant oxidation of POXs. (Author)

  7. Expression and Chloroplast Targeting of Cholesterol Oxidase in Transgenic Tobacco Plants

    Science.gov (United States)

    Corbin, David R.; Grebenok, Robert J.; Ohnmeiss, Thomas E.; Greenplate, John T.; Purcell, John P.

    2001-01-01

    Cholesterol oxidase represents a novel type of insecticidal protein with potent activity against the cotton boll weevil (Anthonomus grandis grandis Boheman). We transformed tobacco (Nicotiana tabacum) plants with the cholesterol oxidase choM gene and expressed cytosolic and chloroplast-targeted versions of the ChoM protein. Transgenic leaf tissues expressing cholesterol oxidase exerted insecticidal activity against boll weevil larvae. Our results indicate that cholesterol oxidase can metabolize phytosterols in vivo when produced cytosolically or when targeted to chloroplasts. The transgenic plants exhibiting cytosolic expression accumulated low levels of saturated sterols known as stanols, and displayed severe developmental aberrations. In contrast, the transgenic plants expressing chloroplast-targeted cholesterol oxidase maintained a greater accumulation of stanols, and appeared phenotypically and developmentally normal. These results are discussed within the context of plant sterol distribution and metabolism. PMID:11457962

  8. Amine oxidase from lentil seedlings: energetic domains and effect of temperature on activity.

    Science.gov (United States)

    Moosavi-Nejad, S Z; Rezaei-Tavirani, M; Padiglia, A; Floris, G; Moosavi-Movahedi, A A

    2001-07-01

    Copper/TPQ amine oxidases from mammalian and plant sources have shown many differences in substrate specificity and molecular properties. In this work the activity of lentil seedling amine oxidase was followed at various temperatures in 100 mM potassium phosphate buffer, pH 7, using benzylamine as substrate. The discontinuous Arrhenius plot of lentil amine oxidase showed two distinct phases with a jump between them. Thermal denaturation of the enzyme, using differential scanning calorimetry under the same experimental conditions, showed a transition at the same temperature ranges in the absence of substrate, indicating the occurrence of conformational changes, with an enthalpy change of about 175.9 kJ/mole. The temperature-induced changes of the activity of lentil amine oxidase are compared with those of bovine serum amine oxidase (taken from the literature).

  9. Functional expression of amine oxidase from Aspergillus niger (AO-I) in Saccharomyces cerevisiae.

    Science.gov (United States)

    Kolaríková, Katerina; Galuszka, Petr; Sedlárová, Iva; Sebela, Marek; Frébort, Ivo

    2009-01-01

    The aim of this work was to prepare recombinant amine oxidase from Aspergillus niger after overexpressing in yeast. The yeast expression vector pDR197 that includes a constitutive PMA1 promoter was used for the expression in Saccharomyces cerevisiae. Recombinant amine oxidase was extracted from the growth medium of the yeast, purified to homogeneity and identified by activity assay and MALDI-TOF peptide mass fingerprinting. Similarity search in the newly published A. niger genome identified six genes coding for copper amine oxidase, two of them corresponding to the previously described enzymes AO-I a methylamine oxidase and three other genes coding for FAD amine oxidases. Thus, A. niger possesses an enormous metabolic gear to grow on amine compounds and thus support its saprophytic lifestyle.

  10. [Experimental rationale for the parameters of a rapid method for oxidase activity determination].

    Science.gov (United States)

    Butorina, N N

    2010-01-01

    Experimental rationale is provided for the parameters of a rapid (1-2-min) test to concurrently determine the oxidase activity of all bacteria grown on the membrane filter after water filtration. Oxidase reagents that are the aqueous solutions of tetramethyl-p-phenylenediamine dihydrochloride and demethyl-p-phenylenediamine dihydrochloride have been first ascertained to exert no effect on the viability and enzymatic activity of bacteria after one-hour contact. An algorithm has been improved for the rapid oxidase activity test: the allowable time for bacteria to contact oxidase reagents and procedures for minimizing the effect on bacterial biochemical activity following the contact. An accelerated method based on lactose medium with tergitol 7 and Endo agar has been devised to determine coliform bacteria, by applying the rapid oxidase test: the time of a final response is 18-24 hours. The method has been included into GOST 52426-2005.

  11. A Biocatalytic One-Pot Approach for the Preparation of Lignin Oligomers Using an Oxidase/Peroxidase Cascade Enzyme System

    NARCIS (Netherlands)

    Habib, Mohamed H. M.; Deuss, Peter J.; Loncar, Nikola; Trajkovic, Milos; Fraaije, Marco W.

    2017-01-01

    Synthetic lignin was prepared biocatalytically in a one-pot, two-step reaction using an oxidase/peroxidase cascade enzyme system. Using eugenol in combination with eugenol oxidase and a peroxidase, lignin-like material was produced. The cascade reaction takes advantage of the ability of the oxidase

  12. Facile direct electron transfer in glucose oxidase modified electrodes

    International Nuclear Information System (INIS)

    Wang Dan; Chen Liwei

    2009-01-01

    Glucose oxidase (GOx) is widely used in the glucose biosensor industry. However, mediatorless direct electron transfer (DET) from GOx to electrode surfaces is very slow. Recently, mediatorless DET has been reported via the incorporation of nanomaterials such as carbon nanotubes and nanoparticles in the modification of electrodes. Here we report GOx electrodes showing DET without the need for any nanomaterials. The enzyme after immobilization with poly-L-lysine (PLL) and Nafion retains the biocatalytic activities and oxidizes glucose efficiently. The amperometric response of Nafion-PLL-GOx modified electrode is linearly proportional to the concentration of glucose up to 10 mM with a sensitivity of 0.75 μA/mM at a low detection potential (-0.460 V vs. Ag/AgCl). The methodology developed in this study will have impact on glucose biosensors and biofuel cells and may potentially simplify enzyme immobilization in other biosensing systems.

  13. Low platelet monoamine oxidase activity in pathological gambling

    International Nuclear Information System (INIS)

    Carrasco, J.L.; Saiz-Ruiz, J.; Hollander, E.; Cesar, J.; Lopez-Ibor, J.J. Jr.

    1994-01-01

    Decreased platelet monoamine oxidase (MAO) activity has been reported in association with sensation-seeking personality type and in some mental disorders associated with a lack of impulse control. Pathological gambling itself has been related with both sensation-seeking and reduced impulse control. Platelet MAO activity was investigated in 15 DSM-III-R pathological gamblers from our outpatient clinic. Gamblers had a significantly lower platelet MAO activity than a group of 25 healthy controls. The range of MAO levels in gamblers was also significantly shorter than in controls. In controls, platelet MAO levels showed the previously described negative correlations with sensation-seeking scores but not in gamblers. The findings are consistent with previous studies showing an association of low platelet MAO activity with impulse control disorders and raise some interesting therapeutic alternatives for pathological gambling. (au) (40 refs.)

  14. Dual oxidase in mucosal immunity and host-microbe homeostasis.

    Science.gov (United States)

    Bae, Yun Soo; Choi, Myoung Kwon; Lee, Won-Jae

    2010-07-01

    Mucosal epithelia are in direct contact with microbes, which range from beneficial symbionts to pathogens. Accordingly, hosts must have a conflicting strategy to combat pathogens efficiently while tolerating symbionts. Recent progress has revealed that dual oxidase (DUOX) plays a key role in mucosal immunity in organisms that range from flies to humans. Information from the genetic model of Drosophila has advanced our understanding of the regulatory mechanism of DUOX and its role in mucosal immunity. Further investigations of DUOX regulation in response to symbiotic or non-symbiotic bacteria and the in vivo consequences in host physiology will give a novel insight into the microbe-controlling system of the mucosa. Copyright 2010 Elsevier Ltd. All rights reserved.

  15. Polyphenol Oxidases in Crops: Biochemical, Physiological and Genetic Aspects

    Directory of Open Access Journals (Sweden)

    Francesca Taranto

    2017-02-01

    Full Text Available Enzymatic browning is a colour reaction occurring in plants, including cereals, fruit and horticultural crops, due to oxidation during postharvest processing and storage. This has a negative impact on the colour, flavour, nutritional properties and shelf life of food products. Browning is usually caused by polyphenol oxidases (PPOs, following cell damage caused by senescence, wounding and the attack of pests and pathogens. Several studies indicated that PPOs play a role in plant immunity, and emerging evidence suggested that PPOs might also be involved in other physiological processes. Genomic investigations ultimately led to the isolation of PPO homologs in several crops, which will be possibly characterized at the functional level in the near future. Here, focusing on the botanic families of Poaceae and Solanaceae, we provide an overview on available scientific literature on PPOs, resulting in useful information on biochemical, physiological and genetic aspects.

  16. The Membrane Modulates Internal Proton Transfer in Cytochrome c Oxidase

    DEFF Research Database (Denmark)

    Öjemyr, Linda Nasvik; Ballmoos, Christoph von; Faxén, Kristina

    2012-01-01

    The functionality of membrane proteins is often modulated by the surrounding membrane. Here, we investigated the effect of membrane reconstitution of purified cytochrome c oxidase (CytcO) on the kinetics and thermodynamics of internal electron and proton-transfer reactions during O-2 reduction...... DOPC lipids. In conclusion, the data show that the membrane significantly modulates internal charge-transfer reactions and thereby the function of the membrane-bound enzyme.......-glycerol) (DOPG). In addition, a small Change in the internal Cu-A-heme a electron equilibrium constant was observed. This effect was lipid-dependent and explained in terms of a lower electrostatic potential within the membrane-spanning part of the protein with the anionic DOPG lipids than with the zwitterionic...

  17. Purification and characterization of amine oxidase from soybean seedlings.

    Science.gov (United States)

    Vianello, F; Di Paolo, M L; Stevanato, R; Gasparini, R; Rigo, A

    1993-11-15

    A simple and rapid procedure for purification of soybean seedling amine oxidase is reported. The crude enzyme, obtained by ammonium sulfate fractionation was purified by ion-exchange chromatography on a cellulose phosphate column and batch affinity chromatography on 6-aminohexyl-Sepharose. Cyclohexylamine, a competitive inhibitor, was utilized to elute the enzyme. A homogeneous enzyme was obtained with a yield higher than 25%, the content of minor components being lauryl sulfate-polyacrylamide gel electrophoresis. The enzyme is a dimer and contains two Cu2+ ion per molecule. Its EPR spectrum is typical of Cu2+ in a tetragonal symmetry. The enzyme oxidizes cadaverine at high rate, the specific activity being 4.3 mukat/mg. Molecular, spectroscopic, and kinetic properties of this enzyme are reported.

  18. Low platelet monoamine oxidase activity in pathological gambling

    Energy Technology Data Exchange (ETDEWEB)

    Carrasco, J.L. [Department of Psychiatry, Centro de Salud Mental, Parla Madrid (Spain); Saiz-Ruiz, J. [Department of Psychiatry and Haematology, Hospital Ramon y Cajal, Madrid (Spain); Hollander, E. [Department of Psychiatry, Mount Sinai School of Medicine, Queens Hospital Center, New York (United States); Cesar, J. [Department of Haematology, Hospital Ramon y Cajal, Madrid (Spain); Lopez-Ibor, J.J. Jr. [Department of Psychiatry, Hospital San Carlos, Complutense University, Madrid (Spain)

    1994-12-01

    Decreased platelet monoamine oxidase (MAO) activity has been reported in association with sensation-seeking personality type and in some mental disorders associated with a lack of impulse control. Pathological gambling itself has been related with both sensation-seeking and reduced impulse control. Platelet MAO activity was investigated in 15 DSM-III-R pathological gamblers from our outpatient clinic. Gamblers had a significantly lower platelet MAO activity than a group of 25 healthy controls. The range of MAO levels in gamblers was also significantly shorter than in controls. In controls, platelet MAO levels showed the previously described negative correlations with sensation-seeking scores but not in gamblers. The findings are consistent with previous studies showing an association of low platelet MAO activity with impulse control disorders and raise some interesting therapeutic alternatives for pathological gambling. (au) (40 refs.).

  19. Optical characterization of porous silicon microcavities for glucose oxidase biosensing

    Science.gov (United States)

    Palestino, G.; Agarwal, V.; Garcia, D. B.; Legros, R.; Pérez, E.; Gergely, C.

    2008-04-01

    PSi microcavity (PSiMc) is characterized by a narrow resonance peak in the optical spectrum that is very sensitive to small changes in the refractive index. We report that the resonant optical cavities of PSi structures can be used to enhance the detection of labeled fluorescent biomolecules. Various PSi configurations were tested in order to compare the optical response of the PSi devices to the capture of organic molecules. Morphological and topographical analyses were performed on PSiMc using Atomic Force (AFM) and Scanning Electron (SEM) microscopies. The heterogeneity in pores lengths resulting from etching process assures a better penetration of larger molecules into the pores and sensor sensitivity depends on the pore size. Molecular detection is monitored by the successive red shifts in the reflectance spectra after the stabilization of PSiMc with 3-aminopropyltriethoxysilane (APTES). The glucose oxidase was cross linked into the PSiMc structures following a silane-glutaraldehyde (GTA) chemistry.

  20. Magnetic field effects on brain monoamine oxidase activity

    Energy Technology Data Exchange (ETDEWEB)

    Borets, V.M.; Ostrovskiy, V.Yu.; Bankovskiy, A.A.; Dudinskaya, T.F.

    1985-03-01

    In view of the increasing use of magnetotherapy, studies were conducted on the effects of 35 mTesla magnetic fields on monoamine oxidase activity in the rat brain. Under in vitro conditions a constant magnetic field in the continuous mode was most effective in inhibiting deamination of dopamine following 1 min exposure, while in vivo studies with 8 min or 10 day exposures showed that inhibition was obtained only with a variable field in the continuous mode. However, inhibition of dopamine deamination was only evident within the first 24 h after exposure was terminated. In addition, in none of the cases was norepinephrine deamination inhibited. The effects of the magnetic fields were, therefore, transient and selective with the CNS as the target system. 9 references.

  1. Suicide attempts, platelet monoamine oxidase and the average evoked response

    International Nuclear Information System (INIS)

    Buchsbaum, M.S.; Haier, R.J.; Murphy, D.L.

    1977-01-01

    The relationship between suicides and suicide attempts and two biological measures, platelet monoamine oxidase levels (MAO) and average evoked response (AER) augmenting was examined in 79 off-medication psychiatric patients and in 68 college student volunteers chosen from the upper and lower deciles of MAO activity levels. In the patient sample, male individuals with low MAO and AER augmenting, a pattern previously associated with bipolar affective disorders, showed a significantly increased incidence of suicide attempts in comparison with either non-augmenting low MAO or high MAO patients. Within the normal volunteer group, all male low MAO probands with a family history of suicide or suicide attempts were AER augmenters themselves. Four completed suicides were found among relatives of low MAO probands whereas no high MAO proband had a relative who committed suicide. These findings suggest that the combination of low platelet MAO activity and AER augmenting may be associated with a possible genetic vulnerability to psychiatric disorders. (author)

  2. Two Domains of Vimentin Are Expressed on the Surface of Lymph Node, Bone and Brain Metastatic Prostate Cancer Lines along with the Putative Stem Cell Marker Proteins CD44 and CD133

    Energy Technology Data Exchange (ETDEWEB)

    Steinmetz, Nicole F. [Case Western Reserve University, Department of Biomedical Engineering, 10900 Euclid Ave, Cleveland, OH 44106 (United States); Maurer, Jochen [Sanford-Burnham, Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037 (United States); Sheng, Huiming [Torrey Pines Institute for Molecular Studies, Division of Immune Regulation, 3550 General Atomics Court, San Diego, CA 92121 (United States); Bensussan, Armand [INSERM U976, Hôpital Saint Louis, F-75475 Paris (France); Department of Immunology, Dermatology and Oncology, Univ Paris Diderot, Sorbonne Paris Cité, UMRS976 F-75475 Paris (France); Maricic, Igor; Kumar, Vipin [Torrey Pines Institute for Molecular Studies, Laboratory of Autoimmunity, 3550 General Atomics Court, San Diego, CA 92121 (United States); Braciak, Todd A., E-mail: tbraciak@tpims.org [Torrey Pines Institute for Molecular Studies, Division of Immune Regulation, 3550 General Atomics Court, San Diego, CA 92121 (United States)

    2011-07-13

    Vimentin was originally identified as an intermediate filament protein present only as an intracellular component in many cell types. However, this protein has now been detected on the surface of a number of different cancer cell types in a punctate distribution pattern. Increased vimentin expression has been indicated as an important step in epithelial-mesenchymal transition (EMT) required for the metastasis of prostate cancer. Here, using two vimentin-specific monoclonal antibodies (SC5 and V9 directed against the coil one rod domain and the C-terminus of the vimentin protein, respectively), we examined whether either of these domains would be displayed on the surface of three commonly studied prostate cancer cell lines isolated from different sites of metastases. Confocal analysis of LNCaP, PC3 and DU145 prostate cancer cell lines (derived from lymph node, bone or brain prostate metastases, respectively) demonstrated that both domains of vimentin are present on the surface of these metastatic cancer cell types. In addition, flow cytometric analysis revealed that vimentin expression was readily detected along with CD44 expression but only a small subpopulation of prostate cancer cells expressed vimentin and the putative stem cell marker CD133 along with CD44. Finally, Cowpea mosaic virus (CPMV) nanoparticles that target vimentin could bind and internalize into tested prostate cancer cell lines. These results demonstrate that at least two domains of vimentin are present on the surface of metastatic prostate cancer cells and suggest that vimentin could provide a useful target for nanoparticle- or antibody- cancer therapeutic agents directed against highly invasive cancer and/or stem cells.

  3. Reengineered glucose oxidase for amperometric glucose determination in diabetes analytics.

    Science.gov (United States)

    Arango Gutierrez, Erik; Mundhada, Hemanshu; Meier, Thomas; Duefel, Hartmut; Bocola, Marco; Schwaneberg, Ulrich

    2013-12-15

    Glucose oxidase is an oxidoreductase exhibiting a high β-D-glucose specificity and high stability which renders glucose oxidase well-suited for applications in diabetes care. Nevertheless, GOx activity is highly oxygen dependent which can lead to inaccuracies in amperometric β-D-glucose determinations. Therefore a directed evolution campaign with two rounds of random mutagenesis (SeSaM followed by epPCR), site saturation mutagenesis studies on individual positions, and one simultaneous site saturation library (OmniChange; 4 positions) was performed. A diabetes care well suited mediator (quinone diimine) was selected and the GOx variant (T30V I94V) served as starting point. For directed GOx evolution a microtiter plate detection system based on the quinone diimine mediator was developed and the well-known ABTS-assay was applied in microtiter plate format to validate oxygen independency of improved GOx variants. Two iterative rounds of random diversity generation and screening yielded to two subsets of amino acid positions which mainly improved activity (A173, A332) and oxygen independency (F414, V560). Simultaneous site saturation of all four positions with a reduced subset of amino acids using the OmniChange method yielded finally variant V7 with a 37-fold decreased oxygen dependency (mediator activity: 7.4 U/mg WT, 47.5 U/mg V7; oxygen activity: 172.3 U/mg WT, 30.1 U/mg V7). V7 is still highly β-D-glucose specific, highly active with the quinone diimine mediator and thermal resistance is retained (prerequisite for GOx coating of diabetes test stripes). The latter properties and V7's oxygen insensitivity make V7 a very promising candidate to replace standard GOx in diabetes care applications. Copyright © 2013 Elsevier B.V. All rights reserved.

  4. Molecular Dynamic Studies of the Complex Polyethylenimine and Glucose Oxidase.

    Science.gov (United States)

    Szefler, Beata; Diudea, Mircea V; Putz, Mihai V; Grudzinski, Ireneusz P

    2016-10-27

    Glucose oxidase (GOx) is an enzyme produced by Aspergillus, Penicillium and other fungi species. It catalyzes the oxidation of β-d-glucose (by the molecular oxygen or other molecules, like quinones, in a higher oxidation state) to form d-glucono-1,5-lactone, which hydrolyses spontaneously to produce gluconic acid. A coproduct of this enzymatic reaction is hydrogen peroxide (H₂O₂). GOx has found several commercial applications in chemical and pharmaceutical industries including novel biosensors that use the immobilized enzyme on different nanomaterials and/or polymers such as polyethylenimine (PEI). The problem of GOx immobilization on PEI is retaining the enzyme native activity despite its immobilization onto the polymer surface. Therefore, the molecular dynamic (MD) study of the PEI ligand (C14N8_07_B22) and the GOx enzyme (3QVR) was performed to examine the final complex PEI-GOx stabilization and the affinity of the PEI ligand to the docking sites of the GOx enzyme. The docking procedure showed two places/regions of major interaction of the protein with the polymer PEI: (LIG1) of -5.8 kcal/mol and (LIG2) of -4.5 kcal/mol located inside the enzyme and on its surface, respectively. The values of enthalpy for the PEI-enzyme complex, located inside of the protein (LIG1) and on its surface (LIG2) were computed. Docking also discovered domains of the GOx protein that exhibit no interactions with the ligand or have even repulsive characteristics. The structural data clearly indicate some differences in the ligand PEI behavior bound at the two places/regions of glucose oxidase.

  5. Apocynin: Chemical and Biophysical Properties of a NADPH Oxidase Inhibitor

    Directory of Open Access Journals (Sweden)

    Valdecir F. Ximenes

    2013-03-01

    Full Text Available Apocynin is the most employed inhibitor of NADPH oxidase (NOX, a multienzymatic complex capable of catalyzing the one-electron reduction of molecular oxygen to the superoxide anion. Despite controversies about its selectivity, apocynin has been used as one of the most promising drugs in experimental models of inflammatory and neurodegenerative diseases. Here, we aimed to study the chemical and biophysical properties of apocynin. The oxidation potential was determined by cyclic voltammetry (Epa = 0.76V, the hydrophobicity index was calculated (logP = 0.83 and the molar absorption coefficient was determined (e275nm = 1.1 × 104 M−1 cm−1. Apocynin was a weak free radical scavenger (as measured using the DPPH, peroxyl radical and nitric oxide assays when compared to protocatechuic acid, used here as a reference antioxidant. On the other hand, apocynin was more effective than protocatechuic acid as scavenger of the non-radical species hypochlorous acid. Apocynin reacted promptly with the non-radical reactive species H2O2 only in the presence of peroxidase. This finding is relevant, since it represents a new pathway for depleting H2O2 in cellular experimental models, besides the direct inhibition of NADPH oxidase. This could be relevant for its application as an inhibitor of NOX4, since this isoform produces H2O2 and not superoxide anion. The binding parameters calculated by fluorescence quenching showed that apocynin binds to human serum albumin (HSA with a binding affinity of 2.19 × 104 M−1. The association did not alter the secondary and tertiary structure of HSA, as verified by synchronous fluorescence and circular dichroism. The displacement of fluorescent probes suggested that apocynin binds to site I and site II of HSA. Considering the current biomedical applications of this phytochemical, the dissemination of these chemical and biophysical properties can be very helpful for scientists and physicians interested in the use of apocynin.

  6. Molecular Dynamic Studies of the Complex Polyethylenimine and Glucose Oxidase

    Directory of Open Access Journals (Sweden)

    Beata Szefler

    2016-10-01

    Full Text Available Glucose oxidase (GOx is an enzyme produced by Aspergillus, Penicillium and other fungi species. It catalyzes the oxidation of β-d-glucose (by the molecular oxygen or other molecules, like quinones, in a higher oxidation state to form d-glucono-1,5-lactone, which hydrolyses spontaneously to produce gluconic acid. A coproduct of this enzymatic reaction is hydrogen peroxide (H2O2. GOx has found several commercial applications in chemical and pharmaceutical industries including novel biosensors that use the immobilized enzyme on different nanomaterials and/or polymers such as polyethylenimine (PEI. The problem of GOx immobilization on PEI is retaining the enzyme native activity despite its immobilization onto the polymer surface. Therefore, the molecular dynamic (MD study of the PEI ligand (C14N8_07_B22 and the GOx enzyme (3QVR was performed to examine the final complex PEI-GOx stabilization and the affinity of the PEI ligand to the docking sites of the GOx enzyme. The docking procedure showed two places/regions of major interaction of the protein with the polymer PEI: (LIG1 of −5.8 kcal/mol and (LIG2 of −4.5 kcal/mol located inside the enzyme and on its surface, respectively. The values of enthalpy for the PEI-enzyme complex, located inside of the protein (LIG1 and on its surface (LIG2 were computed. Docking also discovered domains of the GOx protein that exhibit no interactions with the ligand or have even repulsive characteristics. The structural data clearly indicate some differences in the ligand PEI behavior bound at the two places/regions of glucose oxidase.

  7. The increasing role of monoamine oxidase type B inhibitors in Parkinson's disease therapy.

    Science.gov (United States)

    Elmer, Lawrence W; Bertoni, John M

    2008-11-01

    The role of monoamine oxidase type B inhibitors in the treatment of Parkinson's disease has expanded with the new monoamine oxidase B inhibitor rasagiline and a new formulation, selegiline oral disintegrating tablets. As primary therapy in early disease monoamine oxidase B inhibitors reduce motor disability and delay the need for levodopa. In more advanced disease requiring levodopa, adjunctive monoamine oxidase B inhibitors reduce 'off' time and may improve gait and freezing. Rasagiline and selegiline oral disintegrating tablets may reduce the safety risks associated with the amfetamine and methamfetamine metabolites of conventional oral selegiline while retaining or improving therapeutic efficacy. Articles were identified by searches of PubMed and searches on the Internet and reviewed. All articles and other referenced materials were retrieved using the keywords 'Parkinson's disease', 'treatment' and 'monoamine oxidase B inhibitor' and were published between 1960 and 2007, with older references selected for historical significance. Only papers published in English were reviewed. Accumulating data support the use of monoamine oxidase B inhibitors as monotherapy for early and mild Parkinson's disease and as adjunctive therapy for more advanced Parkinson's disease with levodopa-associated motor fluctuations. The recently released monoamine oxidase B inhibitor rasagiline and a new formulation, selegiline oral disintegrating tablets, have potential advantages over conventional oral selegiline.

  8. Molecular Basis for Converting (2S-Methylsuccinyl-CoA Dehydrogenase into an Oxidase

    Directory of Open Access Journals (Sweden)

    Simon Burgener

    2017-12-01

    Full Text Available Although flavoenzymes have been studied in detail, the molecular basis of their dioxygen reactivity is only partially understood. The members of the flavin adenosine dinucleotide (FAD-dependent acyl-CoA dehydrogenase and acyl-CoA oxidase families catalyze similar reactions and share common structural features. However, both enzyme families feature opposing reaction specificities in respect to dioxygen. Dehydrogenases react with electron transfer flavoproteins as terminal electron acceptors and do not show a considerable reactivity with dioxygen, whereas dioxygen serves as a bona fide substrate for oxidases. We recently engineered (2S-methylsuccinyl-CoA dehydrogenase towards oxidase activity by rational mutagenesis. Here we characterized the (2S-methylsuccinyl-CoA dehydrogenase wild-type, as well as the engineered (2S-methylsuccinyl-CoA oxidase, in detail. Using stopped-flow UV-spectroscopy and liquid chromatography-mass spectrometry (LC-MS based assays, we explain the molecular base for dioxygen reactivity in the engineered oxidase and show that the increased oxidase function of the engineered enzyme comes at a decreased dehydrogenase activity. Our findings add to the common notion that an increased activity for a specific substrate is achieved at the expense of reaction promiscuity and provide guidelines for rational engineering efforts of acyl-CoA dehydrogenases and oxidases.

  9. Angiotensin II inhibits the Na+-K+ pump via PKC-dependent activation of NADPH oxidase.

    Science.gov (United States)

    White, Caroline N; Figtree, Gemma A; Liu, Chia-Chi; Garcia, Alvaro; Hamilton, Elisha J; Chia, Karin K M; Rasmussen, Helge H

    2009-04-01

    The sarcolemmal Na(+)-K(+) pump, pivotal in cardiac myocyte function, is inhibited by angiotensin II (ANG II). Since ANG II activates NADPH oxidase, we tested the hypothesis that NADPH oxidase mediates the pump inhibition. Exposure to 100 nmol/l ANG II increased superoxide-sensitive fluorescence of isolated rabbit ventricular myocytes. The increase was abolished by pegylated superoxide dismutase (SOD), by the NADPH oxidase inhibitor apocynin, and by myristolated inhibitory peptide to epsilon-protein kinase C (epsilonPKC), previously implicated in ANG II-induced Na(+)-K(+) pump inhibition. A role for epsilonPKC was also supported by an ANG II-induced increase in coimmunoprecipitation of epsilonPKC with the receptor for the activated kinase and with the cytosolic p47(phox) subunit of NADPH oxidase. ANG II decreased electrogenic Na(+)-K(+) pump current in voltage-clamped myocytes. The decrease was abolished by SOD, by the gp91ds inhibitory peptide that blocks assembly and activation of NADPH oxidase, and by epsilonPKC inhibitory peptide. Since colocalization should facilitate NADPH oxidase-dependent regulation of the Na(+)-K(+) pump, we examined whether there is physical association between the pump subunits and NADPH oxidase. The alpha(1)-subunit coimmunoprecipitated with caveolin 3 and with membrane-associated p22(phox) and cytosolic p47(phox) NADPH oxidase subunits at baseline. ANG II had no effect on alpha(1)/caveolin 3 or alpha(1)/p22(phox) interaction, but it increased alpha(1)/p47(phox) coimmunoprecipitation. We conclude that ANG II inhibits the Na(+)-K(+) pump via PKC-dependent NADPH oxidase activation.

  10. The Cytochrome bd Oxidase of Porphyromonas gingivalis Contributes to Oxidative Stress Resistance and Dioxygen Tolerance.

    Directory of Open Access Journals (Sweden)

    Julia Leclerc

    Full Text Available Porphyromonas gingivalis is an etiologic agent of periodontal disease in humans. The disease is associated with the formation of a mixed oral biofilm which is exposed to oxygen and environmental stress, such as oxidative stress. To investigate possible roles for cytochrome bd oxidase in the growth and persistence of this anaerobic bacterium inside the oral biofilm, mutant strains deficient in cytochrome bd oxidase activity were characterized. This study demonstrated that the cytochrome bd oxidase of Porphyromonas gingivalis, encoded by cydAB, was able to catalyse O2 consumption and was involved in peroxide and superoxide resistance, and dioxygen tolerance.

  11. Steady-state kinetics of substrate binding and iron release in tomato ACC oxidase.

    Science.gov (United States)

    Thrower, J S; Blalock, R; Klinman, J P

    2001-08-14

    1-Aminocyclopropane-1-carboxylate oxidase (ACC oxidase) catalyzes the last step in the biosynthetic pathway of the plant hormone, ethylene. This unusual reaction results in the oxidative ring cleavage of 1-aminocyclopropane carboxylate (ACC) into ethylene, cyanide, and CO2 and requires ferrous ion, ascorbate, and molecular oxygen for catalysis. A new purification procedure and assay method have been developed for tomato ACC oxidase that result in greatly increased enzymatic activity. This method allowed us to determine the rate of iron release from the enzyme and the effect of the activator, CO2, on this rate. Initial velocity studies support an ordered kinetic mechanism where ACC binds first followed by O2; ascorbate can bind after O2 or possibly before ACC. This kinetic mechanism differs from one recently proposed for the ACC oxidase from avocado.

  12. In vivo oxalate degradation by liposome encapsulated oxalate oxidase in rat model of hyperoxaluria

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    Tulika Dahiya

    2013-01-01

    Interpretation & conclusions: EMA-oxalate oxidase encapsulated liposome caused oxalate degradation in experimental hyperoxaluria indicating that the enzyme could be used as a therapeutic agent in hyperoxaluria leading to urinary stones.

  13. Action of DCCD on the H+/O stoichiometry of mitoplast cytochrome c oxidase.

    Science.gov (United States)

    Lehninger, A L; Reynafarje, B; Costa, L

    1985-01-01

    The mechanistic H+/O ejection stoichiometry of the cytochrome c oxidase reaction in rat liver mitoplasts is close to 4 at level flow when the reduced oxidase is pulsed with O2. Dicyclohexylcarbodiimide (DCCD) up to 30 nmol/mg protein fails to influence the rate of electron flow through the mitoplast oxidase, but inhibits H+ ejection. The inhibition of H+ ejection appears to be biphasic; ejection of 2-3 H+ per O is completely inhibited by very low DCCD, whereas inhibition of the remaining H+ ejection requires very much higher concentrations of DCCD. This effect suggests the occurrence of two types of H+ pumps in the native cytochrome oxidase of mitoplasts.

  14. Production of a new D-amino acid oxidase from the fungus Fusarium oxysporum.

    Science.gov (United States)

    Gabler, M; Fischer, L

    1999-08-01

    The fungus Fusarium oxysporum produced a D-amino acid oxidase (EC 1. 4.3.3) in a medium containing glucose as the carbon and energy source and ammonium sulfate as the nitrogen source. The specific D-amino acid oxidase activity was increased up to 12.5-fold with various D-amino acids or their corresponding derivatives as inducers. The best inducers were D-alanine (2.7 microkat/g of dry biomass) and D-3-aminobutyric acid (2.6 microkat/g of dry biomass). The addition of zinc ions was necessary to permit the induction of peroxisomal D-amino acid oxidase. Bioreactor cultivations were performed on a 50-liter scale, yielding a volumetric D-amino acid oxidase activity of 17 microkat liter(-1) with D-alanine as an inducer. Under oxygen limitation, the volumetric activity was increased threefold to 54 microkat liter(-1) (3,240 U liter(-1)).

  15. Interferon gamma/NADPH oxidase defence system in immunity and cancer

    Czech Academy of Sciences Publication Activity Database

    Hodný, Zdeněk; Reiniš, Milan; Hubáčková, Soňa; Vašicová, Pavla; Bartek, Jiří

    -, 01 Sep (2015) ISSN 2162-4011 Institutional support: RVO:68378050 ; RVO:61388971 Keywords : IFNγ * NADPH oxidase * immunity * cancer Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.266, year: 2014

  16. Metavanadate causes cellular accumulation of copper and decreased lysyl oxidase activity

    International Nuclear Information System (INIS)

    Cui, Changtai T.; Uriu-Adams, Janet Y.; Tchaparian, Eskouhie H.; Keen, Carl L.; Rucker, Robert B.

    2004-01-01

    Selected indices of copper metabolism in weanling rats and fibroblast cultures were progressively altered in response to increased levels of sodium metavanadate. In diets, vanadium was added in amounts ranging from 0 to 80 μg V/g of diet, that is, 0-1.6 μmol V/g of diet. In fibroblast cultures, vanadium ranged from 0 to 400 nmol V/ml. The inhibition of P-ATPase-7A activity by metavanadate, important to copper egress from cells, was a primary focus. In skin, and tendon, the copper concentration was increased in response to increased dietary levels of metavanadate, whereas lysyl oxidase activity, a secreted cuproprotein, was reduced. The reduction in lysyl oxidase activity was also accompanied by reduced redox cycling potential of isolated fractions of lysyl oxidase, presumably due to reduced lysyltyrosyl quinone (LTQ) formation at the active site of lysyl oxidase. In contrast, liver copper concentrations and plasma ceruloplasmin activity were not affected by metavanadate exposure. However, semicarbazide-sensitive benzylamine oxidase (SCBO) activity, which was taken as an indirect measure of vascular adhesive protein-1 (VAP-1), was increased. In cultured fibroblasts, cellular copper was also increased and lysyl oxidase decreased in response to metavanadate. Moreover, the steady-state levels of atp7a and lysyl oxidase mRNAs were not affected by addition of metavanadate to culture medium up to 200 nmol/ml. Taken together, these data suggest that pathways involving copper egress and lysyl oxidase activation are particularly sensitive to metavanadate exposure through processes that are predominately posttranslational

  17. Herbivore-plant interactions: mixed-function oxidases and secondary plant substances.

    Science.gov (United States)

    Brattsten, L B; Wilkinson, C F; Eisner, T

    1977-06-17

    The mixed-function oxidases of a polyphagous insect larva (the southern armyworm, Spodoptera eridania) were found to be induced by a diversity of secondary plant substances. The induction proceeds rapidly and in response to a small quantity of secondary substance. Following induction, the larva is less susceptible to dietary poisoning. It is argued that mixed-function oxidases play a major role in protecting herbivores against chemical stress from secondary plant substances.

  18. Process technology for the application of d-amino acid oxidases in pharmaceutical intermediate manufacturing

    DEFF Research Database (Denmark)

    Tindal, Stuart; Carr, Reuben; Archer, Ian V. J.

    2011-01-01

    Recent advances in biocatalysis have seen increased interest in the use of D-amino acid oxidase to synthesize optically pure amino acids. However, the creation of a genuine oxidase based platform technology will require suitable process technology as well as an understanding of the challenges...... and opportunities of a wider portfolio of synthetic targets. In this article we address some of the recent progress in process technology to enable the future development of a generic platform technology....

  19. Biphenyl Modulates the Expression and Function of Respiratory Oxidases in the Polychlorinated-Biphenyls Degrader Pseudomonas pseudoalcaligenes KF707

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    Federica Sandri

    2017-06-01

    Full Text Available Pseudomonas pseudoalcaligenes KF707 is a soil bacterium which is known for its capacity to aerobically degrade harmful organic compounds such as polychlorinated biphenyls (PCBs using biphenyl as co-metabolite. Here we provide the first genetic and functional analysis of the KF707 respiratory terminal oxidases in cells grown with two different carbon sources: glucose and biphenyl. We identified five terminal oxidases in KF707: two c(caa3 type oxidases (Caa3 and Ccaa3, two cbb3 type oxidases (Cbb31 and Cbb32, and one bd type cyanide-insensitive quinol oxidase (CIO. While the activity and expression of both Cbb31 and Cbb32 oxidases was prevalent in glucose grown cells as compared to the other oxidases, the activity and expression of the Caa3 oxidase increased considerably only when biphenyl was used as carbon source in contrast to the Cbb32 oxidase which was repressed. Further, the respiratory activity and expression of CIO was up-regulated in a Cbb31 deletion strain as compared to W.T. whereas the CIO up-regulation was not present in Cbb32 and C(caa3 deletion mutants. These results, together, reveal that both function and expression of cbb3 and caa3 type oxidases in KF707 are modulated by biphenyl which is the co-metabolite needed for the activation of the PCBs-degradation pathway.

  20. Construction of Mutant Glucose Oxidases with Increased Dye-Mediated Dehydrogenase Activity

    Science.gov (United States)

    Horaguchi, Yohei; Saito, Shoko; Kojima, Katsuhiro; Tsugawa, Wakako; Ferri, Stefano; Sode, Koji

    2012-01-01

    Mutagenesis studies on glucose oxidases (GOxs) were conducted to construct GOxs with reduced oxidase activity and increased dehydrogenase activity. We focused on two representative GOxs, of which crystal structures have already been reported—Penicillium amagasakiense GOx (PDB ID; 1gpe) and Aspergillus niger GOx (PDB ID; 1cf3). We constructed oxygen-interacting structural models for GOxs, and predicted the residues responsible for oxidative half reaction with oxygen on the basis of the crystal structure of cholesterol oxidase as well as on the fact that both enzymes are members of the glucose/methanol/choline (GMC) oxidoreductase family. Rational amino acid substitution resulted in the construction of an engineered GOx with drastically decreased oxidase activity and increased dehydrogenase activity, which was higher than that of the wild-type enzyme. As a result, the dehydrogenase/oxidase ratio of the engineered enzyme was more than 11-fold greater than that of the wild-type enzyme. These results indicate that alteration of the dehydrogenase/oxidase activity ratio of GOxs is possible by introducing a mutation into the putative functional residues responsible for oxidative half reaction with oxygen of these enzymes, resulting in a further increased dehydrogenase activity. This is the first study reporting the alteration of GOx electron acceptor preference from oxygen to an artificial electron acceptor. PMID:23203056

  1. Construction of Mutant Glucose Oxidases with Increased Dye-Mediated Dehydrogenase Activity

    Directory of Open Access Journals (Sweden)

    Koji Sode

    2012-11-01

    Full Text Available Mutagenesis studies on glucose oxidases (GOxs were conducted to construct GOxs with reduced oxidase activity and increased dehydrogenase activity. We focused on two representative GOxs, of which crystal structures have already been reported—Penicillium amagasakiense GOx (PDB ID; 1gpe and Aspergillus niger GOx (PDB ID; 1cf3. We constructed oxygen-interacting structural models for GOxs, and predicted the residues responsible for oxidative half reaction with oxygen on the basis of the crystal structure of cholesterol oxidase as well as on the fact that both enzymes are members of the glucose/methanol/choline (GMC oxidoreductase family. Rational amino acid substitution resulted in the construction of an engineered GOx with drastically decreased oxidase activity and increased dehydrogenase activity, which was higher than that of the wild-type enzyme. As a result, the dehydrogenase/oxidase ratio of the engineered enzyme was more than 11-fold greater than that of the wild-type enzyme. These results indicate that alteration of the dehydrogenase/oxidase activity ratio of GOxs is possible by introducing a mutation into the putative functional residues responsible for oxidative half reaction with oxygen of these enzymes, resulting in a further increased dehydrogenase activity. This is the first study reporting the alteration of GOx electron acceptor preference from oxygen to an artificial electron acceptor.

  2. [The X+ chronic granulomatous disease as a fabulous model to study the NADPH oxidase complex activation].

    Science.gov (United States)

    Stasia, Marie-José

    2007-05-01

    Chronic granulomatous disease (CGD) is a rare inherited disorder in which phagocytes lack NADPH oxidase activity. Patients with CGD suffer from recurrent bacterial and fungal infections because of the absence of superoxide anions (O2- degrees ) generatingsystem. The NADPH oxidase complex is composed of a membranous cytochrome b558, cytosolic proteins p67phox, p47phox, p40phox and two small GTPases Rac2 and Rap1A. Cytochrome b558 consists of two sub-units gp91phox and p22phox. The most common form of CGD is due to mutations in CYBB gene encoding gp91phox. In some rare cases, the mutated gp91phox is normally expressed but is devoided of oxidase activity. These variants called X+ CGD, have provided interesting informations about oxidase activation mechanisms. However modelization of such variants is necessary to obtain enough biological material for studies at the molecular level. A cellular model (knock-out PLB-985 cells) has been developed for expressing recombinant mutated gp91phox for functional analysis of the oxidase complex. Recent works demonstrated that this cell line genetically deficient in gp91phox is a powerful tool for functional analysis of the NADPH oxidase complex activation.

  3. Construction of mutant glucose oxidases with increased dye-mediated dehydrogenase activity.

    Science.gov (United States)

    Horaguchi, Yohei; Saito, Shoko; Kojima, Katsuhiro; Tsugawa, Wakako; Ferri, Stefano; Sode, Koji

    2012-11-02

    Mutagenesis studies on glucose oxidases (GOxs) were conducted to construct GOxs with reduced oxidase activity and increased dehydrogenase activity. We focused on two representative GOxs, of which crystal structures have already been reported—Penicillium amagasakiense GOx (PDB ID; 1gpe) and Aspergillus niger GOx (PDB ID; 1cf3). We constructed oxygen-interacting structural models for GOxs, and predicted the residues responsible for oxidative half reaction with oxygen on the basis of the crystal structure of cholesterol oxidase as well as on the fact that both enzymes are members of the glucose/methanol/choline (GMC) oxidoreductase family. Rational amino acid substitution resulted in the construction of an engineered GOx with drastically decreased oxidase activity and increased dehydrogenase activity, which was higher than that of the wild-type enzyme. As a result, the dehydrogenase/oxidase ratio of the engineered enzyme was more than 11-fold greater than that of the wild-type enzyme. These results indicate that alteration of the dehydrogenase/oxidase activity ratio of GOxs is possible by introducing a mutation into the putative functional residues responsible for oxidative half reaction with oxygen of these enzymes, resulting in a further increased dehydrogenase activity. This is the first study reporting the alteration of GOx electron acceptor preference from oxygen to an artificial electron acceptor.

  4. Ozone affects pollen viability and NAD(P)H oxidase release from Ambrosia artemisiifolia pollen

    International Nuclear Information System (INIS)

    Pasqualini, Stefania; Tedeschini, Emma; Frenguelli, Giuseppe; Wopfner, Nicole; Ferreira, Fatima; D'Amato, Gennaro; Ederli, Luisa

    2011-01-01

    Air pollution is frequently proposed as a cause of the increased incidence of allergy in industrialised countries. We investigated the impact of ozone (O 3 ) on reactive oxygen species (ROS) and allergen content of ragweed pollen (Ambrosia artemisiifolia). Pollen was exposed to acute O 3 fumigation, with analysis of pollen viability, ROS and nitric oxide (NO) content, activity of nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase, and expression of major allergens. There was decreased pollen viability after O 3 fumigation, which indicates damage to the pollen membrane system, although the ROS and NO contents were not changed or were only slightly induced, respectively. Ozone exposure induced a significant enhancement of the ROS-generating enzyme NAD(P)H oxidase. The expression of the allergen Amb a 1 was not affected by O 3 , determined from the mRNA levels of the major allergens. We conclude that O 3 can increase ragweed pollen allergenicity through stimulation of ROS-generating NAD(P)H oxidase. - Highlights: → O 3 reduces the viability of ragweed pollen. → ROS and allergens of ragweed pollen were not affected by O 3 exposure. → O 3 enhances the activity of the ROS-generating enzyme NAD(P)H oxidase. → O 3 increases ragweed pollen allergenicity through NAD(P)H-oxidase stimulation. - This study focuses on the effects of the atmospheric pollutant ozone on ROS content and NAD(P)H oxidase activity of ragweed pollen grains.

  5. A decade of crystallization drops: crystallization of the cbb3 cytochrome c oxidase from Pseudomonas stutzeri.

    Science.gov (United States)

    Buschmann, Sabine; Richers, Sebastian; Ermler, Ulrich; Michel, Hartmut

    2014-04-01

    The cbb3 cytochrome c oxidases are distant members of the superfamily of heme copper oxidases. These terminal oxidases couple O2 reduction with proton transport across the plasma membrane and, as a part of the respiratory chain, contribute to the generation of an electrochemical proton gradient. Compared with other structurally characterized members of the heme copper oxidases, the recently determined cbb3 oxidase structure at 3.2 Å resolution revealed significant differences in the electron supply system, the proton conducting pathways and the coupling of O2 reduction to proton translocation. In this paper, we present a detailed report on the key steps for structure determination. Improvement of the protein quality was achieved by optimization of the number of lipids attached to the protein as well as the separation of two cbb3 oxidase isoenzymes. The exchange of n-dodecyl-β-D-maltoside for a precisely defined mixture of two α-maltosides and decanoylsucrose as well as the choice of the crystallization method had a most profound impact on crystal quality. This report highlights problems frequently encountered in membrane protein crystallization and offers meaningful approaches to improve crystal quality. © 2014 The Protein Society.

  6. Blockade of TGF-β 1 Signalling Inhibits Cardiac NADPH Oxidase Overactivity in Hypertensive Rats

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    José Luis Miguel-Carrasco

    2012-01-01

    Full Text Available NADPH oxidases constitute a major source of superoxide anion (⋅O2 - in hypertension. Several studies suggest an important role of NADPH oxidases in different effects mediated by TGF-β 1. In this study we show that chronic administration of P144, a peptide synthesized from type III TGF-β 1 receptor, significantly reduced the cardiac NADPH oxidase expression and activity as well as in the nitrotyrosine levels observed in control spontaneously hypertensive rats (V-SHR to levels similar to control normotensive Wistar Kyoto rats. In addition, P144 was also able to reduce the significant increases in the expression of collagen type I protein and mRNA observed in hearts from V-SHR. In addition, positive correlations between collagen expression, NADPH oxidase activity, and nitrotyrosine levels were found in all animals. Finally, TGF-β 1-stimulated Rat-2 exhibited significant increases in NADPH oxidase activity that was inhibited in the presence of P144. It could be concluded that the blockade of TGF-β 1 with P144 inhibited cardiac NADPH oxidase in SHR, thus adding new data to elucidate the involvement of this enzyme in the profibrotic actions of TGF-β 1.

  7. Proline Oxidase (POX) as A Target for Cancer Therapy.

    Science.gov (United States)

    Kononczuk, Joanna; Czyzewska, Urszula; Moczydlowska, Joanna; Surażyński, Arkadiusz; Palka, Jerzy; Miltyk, Wojciech

    2015-01-01

    Proline dehydrogenase/proline oxidase (PRODH/POX) is an enzyme catalyzing the first step of proline degradation, during which ROS and/or ATP is generated. POX is widely distributed in living organisms and is responsible for a number of regulatory processes such as redox homeostasis, osmotic adaptation, cell signaling and oxidative stress. Recent data provided evidence that POX plays an important role in carcinogenesis and tumor growth. POX may induce apoptosis in both intrinsic and extrinsic way. Due to ROS generation, POX may induce caspase-9 activity, which mediates mitochondrial apoptosis (intrinsic apoptosis pathway). POX can also stimulate TRAIL (tumor necrosis factorrelated apoptosis inducing ligand) and DR5 (death receptor 5) expression, resulting in cleavage of procaspase-8 and thus extrinsic apoptotic pathway. However, this tumor suppressor in certain environmental conditions may act as a prosurvival factor. Genotoxic, inflammatory and metabolic stress may switch POX from tumor growth inhibiting to tumor growth supporting factor. The potential mechanisms which may regulate switching of POX mode are discussed in this review.

  8. Glucose Oxidase Immobilization on TMAH-Modified Bentonite

    Directory of Open Access Journals (Sweden)

    Ruth Chrisnasari

    2015-03-01

    Full Text Available The influence of bentonite modification by tetramethyl ammonium hydroxide (TMAH on its capability to immobilize glucose oxidase (GOX was studied. Modification of bentonite was conducted by the adding of 0-5% (v/v TMAH. The observed results show that the different concentrations of TMAH affect the percentage of immobilized enzyme. The results of this study show that the best concentration of TMAH is 5% (v/v which can immobilize up to 84.71% of GOX. X-ray diffraction (XRD and Fourier Transforms Infrared Spectroscopy (FTIR studies have been carried out to observe the structural changes in bentonite due to TMAH modification. The obtained immobilized GOX show the optimum catalytic activity on reaction temperature of 40-50 °C and pH of 7. The immobilized GOX kinetics at the optimum conditions determined the Km and Vmax value to be 4.96x10-2 mM and 4.99x10-3 mM.min-1 respectively. In addition, the immobilized GOX on TMAH-modified bentonite is stable enough so it could be re-used six times before its activity decreased by 39.44%.

  9. Introducing inducible fluorescent split cholesterol oxidase to mammalian cells.

    Science.gov (United States)

    Chernov, Konstantin G; Neuvonen, Maarit; Brock, Ivonne; Ikonen, Elina; Verkhusha, Vladislav V

    2017-05-26

    Cholesterol oxidase (COase) is a bacterial enzyme catalyzing the first step in the biodegradation of cholesterol. COase is an important biotechnological tool for clinical diagnostics and production of steroid drugs and insecticides. It is also used for tracking intracellular cholesterol; however, its utility is limited by the lack of an efficient temporal control of its activity. To overcome this we have developed a regulatable fragment complementation system for COase cloned from Chromobacterium sp. The enzyme was split into two moieties that were fused to FKBP (FK506-binding protein) and FRB (rapamycin-binding domain) pair and split GFP fragments. The addition of rapamycin reconstituted a fluorescent enzyme, termed split GFP-COase, the fluorescence level of which correlated with its oxidation activity. A rapid decrease of cellular cholesterol induced by intracellular expression of the split GFP-COase promoted the dissociation of a cholesterol biosensor D4H from the plasma membrane. The process was reversible as upon rapamycin removal, the split GFP-COase fluorescence was lost, and cellular cholesterol levels returned to normal. These data demonstrate that the split GFP-COase provides a novel tool to manipulate cholesterol in mammalian cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Characterization of polyphenol oxidase from blueberry (Vaccinium corymbosum L.).

    Science.gov (United States)

    Siddiq, M; Dolan, K D

    2017-03-01

    Polyphenol oxidase (PPO) was extracted and characterized from high-bush blueberries. PPO showed an optimum activity at pH 6.1-6.3 and 35°C, with the enzyme showing significant activity over a wide temperature range (25-60°C). Catechol was the most readily oxidized substrate followed by 4-methylcatechol, DL-DOPA, and dopamine. Blueberry PPO showed a K m of 15mM and V max of 2.57 ΔA 420 nm/min×10 -1 , determined with catechol. PPO was completely inactivated in 20min at 85°C, however, after 30minat 75°C it showed about 10% residual activity. Thermal treatment at 55 and 65°C for 30min resulted in the partial inactivation of PPO. Ascorbic acid, sodium diethyldithiocarbamic acid, L-cysteine, and sodium metabisulfite were effective inhibitors of PPO at 1.0mM. Benzoic acid and cinnamic acid series inhibitors showed relatively weak inhibition of PPO (21.8-27.6%), even at as high as 2.0mM concentration. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Cataplexy and monoamine oxidase deficiency in Norrie disease.

    Science.gov (United States)

    Vossler, D G; Wyler, A R; Wilkus, R J; Gardner-Walker, G; Vlcek, B W

    1996-05-01

    Norrie disease (ND) is an X-linked recessive disorder causing ocular atrophy, mental retardation, deafness, and dysmorphic features. Virtually absent monoamine oxidase (MAO) type-A and -B activity has been found in some boys with chromosome deletions. We report the coexistence of cataplexy and abnormal REM sleep organization with ND. Three related boys, referred for treatment of medically refractory atonic spells and apneas, underwent extended EEG-video-polysomnographic monitoring. They demonstrated attacks of cataplexy and inappropriate periods of REM sleep during which they were unarousable. One boy also had generalized tonic-clonic seizures. Previous testing revealed that all three have complete ND gene deletions. In all subjects, platelet MAO-B activity was absent, serum serotonin levels were markedly increased, and plasma catecholamine levels were normal. Data from the canine narcolepsy syndrome model implicate abnormal catecholaminergic and cholinergic activities in the pathogenesis of cataplexy. Our findings suggest that abnormal MAO activity or an imbalance between serotonin and other neurotransmitter levels may be involved in the pathogenesis of human cataplexy.

  12. Monoamine oxidase A (MAO A) inhibitors decrease glioma progression

    Science.gov (United States)

    Vaikari, Vijaya Pooja; Kota, Rajesh; Chen, Kevin; Yeh, Tzu-Shao; Jhaveri, Niyati; Groshen, Susan L.; Olenyuk, Bogdan Z.; Chen, Thomas C.; Hofman, Florence M.; Shih, Jean C.

    2016-01-01

    Glioblastoma (GBM) is an aggressive brain tumor which is currently treated with temozolomide (TMZ). Tumors usually become resistant to TMZ and recur; no effective therapy is then available. Monoamine Oxidase A (MAO A) oxidizes monoamine neurotransmitters resulting in reactive oxygen species which cause cancer. This study shows that MAO A expression is increased in human glioma tissues and cell lines. MAO A inhibitors, clorgyline or the near-infrared-dye MHI-148 conjugated to clorgyline (NMI), were cytotoxic for glioma and decreased invasion in vitro. Using the intracranial TMZ-resistant glioma model, clorgyline or NMI alone or in combination with low-dose TMZ reduced tumor growth and increased animal survival. NMI was localized specifically to the tumor. Immunocytochemistry studies showed that the MAO A inhibitor reduced proliferation, microvessel density and invasion, and increased macrophage infiltration. In conclusion, we have identified MAO A inhibitors as potential novel stand-alone drugs or as combination therapy with low dose TMZ for drug-resistant gliomas. NMI can also be used as a non-invasive imaging tool. Thus has a dual function for both therapy and diagnosis. PMID:26871599

  13. Spermine oxidase promotes bile canalicular lumen formation through acrolein production.

    Science.gov (United States)

    Uemura, Takeshi; Takasaka, Tomokazu; Igarashi, Kazuei; Ikegaya, Hiroshi

    2017-11-01

    Spermine oxidase (SMOX) catalyzes oxidation of spermine to generate spermidine, hydrogen peroxide (H 2 O 2 ) and 3-aminopropanal, which is spontaneously converted to acrolein. SMOX is induced by a variety of stimuli including bacterial infection, polyamine analogues and acetaldehyde exposure. However, the physiological functions of SMOX are not yet fully understood. We investigated the physiological role of SMOX in liver cells using human hepatocellular carcinoma cell line HepG2. SMOX localized to the bile canalicular lumen, as determined by F-actin staining. Knockdown of SMOX reduced the formation of bile canalicular lumen. We also found that phospho-Akt (phosphorylated protein kinase B) was localized to canalicular lumen. Treatment with Akt inhibitor significantly reduced the formation of bile canalicular lumen. Acrolein scavenger also inhibited the formation of bile canalicular lumen. PTEN, phosphatase and tensin homolog and an inhibitor of Akt, was alkylated in a SMOX-dependent manner. Our results suggest that SMOX plays a central role in the formation of bile canalicular lumen in liver cells by activating Akt pathway through acrolein production.

  14. Polyphenol Oxidase as a Biochemical Seed Defense Mechanism

    Directory of Open Access Journals (Sweden)

    E. Patrick Fuerst

    2014-12-01

    Full Text Available Seed dormancy and resistance to decay are fundamental survival strategies, which allow a population of seeds to germinate over long periods of time. Seeds have physical, chemical, and biological defense mechanisms that protect their food reserves from decay-inducing organisms and herbivores. Here, we hypothesize that seeds also possess enzyme-based biochemical defenses, based on induction of the plant defense enzyme, polyphenol oxidase (PPO, when wild oat (Avena fatua L. caryopses and seeds were challenged with seed-decaying Fusarium fungi. These studies suggest that dormant seeds are capable of mounting a defense response to pathogens. The pathogen-induced PPO activity from wild oat was attributed to a soluble isoform of the enzyme that appeared to result, at least in part, from proteolytic activation of a latent PPO isoform. PPO activity was also induced in wild oat hulls (lemma and palea, non-living tissues that cover and protect the caryopsis. These results are consistent with the hypothesis that seeds possess inducible enzyme-based biochemical defenses arrayed on the exterior of seeds and these defenses represent a fundamental mechanism of seed survival and longevity in the soil. Enzyme-based biochemical defenses may have broader implications since they may apply to other defense enzymes as well as to a diversity of plant species and ecosystems.

  15. Characterization of active site residues of nitroalkane oxidase.

    Science.gov (United States)

    Valley, Michael P; Fenny, Nana S; Ali, Shah R; Fitzpatrick, Paul F

    2010-06-01

    The flavoenzyme nitroalkane oxidase catalyzes the oxidation of primary and secondary nitroalkanes to the corresponding aldehydes and ketones plus nitrite. The structure of the enzyme shows that Ser171 forms a hydrogen bond to the flavin N5, suggesting that it plays a role in catalysis. Cys397 and Tyr398 were previously identified by chemical modification as potential active site residues. To more directly probe the roles of these residues, the S171A, S171V, S171T, C397S, and Y398F enzymes have been characterized with nitroethane as substrate. The C397S and Y398 enzymes were less stable than the wild-type enzyme, and the C397S enzyme routinely contained a substoichiometric amount of FAD. Analysis of the steady-state kinetic parameters for the mutant enzymes, including deuterium isotope effects, establishes that all of the mutations result in decreases in the rate constants for removal of the substrate proton by approximately 5-fold and decreases in the rate constant for product release of approximately 2-fold. Only the S171V and S171T mutations alter the rate constant for flavin oxidation. These results establish that these residues are not involved in catalysis, but rather are required for maintaining the protein structure. 2009 Elsevier Inc. All rights reserved.

  16. A study of monoamine oxidase activity in fetal membranes.

    Science.gov (United States)

    Sekizawa, A; Ishikawa, H; Morimoto, T; Hirose, K; Suzuki, A; Saito, H; Yanaihara, T; Arai, Y; Oguchi, K

    1996-05-01

    To study the role of decidual monoamine oxidase (MAO)-A and -B activities before delivery, the relationship between MAO activity in fetal membranes and catecholamine (CA) concentration in amniotic fluid (AF) was determined. Fetal membranes and AF were obtained at the time of elective Cesarean section (CS group, n = 11) and Cesarean section due to fetal distress without labor pains (FD group, n = 5). MAO-A and -B activities were radiometrically measured using 14C-5-hydroxytriptamine for MAO-A substrate and 14C-benzylamine for MAO-B substrate. CA concentrations in AF were measured by high performance liquid chromatograph with an electro-chemical detector. Both MAO-A and -B activities in decidua obtained from CS were significantly lower than those obtained from FD. Both norepinephrine (NE) and epinephrine (EP) concentrations were significantly lower in the CS group than the FD group. A significant positive correlation between decidual MAO-A activity and NE concentration in AF was observed. No significant correlation was observed between MAO-B activity and the concentration of NE in AF. There was no correlation between EP concentrations and MAO activities. These results suggest that CA concentration in AF may be related to the activity of MAO in fetal membranes, determined by certain physiological processes during pregnancy. It has been suggested that metabolism of monoamines in fetal membranes also plays an important role in reducing monoamine influx into maternal myometrium from the AF.

  17. ABTS assay of phenol oxidase activity in soil.

    Science.gov (United States)

    Floch, Carine; Alarcon-Gutiérrez, Enrique; Criquet, Stéven

    2007-12-01

    Phenol oxidases (PO) are involved in degradation of many recalcitrant aromatic compounds and may be sensitive to some pollutants. Hence, their activities may be a useful indicator for evaluating soil quality and health. To this end, the aim of this study was to develop a simple method to assay PO activity directly in bulk samples by spectrophotometric test using 2,2'-azinobis-(-3 ethylbenzothiazoline-6-sulfononic acid) diammonium salt (ABTS) as the substrate. Three Mediterranean soils were used as models. For each soil, we studied the kinetic parameters and the effects of certain factors (i.e. amount of soil, pH, temperature, incubation time and substrate concentration) in order to determine the optimum conditions for the ABTS assay. Results showed that PO attain their optimum activities when incubating 0.1 g of soil at 30 degrees C for 5 min with 10 ml of a Modified Universal Buffer (MUB) at pH 2 and 200 microl of a 0.1 M ABTS solution.

  18. Xanthine Oxidase Inhibitory Activity of a Plectranthus saccatus aqueous extract

    Directory of Open Access Journals (Sweden)

    Caldeira F

    2016-12-01

    Full Text Available Gout is a disease with high prevalence in developed countries, resulting from the deposition of uric acid crystals in various locations, particularly at the joints. The pharmacotherapeutic approach to chronic gout essentially consists of administration of uric acid-lowering agents. The main mechanism of action of these agents is the inhibition of xanthine oxidase (XO, the enzyme responsible for the formation of uric acid. The therapeutic alternatives available for this purpose are limited, thus justifying the interest of the discovery of potential new uric acidlowering drugs. In this regard, an aqueous extract of the plant Plectranthus saccatus has been studied for its ability to inhibit XO. The composition of the extract was determined by HPLC and rosmarinic acid was identified as the major constituent. Both the extract and rosmarinic acid have demonstrated the ability to inhibit the production of uric acid by interfering with XO activity. The results obtained herein support the continuation of the study of their uric acid-lowering properties in cell-based and in vivo models to further explore their potential in gout therapy.

  19. The NADPH oxidase inhibitor apocynin (acetovanillone) induces oxidative stress

    International Nuclear Information System (INIS)

    Riganti, Chiara; Costamagna, Costanzo; Bosia, Amalia; Ghigo, Dario

    2006-01-01

    Apocynin (acetovanillone) is often used as a specific inhibitor of NADPH oxidase. In N11 glial cells, apocynin induced, in a dose-dependent way, a significant increase of both malonyldialdehyde level (index of lipid peroxidation) and lactate dehydrogenase release (index of a cytotoxic effect). Apocynin evoked also, in a significant way, an increase of H 2 O 2 concentration and a decrease of the intracellular glutathione/glutathione disulfide ratio, accompanied by augmented efflux of glutathione and glutathione disulfide. Apocynin induced the activation of both pentose phosphate pathway and tricarboxylic acid cycle, which was blocked when the cells were incubated with glutathione together with apocynin. The cell incubation with glutathione prevented also the apocynin-induced increase of malonyldialdehyde generation and lactate dehydrogenase leakage. Apocynin exerted an oxidant effect also in a cell-free system: indeed, in aqueous solution, it evoked a faster oxidation of the thiols glutathione and dithiothreitol, and elicited the generation of reactive oxygen species, mainly superoxide anions. Our results suggest that apocynin per se can induce an oxidative stress and exert a cytotoxic effect in N11 cells and other cell types, and that some effects of apocynin in in vitro and in vivo experimental models should be interpreted with caution

  20. Indanones as high-potency reversible inhibitors of monoamine oxidase.

    Science.gov (United States)

    Mostert, Samantha; Petzer, Anél; Petzer, Jacobus P

    2015-05-01

    Recent reports document that α-tetralone (3,4-dihydro-2H-naphthalen-1-one) is an appropriate scaffold for the design of high-potency monoamine oxidase (MAO) inhibitors. Based on the structural similarity between α-tetralone and 1-indanone, the present study involved synthesis of 34 1-indanone and related indane derivatives as potential inhibitors of recombinant human MAO-A and MAO-B. The results show that C6-substituted indanones are particularly potent and selective MAO-B inhibitors, with IC50 values ranging from 0.001 to 0.030 μM. C5-Substituted indanone and indane derivatives are comparatively weaker MAO-B inhibitors. Although the 1-indanone and indane derivatives are selective inhibitors of the MAO-B isoform, a number of homologues are also potent MAO-A inhibitors, with three homologues possessing IC50 values 1-indanone as a reversible MAO inhibitor with a competitive mode of inhibition. It may be concluded that 1-indanones are promising leads for the design of therapies for neurodegenerative and neuropsychiatric disorders such as Parkinson's disease and depression. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Xanthine oxidase activity regulates human embryonic brain cells growth

    Directory of Open Access Journals (Sweden)

    Kevorkian G. A.

    2011-10-01

    Full Text Available Aim. Involvement of Xanthine Oxidase (XO; EC1.1.3.22 in cellular proliferation and differentiation has been suggested by the numerous investigations. We have proposed that XO might have undoubtedly important role during the development, maturation as well as the death of human embryos brain cells. Methods. Human abortion material was utilized for the cultivation of brain cells (E90. XO activity was measured by the formation of uric acid in tissue. Cell death was detected by the utility of Trypan Blue dye. Results. Allopurinol suppressed the XO activity in the brain tissue (0.12 ± 0.02; 0.20 ± 0.03 resp., p < 0.05. On day 12th the number of cells in the culture treated with the Allopurinol at the early stage of development was higher in comparison with the Control (2350.1 ± 199.0 vs 2123 ± 96 and higher in comparison with the late period of treatment (1479.6 ± 103.8, p < < 0.05. In all groups, the number of the dead cells was less than in Control, indicating the protective nature of Allopurinol as an inhibitor of XO. Conclusions. Allopurinol initiates cells proliferation in case of the early treatment of the human brain derived cell culture whereas at the late stages it has an opposite effect.

  2. Reye's syndrome: salicylate and mitochondrial monoamine oxidase function

    International Nuclear Information System (INIS)

    Faraj, B.A.; Caplan, D.; Lolies, P.

    1986-01-01

    It has been suggested that aspirin is somehow linked with the onset of Reye's syndrome (RS). A general feature of Reye's syndrome is severe impairment of mitochondrial monoamine oxidase (MAO) function. The main objective of this investigation was to study the effect of salicylate on platelet mitochondrial MAO activity in three groups: group A (healthy children, n = 21) and group C (healthy adults, n = 10). Platelet MAO was measured by radio-enzymatic technique with 14 C-tyramine as a substrate. The results showed that salicyclate (10 mM) had a 20 to 60 percent inhibitory effect on platelet MAO function in only 1, 3 and 2 of the subjects in group A, B and C. Furthermore, there was an association between low enzyme activity and salicylate MAO inhibitory effect in these subjects. These preliminary findings suggest that salicylate may induce deterioration in mitochondrial function in susceptible individuals and that the assessment of salicylate MAO inhibitory effect may identify those who may be at risk to develop aspirin poisoning and Reye's syndrome

  3. Assays of D-Amino Acid Oxidase Activity

    Directory of Open Access Journals (Sweden)

    Elena Rosini

    2018-01-01

    Full Text Available D-amino acid oxidase (DAAO is a well-known flavoenzyme that catalyzes the oxidative FAD-dependent deamination of D-amino acids. As a result of the absolute stereoselectivity and broad substrate specificity, microbial DAAOs have been employed as industrial biocatalysts in the production of semi-synthetic cephalosporins and enantiomerically pure amino acids. Moreover, in mammals, DAAO is present in specific brain areas and degrades D-serine, an endogenous coagonist of the N-methyl-D-aspartate receptors (NMDARs. Dysregulation of D-serine metabolism due to an altered DAAO functionality is related to pathological NMDARs dysfunctions such as in amyotrophic lateral sclerosis and schizophrenia. In this protocol paper, we describe a variety of direct assays based on the determination of molecular oxygen consumption, reduction of alternative electron acceptors, or α-keto acid production, of coupled assays to detect the hydrogen peroxide or the ammonium production, and an indirect assay of the α-keto acid production based on a chemical derivatization. These analytical assays allow the determination of DAAO activity both on recombinant enzyme preparations, in cells, and in tissue samples.

  4. Selective inhibition of monoamine oxidase A by purpurin, an anthraquinone.

    Science.gov (United States)

    Lee, Hyun Woo; Ryu, Hyung Won; Kang, Myung-Gyun; Park, Daeui; Oh, Sei-Ryang; Kim, Hoon

    2017-03-01

    Monoamine oxidase (MAO) catalyzes the oxidation of monoamines that act as neurotransmitters. During a target-based screening of natural products using two isoforms of recombinant human MAO-A and MAO-B, purpurin (an anthraquinone derivative) was found to potently and selectively inhibit MAO-A, with an IC 50 value of 2.50μM, and not to inhibit MAO-B. Alizarin (also an anthraquinone) inhibited MAO-A less potently with an IC 50 value of 30.1μM. Furthermore, purpurin was a reversible and competitive inhibitor of MAO-A with a K i value of 0.422μM. A comparison of their chemical structures suggested the 4-hydroxy group of purpurin might play an important role in its inhibition of MAO-A. Molecular docking simulation showed that the binding affinity of purpurin for MAO-A (-40.0kcal/mol) was higher than its affinity for MAO-B (-33.9kcal/mol), and that Ile 207 and Gly 443 of MAO-A were key residues for hydrogen bonding with purpurin. The findings of this study suggest purpurin is a potent, selective, reversible inhibitor of MAO-A, and that it be considered a new potential lead compound for development of novel reversible inhibitors of MAO-A (RIMAs). Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Radio-isotopic determination of platelet monoamine oxidase and regulation of its activity by an indigenous drug

    International Nuclear Information System (INIS)

    Dubey, G.P.; Srivastava, V.K.; Agrawal, A.; Udupa, K.N.

    1988-01-01

    Platelet monoamine oxidase is a mitochondrial enzyme taking part in the deamination reaction of total catecholamine. Recent studies of monoamine oxidase inhibitors have gained its importance in the control of variety of psychosomatic disorders like mental depression, arterial hypertension and anxiety neurosis. 30 apparently normal individuals and 42 diagnosed cases of essential hypertension were selected for the present study. The platelet monoamine oxidase activity was measured by using 14 C-tryptamine bisuccinate. Comparatively low activity of platelet monoamine oxidase was noticed in hypertension cases than in the normal. After oral administration of an indigenous drug 'Geriforte' for three months, a significant rise in platelet monoamine oxidase activity was noticed in hypertension cases. It can be concluded that this indigenous formulation has the capacity to regulate the monoamine oxidase activity, as such, it may provide an alternative remedy in the management of psychosomatic disorders. (author). 11 refs

  6. Significance of membrane bioreactor design on the biocatalytic performance of glucose oxidase and catalase: Free vs. immobilized enzyme systems

    DEFF Research Database (Denmark)

    Morthensen, Sofie Thage; Meyer, Anne S.; Jørgensen, Henning

    2017-01-01

    Membrane separation of xylose and glucose can be accomplished via oxidation of glucose to gluconic acid by enzymatic glucose oxidase catalysis. Oxygen for this reaction can be supplied via decomposition of hydrogen peroxide by enzymatic catalase catalysis. In order to maximize the biocatalytic...... productivity of glucose oxidase and catalase (gluconic acid yield per total amount of enzyme) the following system set-ups were compared: immobilization of glucose oxidase alone; co-immobilization of glucose oxidase and catalase; glucose oxidase and catalase free in the membrane bioreactor. Fouling......-induced enzyme immobilization in the porous support of an ultrafiltration membrane was used as strategy for entrapment of glucose oxidase and catalase. The biocatalytic productivity of the membrane reactor was found to be highly related to the oxygen availability, which in turn depended on the reactor...

  7. Biofabrication Using Pyrrole Electropolymerization for the Immobilization of Glucose Oxidase and Lactate Oxidase on Implanted Microfabricated Biotransducers

    Directory of Open Access Journals (Sweden)

    Christian N. Kotanen

    2014-03-01

    Full Text Available The dual responsive Electrochemical Cell-on-a-Chip Microdisc Electrode Array (ECC MDEA 5037 is a recently developed electrochemical transducer for use in a wireless, implantable biosensor system for the continuous measurement of interstitial glucose and lactate. Fabrication of the biorecognition membrane via pyrrole electropolymerization and both in vitro and in vivo characterization of the resulting biotransducer is described. The influence of EDC-NHS covalent conjugation of glucose oxidase with 4-(3-pyrrolyl butyric acid (monomerization and with 4-sulfobenzoic acid (sulfonization on biosensor performance was examined. As the extent of enzyme conjugation was increased sensitivity decreased for monomerized enzymes but increased for sulfonized enzymes. Implanted biotransducers were examined in a Sprague-Dawley rat hemorrhage model. Resection after 4 h and subsequent in vitro re-characterization showed a decreased sensitivity from 0.68 (±0.40 to 0.22 (±0.17 µA·cm−2·mM−1, an increase in the limit of detection from 0.05 (±0.03 to 0.27 (±0.27 mM and a six-fold increase in the response time from 41 (±18 to 244 (±193 s. This evidence reconfirms the importance of biofouling at the bio-abio interface and the need for mitigation strategies to address the foreign body response.

  8. Interactions of Desmethoxyyangonin, a Secondary Metabolite from Renealmia alpinia, with Human Monoamine Oxidase-A and Oxidase-B

    Directory of Open Access Journals (Sweden)

    Narayan D. Chaurasiya

    2017-01-01

    Full Text Available Renealmia alpinia (Zingiberaceae, a medicinal plant of tropical rainforests, is used to treat snakebites and other injuries and also as a febrifuge, analgesic, antiemetic, antiulcer, and anticonvulsant. The dichloromethane extract of R. alpinia leaves showed potent inhibition of human monoamine oxidases- (MAOs- A and B. Phytochemical studies yielded six known compounds, including pinostrobin 1, 4′-methyl ether sakuranetin 2, sakuranetin 3, pinostrobin chalcone 4, yashabushidiol A 5, and desmethoxyyangonin 6. Compound 6 displayed about 30-fold higher affinity for MAO-B than MAO-A, with Ki values of 31 and 922 nM, respectively. Kinetic analysis of inhibition and equilibrium-dialysis dissociation assay of the enzyme-inhibitor complex showed reversible binding of desmethoxyyangonin 6 with MAO-A and MAO-B. The binding interactions of compound 6 in the active site of the MAO-A and MAO-B isoenzymes, investigated through molecular modeling algorithms, confirmed preferential binding of desmethoxyyangonin 6 with MAO-B compared to MAO-A. Selective reversible inhibitors of MAO-B, like desmethoxyyangonin 6, may have important therapeutic significance for the treatment of neurodegenerative disorders, such as Parkinson’s disease and Alzheimer’s disease.

  9. Presteady-state and steady-state kinetic properties of human cytochrome c oxidase. Identification of rate-limiting steps in mammalian cytochrome c oxidase

    NARCIS (Netherlands)

    van Kuilenburg, A. B.; Gorren, A. C.; Dekker, H. L.; Nieboer, P.; van Gelder, B. F.; Muijsers, A. O.

    1992-01-01

    Human cytochrome c oxidase was purified in a fully active form from heart and skeletal muscle. The enzyme was selectively solubilised with octylglucoside and KCl from submitochondrial particles followed by ammonium sulphate fractionation. The presteady-state and steady-state kinetic properties of

  10. Phospholipid alterations in cardiac sarcoplasmic reticulum induced by xanthine oxidase: contamination of commercial preparations of xanthine oxidase by phospholipase A2

    International Nuclear Information System (INIS)

    Gamache, D.A.; Kornberg, L.J.; Bartolf, M.; Franson, R.C.

    1986-01-01

    Incubation of cardiac sarcoplasmic reticulum with xanthine oxidase alone at pH 7.0 resulted in a loss of lipid phosphorus that was potentiated by the addition of xanthine. Using autoclaved E.coli with 1- 14 C-oleate in the 2-acyl position of membrane phospholipids, the authors demonstrate that many, but not all, commercial preparations of xanthine oxidase contain significant phospholipase A 2 (PLA 2 ) activity (64.3-545.6 nmols/min/mg). The PLA 2 was maximally active in the neutral-alkaline pH range, was Ca 2+ -dependent, and was unaffected by the addition of xanthine. PLA 2 activity was totally inhibited by 1mM EDTA whereas radical production by optimal concentrations of xanthine/xanthine oxidase (X/XO) was unaffected by EDTA. Chromatographically purified xanthine oxidase (Sigma Grade III) contained high levels of PLA 2 activity (64.3 nmols/min/mg) compared to endogenous levels of neutral-active, Ca 2+ -dependent PLA 2 measured in various tissue homogenates (≤ 0.5 nmols/ min/mg). Because X/XO mixtures are used extensively to study oxygen free radical-induced cell injury and membrane phospholipid alterations, the presence of a potent extracellular PLA 2 may have influenced previously published reports, and such studies should be interpreted cautiously

  11. A point mutation of valine-311 to methionine in Bacillus subtilis protoporphyrinogen oxidase does not greatly increase resistance to the diphenyl ether herbicide oxyfluorfen.

    Science.gov (United States)

    Jeong, Eunjoo; Houn, Thavrak; Kuk, Yongin; Kim, Eun-Seon; Chandru, Hema Kumar; Baik, Myunggi; Back, Kyoungwhan; Guh, Ja-Ock; Han, Oksoo

    2003-10-01

    In an effort to asses the effect of Val311Met point mutation of Bacillus subtilis protoporphyrinogen oxidase on the resistance to diphenyl ether herbicides, a Val311Met point mutant of B. subtilis protoporphyrinogen oxidase was prepared, heterologously expressed in Escherichia coli, and the purified recombinant Val311Met mutant protoporphyrinogen oxidase was kinetically characterized. The mutant protoporphyrinogen oxidase showed very similar kinetic patterns to wild type protoporphyrinogen oxidase, with slightly decreased activity dependent on pH and the concentrations of NaCl, Tween 20, and imidazole. When oxyfluorfen was used as a competitive inhibitor, the Val311Met mutant protoporphyrinogen oxidase showed an increased inhibition constant about 1.5 times that of wild type protoporphyrinogen oxidase. The marginal increase of the inhibition constant indicates that the Val311Met point mutation in B. subtilis protoporphyrinogen oxidase may not be an important determinant in the mechanism that protects protoporphyrinogen oxidase against diphenyl ether herbicides.

  12. The substrate oxidation mechanism of pyranose 2-oxidase and other related enzymes in the glucose-methanol-choline superfamily.

    Science.gov (United States)

    Wongnate, Thanyaporn; Chaiyen, Pimchai

    2013-07-01

    Enzymes in the glucose-methanol-choline (GMC) oxidoreductase superfamily catalyze the oxidation of an alcohol moiety to the corresponding aldehyde. In this review, the current understanding of the sugar oxidation mechanism in the reaction of pyranose 2-oxidase (P2O) is highlighted and compared with that of other enzymes in the GMC family for which structural and mechanistic information is available, including glucose oxidase, choline oxidase, cholesterol oxidase, cellobiose dehydrogenase, aryl-alcohol oxidase, and pyridoxine 4-oxidase. Other enzymes in the family that have been newly discovered or for which less information is available are also discussed. A large primary kinetic isotope effect was observed for the flavin reduction when 2-d-D-glucose was used as a substrate, but no solvent kinetic isotope effect was detected for the flavin reduction step. The reaction of P2O is consistent with a hydride transfer mechanism in which there is stepwise formation of d-glucose alkoxide prior to the hydride transfer. Site-directed mutagenesis of P2O and pH-dependence studies indicated that His548 is a catalytic base that facilitates the deprotonation of C2-OH in D-glucose. This finding agrees with the current mechanistic model for aryl-alcohol oxidase, glucose oxidase, cellobiose dehydrogenase, methanol oxidase, and pyridoxine 4-oxidase, but is different from that of cholesterol oxidase and choline oxidase. Although all of the GMC enzymes share similar structural folding and use the hydride transfer mechanism for flavin reduction, they appear to have subtle differences in the fine-tuned details of how they catalyze substrate oxidation. © 2013 The Authors Journal compilation © 2013 FEBS.

  13. Gene cloning and heterologous expression of pyranose 2-oxidase from the brown-rot fungus, Gloeophyllum trabeum

    Science.gov (United States)

    Diane Dietrich; Casey Crooks

    2009-01-01

    A pyranose 2-oxidase gene from the brown-rot basidiomycete Gloeophyllum trabeum was isolated using homology-based degenerate PCR. The gene structure was determined and compared to that of several pyranose 2-oxidases cloned from white-rot fungi. The G. trabeum pyranose 2-oxidase gene consists of 16 coding exons with canonical promoter CAAT and TATA elements in the 5’UTR...

  14. Targeting NADPH oxidase decreases oxidative stress in the transgenic sickle cell mouse penis.

    Science.gov (United States)

    Musicki, Biljana; Liu, Tongyun; Sezen, Sena F; Burnett, Arthur L

    2012-08-01

    Sickle cell disease (SCD) is a state of chronic vasculopathy characterized by endothelial dysfunction and increased oxidative stress, but the sources and mechanisms responsible for reactive oxygen species (ROS) production in the penis are unknown. We evaluated whether SCD activates NADPH oxidase, induces endothelial nitric oxide synthase (eNOS) uncoupling, and decreases antioxidants in the SCD mouse penis. We further tested the hypothesis that targeting NADPH oxidase decreases oxidative stress in the SCD mouse penis. SCD transgenic (sickle) mice were used as an animal model of SCD. Hemizygous (hemi) mice served as controls. Mice received an NADPH oxidase inhibitor apocynin (10 mM in drinking water) or vehicle. Penes were excised at baseline for molecular studies. Markers of oxidative stress (4-hydroxy-2-nonenal [HNE]), sources of ROS (eNOS uncoupling and NADPH oxidase subunits p67(phox) , p47(phox) , and gp91(phox) ), and enzymatic antioxidants (superoxide dismutase [SOD]1, SOD2, catalase, and glutathione peroxidase-1 [GPx1]) were measured by Western blot in penes. Sources of ROS, oxidative stress, and enzymatic antioxidants in the SCD penis. Relative to hemi mice, SCD increased (Ppenis. Apocynin treatment of sickle mice reversed (P0.05) prevented eNOS uncoupling in the penis. Apocynin treatment of hemi mice did not affect any of these parameters. NADPH oxidase and eNOS uncoupling are sources of oxidative stress in the SCD penis; decreased GPx1 further contributes to oxidative stress. Inhibition of NADPH oxidase upregulation decreases oxidative stress, implying a major role for NADPH oxidase as a ROS source and a potential target for improving vascular function in the SCD mouse penis. © 2012 International Society for Sexual Medicine.

  15. Intracellular lysyl oxidase: Effect of a specific inhibitor on nuclear mass in proliferating cells

    Energy Technology Data Exchange (ETDEWEB)

    Saad, Fawzy A. [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Harvard Medical School, Boston, MA 02115 (United States); Torres, Marie [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Wang, Hao [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Harvard Medical School, Boston, MA 02115 (United States); Graham, Lila, E-mail: lilagraham@cs.com [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Harvard Medical School, Boston, MA 02115 (United States)

    2010-06-11

    LOX, the principal enzyme involved in crosslinking of collagen, was the first of several lysyl oxidase isotypes to be characterized. Its active form was believed to be exclusively extracellular. Active LOX was later reported to be present in cell nuclei; its function there is unknown. LOX expression opposes the effect of mutationally activated Ras, which is present in about 30% of human cancers. The mechanism of LOX in countering the action of Ras is also unknown. In the present work, assessment of nuclear protein for possible effects of lysyl oxidase activity led to the discovery that proliferating cells dramatically increase their nuclear protein content when exposed to BAPN ({beta}-aminopropionitrile), a highly specific lysyl oxidase inhibitor that reportedly blocks LOX inhibition of Ras-induced oocyte maturation. In three cell types (PC12 cells, A7r5 smooth muscle cells, and NIH 3T3 fibroblasts), BAPN caused a 1.8-, 1.7-, and 2.1-fold increase in total nuclear protein per cell, respectively, affecting all major components in both nuclear matrix and chromatin fractions. Since nuclear size is correlated with proliferative status, enzyme activity restricting nuclear growth may be involved in the lysyl oxidase tumor suppressive effect. Evidence is also presented for the presence of apparent lysyl oxidase isotype(s) containing a highly conserved LOX active site sequence in the nuclei of PC12 cells, which do not manufacture extracellular lysyl oxidase substrates. Results reported here support the hypothesis that nuclear lysyl oxidase regulates nuclear growth, and thereby modulates cell proliferation.

  16. Alternative oxidase in the branched mitochondrial respiratory network: an overview on structure, function, regulation, and role

    Directory of Open Access Journals (Sweden)

    Sluse F.E.

    1998-01-01

    Full Text Available Plants and some other organisms including protists possess a complex branched respiratory network in their mitochondria. Some pathways of this network are not energy-conserving and allow sites of energy conservation to be bypassed, leading to a decrease of the energy yield in the cells. It is a challenge to understand the regulation of the partitioning of electrons between the various energy-dissipating and -conserving pathways. This review is focused on the oxidase side of the respiratory chain that presents a cyanide-resistant energy-dissipating alternative oxidase (AOX besides the cytochrome pathway. The known structural properties of AOX are described including transmembrane topology, dimerization, and active sites. Regulation of the alternative oxidase activity is presented in detail because of its complexity. The alternative oxidase activity is dependent on substrate availability: total ubiquinone concentration and its redox state in the membrane and O2 concentration in the cell. The alternative oxidase activity can be long-term regulated (gene expression or short-term (post-translational modification, allosteric activation regulated. Electron distribution (partitioning between the alternative and cytochrome pathways during steady-state respiration is a crucial measurement to quantitatively analyze the effects of the various levels of regulation of the alternative oxidase. Three approaches are described with their specific domain of application and limitations: kinetic approach, oxygen isotope differential discrimination, and ADP/O method (thermokinetic approach. Lastly, the role of the alternative oxidase in non-thermogenic tissues is discussed in relation to the energy metabolism balance of the cell (supply in reducing equivalents/demand in energy and carbon and with harmful reactive oxygen species formation.

  17. Comparative study between dimethyl sulfoxide (dmso), allopurinol and urate oxidase administration in nephrotoxic rats induced with

    International Nuclear Information System (INIS)

    Heibashy, M.I.A.; El-Nahla, A.M.; Ibrahim, A.I.; Saleh, Sh.Y.A.

    2010-01-01

    This study was conducted to show whether DMSO, allopurinol and urate oxidase could offer ameliorating effects against abnormal alterations in kidney function tests in gentamicin (GM) induced nephrotoxic rats . Two experiments were carried out, the first one showed that daily injection of 80 mg GM/kg b. wt interapertonealy (I.P) for two weeks induced acute renal failure indicated by significant elevation in serum urea, creatinine, uric acid, potassium, inorganic phosphorus, TBARS and PTH and a significant decline in serum sodium, total and ionized calcium when compared with their corresponding values in saline injected rats. In the second experiment, comparisons were made between GM induced nephrotoxic rats and other nephrotoxic groups received daily pf I.P injection of DMSO (4 ml/kg b.wt), allopurinol (1.5 mg/100 g b.wt) and urate oxidase (10 mg/100 g b.wt) for 30 days after the incidence of nephrotoxicity. At all intervals, 10,20 and 30 days; serum urea, creatinine, uric acid, potassium, inorganic phosphorus, TBARS and PTH in DMSO, allopurinol and urate oxidase treated groups exhibited significant reduction than nephrotoxic untreated rats. During the same intervals, the levels of serum total and ionized calcium showed an opposite trend. serum sodium level did not show any significant difference between all treated groups except after 20 days , it was increased significantly in urate oxidase treated group and after 30 days in both allopurinol and urate oxidase treated groups. in term time intervals, a significant correction was recorded on the level of most measured parameters. in nephritic rats, the administration of DMSO, allopurinol or urate oxidase led to a significant amelioration effects in the kidney function tests and urate oxidase was the best protective.

  18. Prognostic relevance of cytochrome C oxidase in primary glioblastoma multiforme.

    Directory of Open Access Journals (Sweden)

    Corinne E Griguer

    Full Text Available Patients with primary glioblastoma multiforme (GBM have one of the lowest overall survival rates among cancer patients, and reliable biomarkers are necessary to predict patient outcome. Cytochrome c oxidase (CcO promotes the switch from glycolytic to OXPHOS metabolism, and increased CcO activity in tumors has been associated with tumor progression after chemotherapy failure. Thus, we investigated the relationship between tumor CcO activity and the survival of patients diagnosed with primary GBM. A total of 84 patients with grade IV glioma were evaluated in this retrospective cohort study. Cumulative survival was calculated by the Kaplan-Meier method and analyzed by the log-rank test, and univariate and multivariate analyses were performed with the Cox regression model. Mitochondrial CcO activity was determined by spectrophotometrically measuring the oxidation of cytochrome c. High CcO activity was detected in a subset of glioma tumors (∼30%, and was an independent prognostic factor for shorter progression-free survival and overall survival [P = 0.0087 by the log-rank test, hazard ratio = 3.57 for progression-free survival; P<0.001 by the log-rank test, hazard ratio = 10.75 for overall survival]. The median survival time for patients with low tumor CcO activity was 14.3 months, compared with 6.3 months for patients with high tumor CcO activity. High CcO activity occurs in a significant subset of high-grade glioma patients and is an independent predictor of poor outcome. Thus, CcO activity may serve as a useful molecular marker for the categorization and targeted therapy of GBMs.

  19. Scanning tunneling microscopy studies of glucose oxidase on gold surface

    International Nuclear Information System (INIS)

    Losic, D.; Shapter, J.G.; Gooding, J.J.

    2002-01-01

    Full text: Three immobilization methods have been used for scanning tunneling microscopy (STM) studies of glucose oxidase (GOD) on gold. They are based on a) physical adsorption from solution, b) microcontact printing and c) covalent bonding onto self-assembled monolayers (SAM) of 3-mercaptopropionic acid (MPA). The STM images are used to provide information about the organization of individual GOD molecules and more densely packed monolayers of GOD on electrode surfaces, thus providing information of the role of interfacial structure on biosensor performance. The use of atomically flat gold substrates enables easy distinction of deposited enzyme features from the flat gold substrate. Microcontact printing is found to be a more reliable method than adsorption from solution for preparing individual GOD molecules on the gold surface STM images of printed samples reveal two different shapes of native GOD molecules. One is a butterfly shape with dimensions of 10 ± 1 nm x 6 ± 1 nm, assigned to the lying position of molecule while the second is an approximately spherical shape with dimensions of 6.5 ± 1 nm x 5 ± 1nm assigned to a standing position. Isolated clusters of 5 to 6 GOD molecules are also observed. With monolayer coverage, GOD molecules exhibit a tendency to organize themselves into a two dimensional array with adequate sample stability to obtain high-resolution STM images. Within these two-dimensional arrays are clearly seen repeating clusters of five to six enzyme molecules in a unit STM imaging of GOD monolayers covalently immobilized onto SAM (MPA) are considerably more difficult than when the enzyme is adsorbed directly onto the metal. Cluster structures are observed both high and low coverage despite the fact that native GOD is a negatively charged molecule. Copyright (2002) Australian Society for Electron Microscopy Inc

  20. Development of new radiopharmaceuticals for imaging monoamine oxidase B

    International Nuclear Information System (INIS)

    Vasdev, Neil; Sadovski, Oleg; Moran, Matthew D.; Parkes, Jun; Meyer, Jeffrey H.; Houle, Sylvain; Wilson, Alan A.

    2011-01-01

    Introduction: Imaging monoamine oxidase B (MAO-B) in the central nervous system with PET is an important goal for psychiatric studies. We here report an improved and automated radiosynthesis of N-(6-[ 18 F]-fluorohexyl)-N-methylpropargylamine ([ 18 F]FHMP; [ 18 F]-1), as well as the radiosynthesis of two new promising candidates for imaging cerebral MAO-B, namely, carbon-11-labeled 3-(4-[ 11 C]-methoxyphenyl)-6-methyl-2H-1-benzopyran-2-one ([ 11 C]-2) and N-((1H-pyrrol-2-yl)methyl)-N-[ 11 C]-methyl-1-phenylmethanamine ([ 11 C]-3). Methods: Fluorine-18-labeled 1 was prepared via a tosyloxy precursor in 29%±5% uncorrected radiochemical yield, relative to [ 18 F]-fluoride. Both carbon-11-labeled compounds were prepared with [ 11 C]CH 3 I using the 'LOOP' method in 11% and 18% uncorrected radiochemical yields, respectively, relative to starting [ 11 C]CO 2 . All radiotracers had specific activities >37 GBq/μmol and were >98% radiochemically pure at end of synthesis ( 18 F]-1. While [ 11 C]-2 had moderate brain penetration and good clearance from normal brain tissue, distribution of radioactivity in brain was indicative of free and nonspecific binding. Good brain uptake was observed with [ 11 C]-3 (0.8%-1.4% injected dose per gram at 5 min postinjection), binding appeared to be reversible and distribution conformed with regional distribution of MAO-B in the rat brain. Preinjection of 3 or L-deprenyl showed a modest reduction (up to 25%) of brain activity. Conclusion: Carbon-11-labeled 3 was found to have the most favorable properties of the radiotracers evaluated; however, the signal-to-noise ratio was too low to warrant further in vivo imaging studies. Alternative radiotracers for imaging MAO-B are under development.

  1. Monamine oxidase inhibitors: current and emerging agents for Parkinson disease.

    Science.gov (United States)

    Fernandez, Hubert H; Chen, Jack J

    2007-01-01

    Monoamine oxidase type B (MAO-B) is the predominant isoform responsible for the metabolic breakdown of dopamine in the brain. Selective inhibition of brain MAO-B results in elevation of synaptosomal dopamine concentrations. Data have been reported regarding the selective MAO-B inhibitors, rasagiline and selegiline, for the symptomatic treatment of Parkinson disease (PD). Selegiline has demonstrated efficacy as monotherapy in patients with early PD (Deprenyl and Tocopherol Antioxidative Therapy of Parkinsonism study), but evidence of selegiline efficacy as adjunctive treatment in levodopa-treated PD patients with motor fluctuations is equivocal. A new formulation of selegiline (Zydis selegiline) has been evaluated in 2 small, placebo-controlled studies as adjunctive therapy to levodopa. The Zydis formulation allows pregastric absorption of selegiline, minimizing first-pass metabolism, and thereby increasing selegiline bioavailability and reducing the concentration of amphetamine metabolites. Rasagiline is a selective, second-generation, irreversible MAO-B inhibitor, with at least 5 times the potency of selegiline in vitro and in animal models. Rasagiline has demonstrated efficacy in 1 large, randomized, double-blind, placebo-controlled trial (TVP-1012 in Early Monotherapy for Parkinson's Disease Outpatients) as initial monotherapy in patients with early PD, and in 2 large, controlled trials (Parkinson's Rasagiline: Efficacy and Safety in the Treatment of "Off," Lasting Effect in Adjunct Therapy With Rasagiline Given Once Daily) as adjunctive treatment in levodopa-treated PD patients with motor fluctuations. Unlike selegiline, rasagiline is an aminoindan derivative with no amphetamine metabolites. A randomized clinical trial is underway to confirm preclinical and preliminary clinical data suggesting rasagiline has disease-modifying effects.

  2. Decoding NADPH oxidase 4 expression in human tumors

    Directory of Open Access Journals (Sweden)

    Jennifer L. Meitzler

    2017-10-01

    Full Text Available NADPH oxidase 4 (NOX4 is a redox active, membrane-associated protein that contributes to genomic instability, redox signaling, and radiation sensitivity in human cancers based on its capacity to generate H2O2 constitutively. Most studies of NOX4 in malignancy have focused on the evaluation of a small number of tumor cell lines and not on human tumor specimens themselves; furthermore, these studies have often employed immunological tools that have not been well characterized. To determine the prevalence of NOX4 expression across a broad range of solid tumors, we developed a novel monoclonal antibody that recognizes a specific extracellular region of the human NOX4 protein, and that does not cross-react with any of the other six members of the NOX gene family. Evaluation of 20 sets of epithelial tumors revealed, for the first time, high levels of NOX4 expression in carcinomas of the head and neck (15/19 patients, esophagus (12/18 patients, bladder (10/19 patients, ovary (6/17 patients, and prostate (7/19 patients, as well as malignant melanoma (7/15 patients when these tumors were compared to histologically-uninvolved specimens from the same organs. Detection of NOX4 protein upregulation by low levels of TGF-β1 demonstrated the sensitivity of this new probe; and immunofluorescence experiments found that high levels of endogenous NOX4 expression in ovarian cancer cells were only demonstrable associated with perinuclear membranes. These studies suggest that NOX4 expression is upregulated, compared to normal tissues, in a well-defined, and specific group of human carcinomas, and that its expression is localized on intracellular membranes in a fashion that could modulate oxidative DNA damage.

  3. NADPH Oxidase, NOX1, Mediates Vascular Injury in Ischemic Retinopathy

    Science.gov (United States)

    Deliyanti, Devy; Rana, Indrajeetsinh; Miller, Antonia G.; Agrotis, Alex; Armani, Roksana; Szyndralewiez, Cédric; Wingler, Kirstin; Touyz, Rhian M.; Cooper, Mark E.; Jandeleit-Dahm, Karin A.; Schmidt, Harald H.H.W.

    2014-01-01

    Abstract Aims: Ischemic retinal diseases such as retinopathy of prematurity are major causes of blindness due to damage to the retinal microvasculature. Despite this clinical situation, retinopathy of prematurity is mechanistically poorly understood. Therefore, effective preventative therapies are not available. However, hypoxic-induced increases in reactive oxygen species (ROS) have been suggested to be involved with NADPH oxidases (NOX), the only known dedicated enzymatic source of ROS. Our major aim was to determine the contribution of NOX isoforms (1, 2, and 4) to a rodent model of retinopathy of prematurity. Results: Using a genetic approach, we determined that only mice with a deletion of NOX1, but not NOX2 or NOX4, were protected from retinal neovascularization and vaso-obliteration, adhesion of leukocytes, microglial accumulation, and the increased generation of proangiogenic and proinflammatory factors and ROS. We complemented these studies by showing that the specific NOX inhibitor, GKT137831, reduced vasculopathy and ROS levels in retina. The source of NOX isoforms was evaluated in retinal vascular cells and neuro-glial elements. Microglia, the immune cells of the retina, expressed NOX1, 2, and 4 and responded to hypoxia with increased ROS formation, which was reduced by GKT137831. Innovation: Our studies are the first to identify the NOX1 isoform as having an important role in the pathogenesis of retinopathy of prematurity. Conclusions: Our findings suggest that strategies targeting NOX1 have the potential to be effective treatments for a range of ischemic retinopathies. Antioxid. Redox Signal. 20, 2726–2740. PMID:24053718

  4. Adenine nucleotide depletion from endothelial cells exposed to xanthine oxidase

    International Nuclear Information System (INIS)

    Aalto, T.K.; Raivio, K.O.

    1990-01-01

    Hypoxia causes breakdown of cellular nucleotides, accumulation of hypoxanthine (HX), and conversion of xanthine dehydrogenase into xanthine oxidase (XO). Upon reoxygenation, the HX-XO reaction generates free radicals, one potential mechanism of tissue damage. Because endothelial cells contain XO and are exposed to circulating HX, they are a likely target for damage. We studied the effect of XO and/or HX at physiologically relevant concentrations on nucleotide metabolism of cultured endothelial cells from human umbilical veins. Cells were labeled with [14C]adenine and incubated for up to 6 h with HX, XO, or both, in the absence or presence of serum. Adenine nucleotides from cell extracts and nucleotide breakdown products (HX, xanthine, and urate) from the medium were separated and counted. HX alone had no effect. XO (80 mU/ml) alone caused a 70% (no serum) or 40% (with serum) fall in adenine nucleotides and an equivalent increase of xanthine and urate. The combination of HX and XO caused a 90% (no serum) or 70% (with serum) decrease in nucleotides, decrease in energy charge, and detachment of cells from the culture plate. Nucleotide depletion was not accounted for by proteolytic activity in the XO preparation. Albumin was only half as effective as serum in preventing nucleotide loss. Thus exogenous XO, in the presence of endogenous HX, triggers adenine nucleotide catabolism, but endogenous XO activity is too low to influence nucleotide levels even at high exogenous HX concentrations. Serum limits the catabolic effect of XO and thus protects cells from free radical damage

  5. Novel thidiazuron-derived inhibitors of cytokinin oxidase/dehydrogenase.

    Science.gov (United States)

    Nisler, Jaroslav; Kopečný, David; Končitíková, Radka; Zatloukal, Marek; Bazgier, Václav; Berka, Karel; Zalabák, David; Briozzo, Pierre; Strnad, Miroslav; Spíchal, Lukáš

    2016-09-01

    Two new TDZ derivatives (HETDZ and 3FMTDZ) are very potent inhibitors of CKX and are promising candidates for in vivo studies. Cytokinin hormones regulate a wide range of essential processes in plants. Thidiazuron (N-phenyl-N'-1,2,3-thiadiazol-5-yl urea, TDZ), formerly registered as a cotton defoliant, is a well known inhibitor of cytokinin oxidase/dehydrogenase (CKX), an enzyme catalyzing the degradation of cytokinins. TDZ thus increases the lifetime of cytokinins and their effects in plants. We used in silico modeling to design, synthesize and characterize twenty new TDZ derivatives with improved inhibitory properties. Two compounds, namely 1-[1,2,3]thiadiazol-5-yl-3-(3-trifluoromethoxy-phenyl)urea (3FMTDZ) and 1-[2-(2-hydroxyethyl)phenyl]-3-(1,2,3-thiadiazol-5-yl)urea (HETDZ), displayed up to 15-fold lower IC 50 values compared with TDZ for AtCKX2 from Arabidopsis thaliana and ZmCKX1 and ZmCKX4a from Zea mays. Binding modes of 3FMTDZ and HETDZ were analyzed by X-ray crystallography. Crystal structure complexes, solved at 2.0 Å resolution, revealed that HETDZ and 3FMTDZ bound differently in the active site of ZmCKX4a: the thiadiazolyl ring of 3FMTDZ was positioned over the isoalloxazine ring of FAD, whereas that of HETDZ had the opposite orientation, pointing toward the entrance of the active site. The compounds were further tested for cytokinin activity in several cytokinin bioassays. We suggest that the combination of simple synthesis, lowered cytokinin activity, and enhanced inhibitory effects on CKX isoforms, makes 3FMTDZ and HETDZ suitable candidates for in vivo studies.

  6. Alterations of sirtuins in mitochondrial cytochrome c-oxidase deficiency.

    Directory of Open Access Journals (Sweden)

    Arne Björn Potthast

    Full Text Available Sirtuins are NAD+ dependent deacetylases, which regulate mitochondrial energy metabolism as well as cellular response to stress. The NAD/NADH-system plays a crucial role in oxidative phosphorylation linking sirtuins and the mitochondrial respiratory chain. Furthermore, sirtuins are able to directly deacetylate and activate different complexes of the respiratory chain. This prompted us to analyse sirtuin levels in skin fibroblasts from patients with cytochrome c-oxidase (COX deficiency and to test the impact of different pharmaceutical activators of sirtuins (SRT1720, paeonol to modulate sirtuins and possibly respiratory chain enzymes in patient cells in vitro.We assayed intracellular levels of sirtuin 1 and the mitochondrial sirtuins SIRT3 and SIRT4 in human fibroblasts from patients with COX- deficiency. Furthermore, sirtuins were measured after inhibiting complex IV in healthy control fibroblasts by cyanide and after incubation with activators SRT1720 and paeonol. To determine the effect of sirtuin inhibition at the cellular level we measured total cellular acetylation (control and patient cells, with and without treatment by Western blot.We observed a significant decrease in cellular levels of all three sirtuins at the activity, protein and transcriptional level (by 15% to 50% in COX-deficient cells. Additionally, the intracellular concentration of NAD+ was reduced in patient cells. We mimicked the biochemical phenotype of COX- deficiency by incubating healthy fibroblasts with cyanide and observed reduced sirtuin levels. A pharmacological activation of sirtuins resulted in normalized sirtuin levels in patient cells. Hyper acetylation was also reversible after treatment with sirtuin activators. Pharmacological modulation of sirtuins resulted in altered respiratory chain complex activities.We found inhibition of situins 1, 3 and 4 at activity, protein and transcriptional levels in fibroblasts from patient with COX-deficiency. Pharmacological

  7. Insomnia, platelet serotonin and platelet monoamine oxidase in chronic alcoholism.

    Science.gov (United States)

    Nenadic Sviglin, Korona; Nedic, Gordana; Nikolac, Matea; Mustapic, Maja; Muck-Seler, Dorotea; Borovecki, Fran; Pivac, Nela

    2011-08-18

    Insomnia is a common sleep disorder frequently occurring in chronic alcoholic patients. Neurobiological basis of insomnia, as well as of alcoholism, is associated with disrupted functions of the main neurotransmitter systems, including the serotonin (5-hydroxytryptamine, 5-HT) system. Blood platelets are considered a limited peripheral model for the central 5-HT neurons, since both platelets and central 5-HT synaptosomes have similar dynamics of 5-HT. Platelet 5-HT concentration and platelet monoamine oxidase type B (MAO-B) are assumed to represent biomarkers for particular symptoms and behaviors in psychiatric disorders. The hypothesis of this study was that platelet 5-HT concentration and platelet MAO-B activity will be altered in chronic alcoholic patients with insomnia compared to comparable values in patients without insomnia. The study included 498 subjects: 395 male and 103 female medication-free patients with alcohol dependence and 502 healthy control subjects: 325 men and 177 women. The effects of early, middle and late insomnia (evaluated using the Hamilton Depression Rating Scale), as well as sex, age and smoking on platelet 5-HT concentration and platelet MAO-B activity were evaluated using one-way ANOVA and multiple regression analysis by the stepwise method. Platelet 5-HT concentration, but not platelet MAO-B activity, was significantly reduced in alcoholic patients with insomnia compared to patients without insomnia. Multiple regression analysis revealed that platelet 5-HT concentration was affected by middle insomnia, smoking and sex, while platelet MAO activity was affected only by sex and age. The present and previous data suggest that platelet 5-HT concentration might be used, after controlling for sex and smoking, as a biomarker for insomnia in alcoholism, PTSD and in rotating shift workers. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  8. Ultrafine carbon particles promote rotenone-induced dopamine neuronal loss through activating microglial NADPH oxidase

    International Nuclear Information System (INIS)

    Wang, Yinxi; Liu, Dan; Zhang, Huifeng; Wang, Yixin; Wei, Ling; Liu, Yutong; Liao, Jieying; Gao, Hui-Ming; Zhou, Hui

    2017-01-01

    Background: Atmospheric ultrafine particles (UFPs) and pesticide rotenone were considered as potential environmental risk factors for Parkinson's disease (PD). However, whether and how UFPs alone and in combination with rotenone affect the pathogenesis of PD remains largely unknown. Methods: Ultrafine carbon black (ufCB, a surrogate of UFPs) and rotenone were used individually or in combination to determine their roles in chronic dopaminergic (DA) loss in neuron-glia, and neuron-enriched, mix-glia cultures. Immunochemistry using antibody against tyrosine hydroxylase was performed to detect DA neuronal loss. Measurement of extracellular superoxide and intracellular reactive oxygen species (ROS) were performed to examine activation of NADPH oxidase. Genetic deletion and pharmacological inhibition of NADPH oxidase and MAC-1 receptor in microglia were employed to examine their role in DA neuronal loss triggered by ufCB and rotenone. Results: In rodent midbrain neuron-glia cultures, ufCB and rotenone alone caused neuronal death in a dose-dependent manner. In particularly, ufCB at doses of 50 and 100 μg/cm 2 induced significant loss of DA neurons. More importantly, nontoxic doses of ufCB (10 μg/cm 2 ) and rotenone (2 nM) induced synergistic toxicity to DA neurons. Microglial activation was essential in this process. Furthermore, superoxide production from microglial NADPH oxidase was critical in ufCB/rotenone-induced neurotoxicity. Studies in mix-glia cultures showed that ufCB treatment activated microglial NADPH oxidase to induce superoxide production. Firstly, ufCB enhanced the expression of NADPH oxidase subunits (gp91 phox , p47 phox and p40 phox ); secondly, ufCB was recognized by microglial surface MAC-1 receptor and consequently promoted rotenone-induced p47 phox and p67 phox translocation assembling active NADPH oxidase. Conclusion: ufCB and rotenone worked in synergy to activate NADPH oxidase in microglia, leading to oxidative damage to DA neurons. Our

  9. Ultrafine carbon particles promote rotenone-induced dopamine neuronal loss through activating microglial NADPH oxidase

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yinxi; Liu, Dan; Zhang, Huifeng; Wang, Yixin [Department of Occupational and Environmental Health Sciences, School of Public Health, Peking University, 100191 (China); Wei, Ling [Beijing Center for Physical & Chemical Analysis, Beijing 100089 (China); Liu, Yutong [School of Life Science, Beijing Normal University, Beijing 100875 (China); Liao, Jieying [Department of Translational Medicine, Xiamen Institute of Rare Earth Materials, Chinese Academy of Sciences, Xiamen 361024 (China); Gao, Hui-Ming [Model Animal Research Center of Nanjing University, Nanjing 211800 (China); Zhou, Hui, E-mail: hardhui@gmail.com [Department of Occupational and Environmental Health Sciences, School of Public Health, Peking University, 100191 (China)

    2017-05-01

    Background: Atmospheric ultrafine particles (UFPs) and pesticide rotenone were considered as potential environmental risk factors for Parkinson's disease (PD). However, whether and how UFPs alone and in combination with rotenone affect the pathogenesis of PD remains largely unknown. Methods: Ultrafine carbon black (ufCB, a surrogate of UFPs) and rotenone were used individually or in combination to determine their roles in chronic dopaminergic (DA) loss in neuron-glia, and neuron-enriched, mix-glia cultures. Immunochemistry using antibody against tyrosine hydroxylase was performed to detect DA neuronal loss. Measurement of extracellular superoxide and intracellular reactive oxygen species (ROS) were performed to examine activation of NADPH oxidase. Genetic deletion and pharmacological inhibition of NADPH oxidase and MAC-1 receptor in microglia were employed to examine their role in DA neuronal loss triggered by ufCB and rotenone. Results: In rodent midbrain neuron-glia cultures, ufCB and rotenone alone caused neuronal death in a dose-dependent manner. In particularly, ufCB at doses of 50 and 100 μg/cm{sup 2} induced significant loss of DA neurons. More importantly, nontoxic doses of ufCB (10 μg/cm{sup 2}) and rotenone (2 nM) induced synergistic toxicity to DA neurons. Microglial activation was essential in this process. Furthermore, superoxide production from microglial NADPH oxidase was critical in ufCB/rotenone-induced neurotoxicity. Studies in mix-glia cultures showed that ufCB treatment activated microglial NADPH oxidase to induce superoxide production. Firstly, ufCB enhanced the expression of NADPH oxidase subunits (gp91{sup phox}, p47{sup phox} and p40{sup phox}); secondly, ufCB was recognized by microglial surface MAC-1 receptor and consequently promoted rotenone-induced p47{sup phox} and p67{sup phox} translocation assembling active NADPH oxidase. Conclusion: ufCB and rotenone worked in synergy to activate NADPH oxidase in microglia, leading to

  10. A new methodology for the determination of enzyme activity based on carbon nanotubes and glucose oxidase.

    Science.gov (United States)

    Yeşiller, Gülden; Sezgintürk, Mustafa Kemal

    2015-11-10

    In this research, a novel enzyme activity analysis methodology is introduced as a new perspective for this area. The activity of elastase enzyme, which is a digestive enzyme mostly of found in the digestive system of vertebrates, was determined by an electrochemical device composed of carbon nanotubes and a second enzyme, glucose oxidase, which was used as a signal generator enzyme. In this novel methodology, a complex bioactive layer was constructed by using carbon nanotubes, glucose oxidase and a supporting protein, gelatin on a solid, conductive substrate. The activity of elastase was determined by monitoring the hydrolysis rate of elastase enzyme in the bioactive layer. As a result of this hydrolysis of elastase, glucose oxidase was dissociated from the bioactive layer, and following this the electrochemical signal due to glucose oxidase was decreased. The progressive elastase-catalyzed digestion of the bioactive layer containing glucose oxidase decreased the layer's enzymatic efficiency, resulting in a decrease of the glucose oxidation current as a function of the enzyme activity. The ratio of the decrease was correlated to elastase activity level. In this study, optimization experiments of bioactive components and characterization of the resulting new electrochemical device were carried out. A linear calibration range from 0.0303U/mL to 0.0729U/mL of elastase was reported. Real sample analyses were also carried out by the new electrochemical device. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Lower growth temperature increases alternative pathway capacity and alternative oxidase protein in tobacco.

    Science.gov (United States)

    Vanlerberghe, G C; McIntosh, L

    1992-09-01

    Suspension cells of NT1 tobacco (Nicotiana tabacum L. cv bright yellow) have been used to study the effect of growth temperature on the CN-resistant, salicylhydroxamic acid-sensitive alternative pathway of respiration. Mitochondria isolated from cells maintained at 30 degrees C had a low capacity to oxidize succinate via the alternative pathway, whereas mitochondria isolated from cells 24 h after transfer to 18 degrees C displayed, on average, a 5-fold increase in this capacity (from 7 to 32 nanoatoms oxygen per milligram protein per minute). This represented an increase in alternative pathway capacity from 18 to 45% of the total capacity of electron transport. This increased capacity was lost upon transfer of cells back to 30 degrees C. A monoclonal antibody to the terminal oxidase of the alternative pathway (the alternative oxidase) from Sauromatum guttatum (T.E. Elthon, R.L. Nickels, L. McIntosh [1989] Plant Physiology 89: 1311-1317) recognized a 35-kilodalton mitochondrial protein in tobacco. There was an excellent correlation between the capacity of the alternative path in isolated tobacco mitochondria and the levels of this 35-kilodalton alternative oxidase protein. Cycloheximide could inhibit both the increased level of the 35-kilodalton alternative oxidase protein and the increased alternative pathway capacity normally seen upon transfer to 18 degrees C. We conclude that transfer of tobacco cells to the lower temperature increases the capacity of the alternative pathway due, at least in part, to de novo synthesis of the 35-kilodalton alternative oxidase protein.

  12. NADPH oxidases as novel pharmacologic targets against influenza A virus infection.

    Science.gov (United States)

    Vlahos, Ross; Selemidis, Stavros

    2014-12-01

    Influenza A viruses represent a major global health care challenge, with imminent pandemics, emerging antiviral resistance, and long lag times for vaccine development, raising a pressing need for novel pharmacologic strategies that ideally target the pathology irrespective of the infecting strain. Reactive oxygen species (ROS) pervade all facets of cell biology with both detrimental and protective properties. Indeed, there is compelling evidence that activation of the NADPH oxidase 2 (NOX2) isoform of the NADPH oxidase family of ROS-producing enzymes promotes lung oxidative stress, inflammation, injury, and dysfunction resulting from influenza A viruses of low to high pathogenicity, as well as impeding virus clearance. By contrast, the dual oxidase isoforms produce ROS that provide vital protective antiviral effects for the host. In this review, we propose that inhibitors of NOX2 are better alternatives than broad-spectrum antioxidant approaches for treatment of influenza pathologies, for which clinical efficacy may have been limited owing to poor bioavailability and inadvertent removal of beneficial ROS. Finally, we briefly describe the current suite of NADPH oxidase inhibitors and the molecular features of the NADPH oxidase enzymes that could be exploited by drug discovery for development of more specific and novel inhibitors to prevent or treat disease caused by influenza. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

  13. Characterization of Two VAO-Type Flavoprotein Oxidases from Myceliophthora thermophila

    Directory of Open Access Journals (Sweden)

    Alessandro R. Ferrari

    2018-01-01

    Full Text Available The VAO flavoprotein family consists mostly of oxidoreductases harboring a covalently linked flavin cofactor. The linkage can be either monocovalent at position 8 with a histidine or tyrosine or bicovalent at position 8 with a histidine and at position 6 with a cysteine. Bicovalently bound flavoproteins show a preference for bulkier substrates such as oligosaccharides or secondary metabolites. The genome of the thermophilic fungus Myceliophthora thermophila C1 was found to be rich in genes encoding putative covalent VAO-type flavoproteins. Enzymes from this fungus have the advantage of being rather thermostable and homologous overexpression in M. thermophila C1 is feasible. Recently we discovered a new and VAO-type carbohydrate oxidase from this fungus: xylooligosaccharide oxidase. In this study, two other putative VAO-type oxidases, protein sequence XP_003663615 (MtVAO615 and XP_003665713 (MtVAO713, were expressed in M. thermophila C1, purified and characterized. Enzyme MtVAO615 was found to contain a bicovalently bound FAD, while enzyme MtVAO713 contained a monocovalent histidyl-bound FAD. The crystal structures of both proteins were obtained which revealed atypical active site architectures. It could be experimentally verified that both proteins, when reduced, rapidly react with molecular oxygen, a hallmark of flavoprotein oxidases. A large panel of alcohols, including carbohydrates, steroids and secondary alcohols were tested as potential substrates. For enzyme MtVAO713 low oxidase activity was discovered towards ricinoleic acid.

  14. Functional Analysis of Polyphenol Oxidases by Antisense/Sense Technology

    Directory of Open Access Journals (Sweden)

    Jutharat Attajarusit

    2007-07-01

    Full Text Available Polyphenol oxidases (PPOs catalyze the oxidation of phenolics to quinones, the secondary reactions of which lead to oxidative browning and postharvest losses of many fruits and vegetables. PPOs are ubiquitous in angiosperms, are inducible by both biotic and abiotic stresses, and have been implicated in several physiological processes including plant defense against pathogens and insects, the Mehler reaction, photoreduction of molecular oxygen by PSI, regulation of plastidic oxygen levels, aurone biosynthesis and the phenylpropanoid pathway. Here we review experiments in which the roles of PPO in disease and insect resistance as well as in the Mehler reaction were investigated using transgenic tomato (Lycopersicon esculentum plants with modified PPO expression levels (suppressed PPO and overexpressing PPO. These transgenic plants showed normal growth, development and reproduction under laboratory, growth chamber and greenhouse conditions. Antisense PPO expression dramatically increased susceptibility while PPO overexpression increased resistance of tomato plants to Pseudomonas syringae. Similarly, PPO-overexpressing transgenic plants showed an increase in resistance to various insects, including common cutworm (Spodoptera litura (F., cotton bollworm (Helicoverpa armigera (Hübner and beet army worm (Spodoptera exigua (Hübner, whereas larvae feeding on plants with suppressed PPO activity had higher larval growth rates and consumed more foliage. Similar increases in weight gain, foliage consumption, and survival were also observed with Colorado potato beetles (Leptinotarsa decemlineata (Say feeding on antisense PPO transgenic tomatoes. The putative defensive mechanisms conferred by PPO and its interaction with other defense proteins are discussed. In addition, transgenic plants with suppressed PPO exhibited more favorable water relations and decreased photoinhibition compared to nontransformed controls and transgenic plants

  15. Sulfite oxidase activity of cytochrome c: Role of hydrogen peroxide

    Directory of Open Access Journals (Sweden)

    Murugesan Velayutham

    2016-03-01

    occur to humans born with molybdenum cofactor and sulfite oxidase deficiencies.

  16. Development of new radiopharmaceuticals for imaging monoamine oxidase B

    Energy Technology Data Exchange (ETDEWEB)

    Vasdev, Neil, E-mail: neil.vasdev@utoronto.ca; Sadovski, Oleg; Moran, Matthew D.; Parkes, Jun; Meyer, Jeffrey H.; Houle, Sylvain; Wilson, Alan A.

    2011-10-15

    Introduction: Imaging monoamine oxidase B (MAO-B) in the central nervous system with PET is an important goal for psychiatric studies. We here report an improved and automated radiosynthesis of N-(6-[{sup 18}F]-fluorohexyl)-N-methylpropargylamine ([{sup 18}F]FHMP; [{sup 18}F]-1), as well as the radiosynthesis of two new promising candidates for imaging cerebral MAO-B, namely, carbon-11-labeled 3-(4-[{sup 11}C]-methoxyphenyl)-6-methyl-2H-1-benzopyran-2-one ([{sup 11}C]-2) and N-((1H-pyrrol-2-yl)methyl)-N-[{sup 11}C]-methyl-1-phenylmethanamine ([{sup 11}C]-3). Methods: Fluorine-18-labeled 1 was prepared via a tosyloxy precursor in 29%{+-}5% uncorrected radiochemical yield, relative to [{sup 18}F]-fluoride. Both carbon-11-labeled compounds were prepared with [{sup 11}C]CH{sub 3}I using the 'LOOP' method in 11% and 18% uncorrected radiochemical yields, respectively, relative to starting [{sup 11}C]CO{sub 2}. All radiotracers had specific activities >37 GBq/{mu}mol and were >98% radiochemically pure at end of synthesis (<40 min). All radiotracers were evaluated by ex vivo biodistribution studies in conscious rodents. Results: A major radioactive metabolite in the rodent brain was observed following administration of [{sup 18}F]-1. While [{sup 11}C]-2 had moderate brain penetration and good clearance from normal brain tissue, distribution of radioactivity in brain was indicative of free and nonspecific binding. Good brain uptake was observed with [{sup 11}C]-3 (0.8%-1.4% injected dose per gram at 5 min postinjection), binding appeared to be reversible and distribution conformed with regional distribution of MAO-B in the rat brain. Preinjection of 3 or L-deprenyl showed a modest reduction (up to 25%) of brain activity. Conclusion: Carbon-11-labeled 3 was found to have the most favorable properties of the radiotracers evaluated; however, the signal-to-noise ratio was too low to warrant further in vivo imaging studies. Alternative radiotracers for imaging MAO

  17. Surface reconstitution of glucose oxidase onto a norbornylogous bridge self-assembled monolayer

    International Nuclear Information System (INIS)

    Liu Jingquan; Paddon-Row, Michael N.; Gooding, J. Justin

    2006-01-01

    An electrode construct was fabricated in which a self-assembled monolayer containing a novel norbornylogous bridge was covalently attached to flavin adenine dinucleotide (FAD), the redox active centre of several oxidase enzymes. The electrochemistry of the construct was investigated before and after the reconstitution of glucose oxidase around the surface bound FAD. Rapid rates of electron transfer were observed both before and after the reconstitution of biocatalytically active enzyme. However, no biocatalytic activity was observed under anaerobic conditions suggesting the a lack of enzyme turnover through direct electron transfer. It is proposed that a decrease in the electronic coupling between the redox active FAD and the electrode following reconstitution of the glucose oxidase - a probable consequence of the FAD being immersed in a protein environment - was responsible for the inability of the enzyme to be turned over under anaerobic conditions

  18. Carbon Nanotube Modified Screen Printed Electrodes: Pyranose Oxidase Immobilization Platform for Amperometric Enzyme Sensors

    Directory of Open Access Journals (Sweden)

    Dilek ODACI DEMIRKOL

    2017-03-01

    Full Text Available Here, a novel enzymatic biosensor was developed using multiwalled carbon nanotube including screen printed electrodes (MWCNT-SPE. Pyranose oxidase (PyOx was immobilized on the electrode surface by way of gelatin membrane and then cross-linked using glutaraldehyde. Glucose was detected at -0.7 V (vs. Ag/AgCl by watching consumed oxygen in enzymatic reaction after addition substrate. After optimization of pH and enzyme loading, the linearity was found in the range of 0.1–1.0 mM of glucose. After that, the effect of MCNT on the current was tested. Also the enzymatic biosensor including glucose oxidase instead of pyranose oxidase was prepared and the biosensor response followed for glucose. Furthermore, this system was tested for glucose analysis in soft drinks.

  19. Ratiometric glucose sensing based on fluorescent oxygen films and glucose oxidase

    Directory of Open Access Journals (Sweden)

    Fengyu Su

    2017-06-01

    Full Text Available A new two-layer sensor film was constructed for sensing glucose based on glucose oxidase and oxygen sensing material. The first layer of film containing the oxygen sensor and intra-reference material was polymerized, then the second layer of glucose oxidase and glutaraldehyde was formed on the oxygen sensor layer. The two-layer sensor film has a resolution up to 0.05 mM and a detection range from 0 to 5 mM to glucose. The effects of pH and temperature on the sensing performance were systematically investigated. The selective detection of glucose among other monosaccharides, such as fructose, mannose and galactose indicated that the sensing film has excellent selectivity. The prepared sensor was successfully applied for glucose sample detection of glucose concentration in artificial tears. Keywords: Glucose sensor, Glucose oxidase, Fluorescence, Oxygen film, Diabetes

  20. Layer-by-Layer Assembly of Glucose Oxidase on Carbon Nanotube Modified Electrodes.

    Science.gov (United States)

    Suroviec, Alice H

    2017-01-01

    The use of enzymatically modified electrodes for the detection of glucose or other non-electrochemically active analytes is becoming increasingly common. Direct heterogeneous electron transfer to glucose oxidase has been shown to be kinetically difficult, which is why electron transfer mediators or indirect detection is usually used for monitoring glucose with electrochemical sensors. It has been found, however, that electrodes modified with single or multi-walled carbon nanotubes (CNTs) demonstrate fast heterogeneous electron transfer kinetics as compared to that found for traditional electrodes. Incorporating CNTs into the assembly of electrochemical glucose sensors, therefore, affords the possibility of facile electron transfer to glucose oxidase, and a more direct determination of glucose. This chapter describes the methods used to use CNTs in a layer-by-layer structure along with glucose oxidase to produce an enzymatically modified electrode with high turnover rates, increased stability and shelf-life.

  1. Xanthine oxidase activity and free radical generation in patients with sepsis syndrome

    DEFF Research Database (Denmark)

    Galley, H F; Davies, Michael Jonathan; Webster, N R

    1996-01-01

    OBJECTIVE: To determine xanthine oxidase activity, free radical concentrations, and lipid peroxidation in patients with sepsis syndrome compared with noninfected critically ill patients. DESIGN: A prospective observational study. SETTING: A nine-bed intensive care unit in a university teaching......). CONCLUSIONS: Patients with sepsis have xanthine oxidase activation, high free-radical concentrations, and evidence of free radical damage. The finding that xanthine oxidase activity was lower in those patients who died, coupled with increased lactate concentrations implies more severe ischemia with incomplete...... to the Acute Physiology and Chronic Health Evaluation (APACHE) II score or to the presence of organ dysfunction. The mean ascorbyl radical concentration (arbitrary units) determined by electron paramagnetic resonance following spin trapping was increased in patients compared with healthy subjects (p

  2. Erv2p: characterization of the redox behavior of a yeast sulfhydryl oxidase

    DEFF Research Database (Denmark)

    Wang, Wenzhong; Winther, Jakob R; Thorpe, Colin

    2007-01-01

    centers that facilitate the transfer of reducing equivalents from the dithiol substrates of these oxidases to the isoalloxazine ring where the reaction with molecular oxygen occurs. The present study examines yeast Erv2p and compares the redox behavior of this ER luminal protein with the augmenter...... and with unfolded proteins. Rapid reaction studies confirm that reduction of the flavin center of Erv2p is rate-limiting during turnover with molecular oxygen. This comparison of the redox properties between members of the ERV/ALR family of sulfhydryl oxidases provides insights into their likely roles in oxidative......The FAD prosthetic group of the ERV/ALR family of sulfhydryl oxidases is housed at the mouth of a 4-helix bundle and communicates with a pair of juxtaposed cysteine residues that form the proximal redox active disulfide. Most of these enzymes have one or more additional distal disulfide redox...

  3. Functional Assembly of Soluble and Membrane Recombinant Proteins of Mammalian NADPH Oxidase Complex.

    Science.gov (United States)

    Souabni, Hajer; Ezzine, Aymen; Bizouarn, Tania; Baciou, Laura

    2017-01-01

    Activation of phagocyte cells from an innate immune system is associated with a massive consumption of molecular oxygen to generate highly reactive oxygen species (ROS) as microbial weapons. This is achieved by a multiprotein complex, the so-called NADPH oxidase. The activity of phagocyte NADPH oxidase relies on an assembly of more than five proteins, among them the membrane heterodimer named flavocytochrome b 558 (Cytb 558 ), constituted by the tight association of the gp91 phox (also named Nox2) and p22 phox proteins. The Cytb 558 is the membrane catalytic core of the NADPH oxidase complex, through which the reducing equivalent provided by NADPH is transferred via the associated prosthetic groups (one flavin and two hemes) to reduce dioxygen into superoxide anion. The other major proteins (p47 phox , p67 phox , p40 phox , Rac) requisite for the complex activity are cytosolic proteins. Thus, the NADPH oxidase functioning relies on a synergic multi-partner assembly that in vivo can be hardly studied at the molecular level due to the cell complexity. Thus, a cell-free assay method has been developed to study the NADPH oxidase activity that allows measuring and eventually quantifying the ROS generation based on optical techniques following reduction of cytochrome c. This setup is a valuable tool for the identification of protein interactions, of crucial components and additives for a functional enzyme. Recently, this method was improved by the engineering and the production of a complete recombinant NADPH oxidase complex using the combination of purified proteins expressed in bacterial and yeast host cells. The reconstitution into artificial membrane leads to a fully controllable system that permits fine functional studies.

  4. Functional heterogeneity of NADPH oxidase-mediated contractions to endothelin with vascular aging.

    Science.gov (United States)

    Meyer, Matthias R; Barton, Matthias; Prossnitz, Eric R

    2014-11-24

    Aging, a physiological process and main risk factor for cardiovascular and renal diseases, is associated with endothelial cell dysfunction partly resulting from NADPH oxidase-dependent oxidative stress. Because increased formation of endothelium-derived endothelin-1 (ET-1) may contribute to vascular aging, we studied the role of NADPH oxidase function in age-dependent contractions to ET-1. Renal arteries and abdominal aortas from young and old C57BL6 mice (4 and 24 months of age) were prepared for isometric force measurements. Contractions to ET-1 (0.1-100 nmol/L) were determined in the presence and absence of the NADPH oxidase-selective inhibitor gp91ds-tat (3 μmol/L). To exclude age-dependent differential effects of NO bioactivity between vascular beds, all experiments were conducted in the presence of the NO synthase inhibitor L-NAME (300 μmol/L). In young animals, ET-1-induced contractions were 6-fold stronger in the renal artery than in the aorta (prenal artery and aorta, respectively (pAging had no effect on NADPH oxidase-dependent and -independent contractions to ET-1 in the renal artery. In contrast, contractions to ET-1 were markedly reduced in the aged aorta (5-fold, page-dependent heterogeneity of NADPH oxidase-mediated vascular contractions to ET-1, demonstrating an inherent resistance to functional changes in the renal artery but not in the aorta with aging. Thus, local activity of NADPH oxidase differentially modulates responses to ET-1 with aging in distinct vascular beds. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  5. Ozone affects pollen viability and NAD(P)H oxidase release from Ambrosia artemisiifolia pollen

    Energy Technology Data Exchange (ETDEWEB)

    Pasqualini, Stefania, E-mail: spas@unipg.it [Department of Applied Biology, University of Perugia, Perugia (Italy); Tedeschini, Emma; Frenguelli, Giuseppe [Department of Applied Biology, University of Perugia, Perugia (Italy); Wopfner, Nicole; Ferreira, Fatima [Department of Molecular Biology, CD Laboratory for Allergy Diagnosis and Therapy, University of Salzburg, Salzburg (Austria); D' Amato, Gennaro [Division of Respiratory and Allergic Diseases, ' A. Cardarelli' High Speciality Hospital, Naples (Italy); Ederli, Luisa [Department of Applied Biology, University of Perugia, Perugia (Italy)

    2011-10-15

    Air pollution is frequently proposed as a cause of the increased incidence of allergy in industrialised countries. We investigated the impact of ozone (O{sub 3}) on reactive oxygen species (ROS) and allergen content of ragweed pollen (Ambrosia artemisiifolia). Pollen was exposed to acute O{sub 3} fumigation, with analysis of pollen viability, ROS and nitric oxide (NO) content, activity of nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase, and expression of major allergens. There was decreased pollen viability after O{sub 3} fumigation, which indicates damage to the pollen membrane system, although the ROS and NO contents were not changed or were only slightly induced, respectively. Ozone exposure induced a significant enhancement of the ROS-generating enzyme NAD(P)H oxidase. The expression of the allergen Amb a 1 was not affected by O{sub 3}, determined from the mRNA levels of the major allergens. We conclude that O{sub 3} can increase ragweed pollen allergenicity through stimulation of ROS-generating NAD(P)H oxidase. - Highlights: > O{sub 3} reduces the viability of ragweed pollen. > ROS and allergens of ragweed pollen were not affected by O{sub 3} exposure. > O{sub 3} enhances the activity of the ROS-generating enzyme NAD(P)H oxidase. > O{sub 3} increases ragweed pollen allergenicity through NAD(P)H-oxidase stimulation. - This study focuses on the effects of the atmospheric pollutant ozone on ROS content and NAD(P)H oxidase activity of ragweed pollen grains.

  6. Systemic Manifestations in Pyridox(am)ine 5'-Phosphate Oxidase Deficiency.

    Science.gov (United States)

    Guerriero, Réjean M; Patel, Archana A; Walsh, Brian; Baumer, Fiona M; Shah, Ankoor S; Peters, Jurriaan M; Rodan, Lance H; Agrawal, Pankaj B; Pearl, Phillip L; Takeoka, Masanori

    2017-11-01

    Pyridoxine is converted to its biologically active form pyridoxal-5-phosphate (P5P) by the enzyme pyridox(am)ine 5'-phosphate oxidase and serves as a cofactor in nearly 200 reactions in the central nervous system. Pyridox(am)ine 5'-phosphate oxidase deficiency leads to P5P dependent epilepsy, typically a neonatal- or infantile-onset epileptic encephalopathy treatable with P5P or in some cases, pyridoxine. Following identification of retinopathy in a patient with pyridox(am)ine 5'-phosphate oxidase deficiency that was reversible with P5P therapy, we describe the systemic manifestations of pyridox(am)ine 5'-phosphate oxidase deficiency. A series of six patients with homozygous mutations of PNPO, the gene coding pyridox(am)ine 5'-phosphate oxidase, were evaluated in our center over the course of two years for phenotyping of neurological and systemic manifestations. Five of six were born prematurely, three had anemia and failure to thrive, and two had elevated alkaline phosphatase. A movement disorder was observed in two children, and a reversible retinopathy was observed in the most severely affected infant. All patients had neonatal-onset epilepsy and were on a continuum of developmental delay to profound encephalopathy. Electroencephalographic features included background slowing and disorganization, absent sleep features, and multifocal and generalized epileptiform discharges. All the affected probands carried a homozygous PNPO mutation (c.674 G>T, c.686 G>A and c.352G>A). In addition to the well-described epileptic encephalopathy, pyridox(am)ine 5'-phosphate oxidase deficiency causes a range of neurological and systemic manifestations. A movement disorder, developmental delay, and encephalopathy, as well as retinopathy, anemia, and failure to thrive add to the broadening clinical spectrum of P5P dependent epilepsy. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Ratiometric glucose sensing based on fluorescent oxygen films and glucose oxidase

    OpenAIRE

    Fengyu Su; Liqiang Zhang; Xiangxing Kong; Fred Lee; Yanqing Tian; Deirdre R. Meldrum

    2017-01-01

    A new two-layer sensor film was constructed for sensing glucose based on glucose oxidase and oxygen sensing material. The first layer of film containing the oxygen sensor and intra-reference material was polymerized, then the second layer of glucose oxidase and glutaraldehyde was formed on the oxygen sensor layer. The two-layer sensor film has a resolution up to 0.05 mM and a detection range from 0 to 5 mM to glucose. The effects of pH and temperature on the sensing performance were systemati...

  8. A new procedure for the purification of monodisperse highly active cytochrome c oxidase from bovine heart.

    OpenAIRE

    Li, Y; Naqui, A; Frey, T G; Chance, B

    1987-01-01

    A simple and rapid method for the isolation of a large quantity of cytochrome c oxidase from bovine heart mitochondria was developed, based on selective solubilization of mitochondrial protein with first Triton and then lauryl maltoside. Gel filtration shows that the lauryl maltoside-solubilized oxidase preparation is in a hydrodynamically homogeneous state with a Stokes radius of 7.5 +/- 0.2 nm. It contains 8.0 mumol of haem (with an a/a3 ratio of 1)/g of protein. The catalytic constant (max...

  9. Cloning and Functional Analysis of the Promoter of an Ascorbate Oxidase Gene from Gossypium hirsutum

    OpenAIRE

    Xin, Shan; Tao, Chengcheng; Li, Hongbin

    2016-01-01

    Apoplastic ascorbate oxidase (AO) plays significant roles in plant cell growth. However, the mechanism of underlying the transcriptional regulation of AO in Gossypium hirsutum remains unclear. Here, we obtained a 1,920-bp promoter sequence from the Gossypium hirsutum ascorbate oxidase (GhAO1) gene, and this GhAO1 promoter included a number of known cis-elements. Promoter activity analysis in overexpressing pGhAO1::GFP-GUS tobacco (Nicotiana benthamiana) showed that the GhAO1 promoter exhibite...

  10. Cytochrome oxidase as an indicator of ice storage and frozen storage

    DEFF Research Database (Denmark)

    Godiksen, Helene; Jessen, Flemming

    2001-01-01

    in different cods was 21%, and the coefficient of variation of different analyses on the same homogenate was 5%. It was shown that ice storage of muscle samples before they were frozen and thawed resulted in a major freezing-induced activation of cytochrome oxidase activity. The enzyme may therefore be used...... as an indicator of frozen fish to determine if the fish has been stored on ice before freezing. Cytochrome oxidase activity showed also potential as an indicator of frozen storage, as it was possible to distinguish between the frozen storage temperatures -9, -20, and -40 degreesC....

  11. Immunological and molecular comparison of polyphenol oxidase in Rosaceae fruit trees.

    Science.gov (United States)

    Haruta, M; Murata, M; Kadokura, H; Homma, S

    1999-03-01

    An antibody raised against apple polyphenol oxidase (PPO) cross-reacted with PPOs from Japanese pear (Pyrus pyrifolia), pear (Pyrus communis), peach (Prunus persica), Chinese quince (Pseudocydonia sinensis) and Japanese loquat (Eriobotrya japonica). Core fragments (681 bp) of the corresponding PPO genes were amplified and characterized. The deduced protein sequences showed identities of 85.3 to 97.5%. Chlorogenic acid oxidase activity of these PPOs showed higher activities when assayed at pH 4 than at pH 6. These results indicate that PPOs in Rosaceae plants are structurally and enzymatically similar.

  12. Kinetic Resolution of sec-Thiols via Enantioselective Oxidation with Rationally Engineered 5-(Hydroxymethyl)furfural Oxidase

    NARCIS (Netherlands)

    Pickl, Mathias; Swoboda, Alexander; Romero, Elvira; Winkler, Christoph; Binda, Claudia; Mattevi, Andrea; Faber, Kurt; Fraaije, Marco

    2018-01-01

    Various flavoprotein oxidases were recently shown to oxidize prim-thiols. Here we extend this reactivity towards sec-thiols via structure-guided engineering of 5-(hydroxymethyl)furfural oxidase (HMFO). The variants obtained were employed for the oxidative kinetic resolution of rac-sec-thiols

  13. Isolation and purification of pyranose 2-oxidase from Phanerochaete chrysosporium and characterization of gene structure and regulation

    Science.gov (United States)

    Theodorus H. de Koker; Michael D. Mozuch; Daniel Cullen; Jill Gaskell; Philip J. Kersten

    2004-01-01

    Pyranose 2-oxidase (POX) was recovered from Phanerochaete chrysosporium BKM-F-1767 solid substrate culture using mild extraction conditions and was purified. 13C-nuclear magnetic resonance confirmed production of D- arabino -hexos-2-ulose (glucosone) from D-glucose with the oxidase. Peptide fingerprints generated by liquid chromatography-tandem mass spectrometry of...

  14. The cytochemical demonstration of catalase and D-amino acid oxidase in the microbodies of teleost kidney cells

    NARCIS (Netherlands)

    Veenhuis, M.; Wendelaar Bonga, S.D.

    1977-01-01

    The distribution of catalase and D-amino acid oxidase, marker enzymes for peroxisomes, was determined cytochemically in the kidney tubules of an euryhaline teleost, the three-spined stickleback. Catalase activity was localized with the diaminobenzidine technique. The presence of D-amino acid oxidase

  15. Biocatalytic properties and structural analysis of eugenol oxidase from Rhodococcus jostii RHA1 : a versatile oxidative biocatalyst

    NARCIS (Netherlands)

    Nguyen, Quoc-Thai; De Gonzalo, Gonzalo; Binda, Claudia; Rioz, Ana; Mattevi, Andrea; Fraaije, Marco

    2016-01-01

    Eugenol oxidase (EUGO) from Rhodococcus jostii RHA1 was previously shown to convert only a limited set of phenolic compounds. In this study, we have explored the biocatalytic potential of this flavoprotein oxidase resulting in a broadened substrate scope and a deeper insight into its structural

  16. Substrate specificity of flavin-dependent vanillyl-alcohol oxidase from Penicillium simplicissimum.Evidence for the production of 4-hydroxycinnamyl alcohols from 4-allylphenols

    NARCIS (Netherlands)

    Fraaije, Marco W.; Veeger, Cees; Berkel, Willem J.H. van

    1995-01-01

    The substrate specificity of the flavoprotein vanillyl-alcohol oxidase from Penicillium simplicissimum was investigated. Vanillyl-alcohol oxidase catalyzes besides the oxidation of 4-hydroxybenzyl alcohols, the oxidative deamination of 4-hydroxybenzylamines and the oxidative demethylation of

  17. Identification and statistical optimization of fermentation conditions for a newly isolated extracellular cholesterol oxidase-producing Streptomyces cavourensis strain NEAE-42

    OpenAIRE

    El-Naggar, Noura El-Ahmady; El-Shweihy, Nancy M.; El-Ewasy, Sara M.

    2016-01-01

    Background Due to broad range of clinical and industrial applications of cholesterol oxidase, isolation and screening of bacterial strains producing extracellular form of cholesterol oxidase is of great importance. Results One hundred and thirty actinomycete isolates were screened for their cholesterol oxidase activity. Among them, a potential culture, strain NEAE-42 is displayed the highest extracellular cholesterol oxidase activity. It was selected and identified as Streptomyces cavourensis...

  18. Cloning and sequencing of the peroxisomal amine oxidase gene from Hansenula polymorpha

    NARCIS (Netherlands)

    Bruinenberg, P. G.; Evers, M.; Waterham, H. R.; Kuipers, J.; Arnberg, A. C.; AB, G.

    1989-01-01

    We have cloned the AMO gene, encoding the microbody matrix enzyme amine oxidase (EC 1.4.3.6) from the yeast Hansenula polymorpha. The gene was isolated by differential screening of a cDNA library, immunoselection, and subsequent screening of a H. polymorpha genomic library. The nucleotide sequence

  19. In vitro oxidation of indoleacetic acid by soluble auxin-oxidases and peroxidases from maize roots

    International Nuclear Information System (INIS)

    Beffa, R.; Martin, H.V.; Pilet, P.E.

    1990-01-01

    Soluble auxin-oxidases were extracted from Zea mays L. cv LG11 apical root segments and partially separated from peroxidases (EC 1.11.1.7) by size-exclusion chromatography. Auxin-oxidases were resolved into one main peak corresponding to a molecular mass of 32.5 kilodaltons and a minor peak at 54.5 kilodaltons. Peroxidases were separated into at least four peaks, with molecular masses from 32.5 to 78 kilodaltons. In vitro activity of indoleacetic acid-oxidases was dependent on the presence of MnCl 2 and p-coumaric acid. Compound(s) present in the crude extract and several synthetic auxin transport inhibitors (including 2,3,5-triiodobenzoic acid and N-1-naphthylphthalamic acid) inhibited auxin-oxidase activity, but had no effect on peroxidases. The products resulting from the in vitro enzymatic oxidation of [ 3 H]indoleacetic acid were separated by HPLC and the major metabolite was found to cochromatograph with indol-3yl-methanol

  20. Random mutagenesis of aspergillus niger and process optimization for enhanced production of glucose oxidase

    International Nuclear Information System (INIS)

    Haq, I.; Nawaz, A.; Mukhtar, A.N.H.; Mansoor, H.M.Z.; Ameer, S.M.

    2014-01-01

    The study deals with the improvement of wild strain Aspergillus niger IIB-31 through random mutagenesis using chemical mutagens. The main aim of the work was to enhance the glucose oxidase (GOX) yield of wild strain (24.57+-0.01 U/g of cell mass) through random mutagenesis and process optimization. The wild strain of Aspergillus niger IIB-31 was treated with chemical mutagens such as Ethyl methane sulphonate (EMS) and nitrous acid for this purpose. Mutagen treated 98 variants indicating the positive results were picked and screened for the glucose oxidase production using submerged fermentation. EMS treated E45 mutant strain gave the highest glucose oxidase production (69.47 + 0.01 U/g of cell mass), which was approximately 3-folds greater than the wild strain IIB-31. The preliminary cultural conditions for the production of glucose oxidase using submerged fermentation from strain E45 were also optimized. The highest yield of GOD was obtained using 8% glucose as carbon and 0.3% peptone as nitrogen source at a medium pH of 7.0 after an incubation period of 72 hrs at 30 degree. (author)

  1. A low perfusion rate microreactor for the continous monitoring of enzyme characteristics: applications for glucose oxidase.

    NARCIS (Netherlands)

    Posthuma-Trumpie, G.A.; Venema, K.; Berkel, van W.J.H.; Korf, J.

    2007-01-01

    This report describes a versatile and robust microreactor for bioactive proteins physically immobilized on a polyether sulfone filter. The potential of the reactor is illustrated with glucose oxidase immobilized on a filter with a cut-off value of 30 kDa. A flow-injection system was used to deliver

  2. A kinetic study of soluble glucose oxidase using a rotating-disc electrode

    NARCIS (Netherlands)

    Stroe-Biezen, van S.A.M.; Janssen, A.P.M.; Janssen, L.J.J.

    1994-01-01

    In order to determine the kinetic parameters of glucose oxidation catalysed by the enzyme glucose oxidase (GO) the initial velocity of hydrogen peroxide formation was measured using a rotating disc electrode (RDE). The major advantage of this method is the possibility of continuous measurement of

  3. An explanation for the combined effect of xylanase-glucose oxidase in dough systems

    NARCIS (Netherlands)

    Primo-Martín, C.; Wang, M.; Lichtendonk, W.J.; Plijter, J.J.; Hamer, R.J.

    2005-01-01

    In the bakery industry, glucose oxidase is usually used in combination with xylanase. Although many theories exist on the mechanism of action of each enzyme, the positive effect of combining the two is as yet unexplained. In this paper we studied a possible basis for this synergy by focusing on the

  4. Enhancing the rheological performance of wheat flour dough with glucose oxidase, transglutaminase or supplementary gluten

    NARCIS (Netherlands)

    Meerts, Mathieu; Van Ammel, Helene; Meeus, Yannick; Van Engeland, Sarah; Cardinaels, Ruth; Oosterlinck, Filip; Courtin, Christophe M.; Moldenaers, Paula

    2017-01-01

    The enzymes glucose oxidase and transglutaminase are frequently used to improve the breadmaking performance of wheat flours, as they have the ability to considerably alter the viscoelastic nature of the gluten network. To evaluate a flour’s breadmaking performance, rheological tests offer an

  5. A low perfusion rate microreactor for continuous monitoring of enzyme characteristics : application to glucose oxidase

    NARCIS (Netherlands)

    Posthuma-Trumpie, G. A.; Venema, K.; van Berkel, W. J. H.; Korf, J.

    This report describes a versatile and robust microreactor for bioactive proteins physically immobilized on a polyether sulfone filter. The potential of the reactor is illustrated with glucose oxidase immobilized on a filter with a cut-off value of 30 kDa. A flow-injection system was used to deliver

  6. Glucose oxidase-functionalized fluorescent gold nanoclusters as probes for glucose

    International Nuclear Information System (INIS)

    Xia, Xiaodong; Long, Yunfei; Wang, Jianxiu

    2013-01-01

    Highlights: ► A glucose oxidase/gold nanocluster conjugates formed by etching chemistry. ► Integration of the bioactivities and fluorescence properties within a single unit. ► These conjugates serve as novel fluorescent probe for glucose. -- Abstract: Creation and application of noble metal nanoclusters have received continuous attention. By integrating enzyme activity and fluorescence for potential applications, enzyme-capped metal clusters are more desirable. This work demonstrated a glucose oxidase (an enzyme for glucose)-functionalized gold cluster as probe for glucose. Under physiological conditions, such bioconjugate was successfully prepared by an etching reaction, where tetrakis (hydroxylmethyl) phosphonium-protected gold nanoparticle and thioctic acid-modified glucose oxidase were used as precursor and etchant, respectively. These bioconjugates showed unique fluorescence spectra (λ em max = 650 nm, λ ex max = 507 nm) with an acceptable quantum yield (ca. 7%). Moreover, the conjugated glucose oxidase remained active and catalyzed reaction of glucose and dissolved O 2 to produce H 2 O 2 , which quenched quantitatively the fluorescence of gold clusters and laid a foundation of glucose detection. A linear range of 2.0 × 10 −6 –140 × 10 −6 M and a detection limit of 0.7 × 10 −6 M (S/N = 3) were obtained. Also, another horseradish peroxidase/gold cluster bioconjugate was produced by such general synthesis method. Such enzyme/metal cluster bioconjugates represented a promising class of biosensors for biologically important targets in organelles or cells

  7. Addition of glucose oxidase for the improvement of refrigerated dough quality

    Science.gov (United States)

    Refrigerated dough encompasses a wide range of products and is a very popular choice for consumers. Two of the largest problems that occur during refrigerated dough storage are dough syruping and loss of dough strength. The goal of this study was to evaluate glucose oxidase as an additive to refri...

  8. Covalent immobilization of glucose oxidase on amino MOFs via post-synthetic modification

    NARCIS (Netherlands)

    Tudisco, C.G.; Zolubas, G.; Seoane de la Cuesta, B.; Zafarani, H.; Kazemzad Asiabi, M.; Gascon Sabate, J.; Hagedoorn, P.L.; Rassaei, L.

    2016-01-01

    The post-synthetic modification (PSM) of two amino-MOFs with glucose oxidase is reported in this study. The multi-step approach preserved the MOFs' structure and allowed the production of enzyme-functionalized MOFs (MOFs@GOx), which retained the enzymatic activity and showed selective properties

  9. Cytochrome c oxidase loses catalytic activity and structural integrity during the aging process in Drosophila melanogaster

    Energy Technology Data Exchange (ETDEWEB)

    Ren, Jian-Ching; Rebrin, Igor [Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90033 (United States); Klichko, Vladimir; Orr, William C. [Department of Biological Sciences, Southern Methodist University, Dallas, TX 75275 (United States); Sohal, Rajindar S., E-mail: sohal@usc.edu [Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90033 (United States)

    2010-10-08

    Research highlights: {yields} Cytochrome c oxidase loses catalytic activity during the aging process. {yields} Abundance of seven nuclear-encoded subunits of cytochrome c oxidase decreased with age in Drosophila. {yields} Cytochrome c oxidase is specific intra-mitochondrial site of age-related deterioration. -- Abstract: The hypothesis, that structural deterioration of cytochrome c oxidase (CcO) is a causal factor in the age-related decline in mitochondrial respiratory activity and an increase in H{sub 2}O{sub 2} generation, was tested in Drosophila melanogaster. CcO activity and the levels of seven different nuclear DNA-encoded CcO subunits were determined at three different stages of adult life, namely, young-, middle-, and old-age. CcO activity declined progressively with age by 33%. Western blot analysis, using antibodies specific to Drosophila CcO subunits IV, Va, Vb, VIb, VIc, VIIc, and VIII, indicated that the abundance these polypeptides decreased, ranging from 11% to 40%, during aging. These and previous results suggest that CcO is a specific intra-mitochondrial site of age-related deterioration, which may have a broad impact on mitochondrial physiology.

  10. Impacts on the metabolome of down-regulating polyphenol oxidase in transgenic potato tubers

    Science.gov (United States)

    Tubers of potato (Solanum tuberosum L. cv. Estima) genetically modified (GM) to reduce polyphenol oxidase (PPO) activity and enzymatic discolouration were assessed for changes in the metabolome using Liquid Chromatography-Mass Spectrometry (LC-MS) and Gas Chromatography (GC)-MS. Metabolome changes ...

  11. Pyranose 2-oxidase from Phanerochaete chrysosporium : expression in E. coli and biochemical characterization

    Science.gov (United States)

    Ines Pisanelli; Magdalena Kujawa; Oliver Spadiut; Roman Kittl; Petr Halada; Jindrich Volc; Michael D. Mozuch; Philip Kersten; Dietmar Haltrich; Clemens Peterbauer

    2009-01-01

    The presented work reports the isolation and heterologous expression of the p2ox gene encoding the flavoprotein pyranose 2-oxidase (P2Ox) from the basidiomycete Phanerochaete chrysosporium. The p2ox cDNA was inserted into the bacterial expression vector pET21a(+) and successfully expressed in Escherichia coli. We obtained active, fully flavinylated recombinant P2Ox in...

  12. Expanding the substrate scope of chitooligosaccharide oxidase from Fusarium graminearum by structure-inspired mutagenesis

    NARCIS (Netherlands)

    Ferrari, Alessandro; Lee, Misun; Fraaije, Marco

    2015-01-01

    Chitooligosaccharide oxidase from Fusarium graminearum (ChitO) oxidizes N-acetyl-D-glucosamine (GlcNAc) and its oligomers with high efficiency at the C1-hydroxyl moiety while it shows poor or no activity with other carbohydrates. By sequence and structural comparison with other known carbohydrate

  13. Xylem occlusion in Bouvardia flowers : evidence for a role of peroxidase and catechol oxidase

    NARCIS (Netherlands)

    Vaslier, N.; Doorn, van W.G.

    2003-01-01

    During vase life, Bouvardia flowers show rapid leaf wilting, especially if they are stored dry prior to placement in water. Wilting is due to a blockage in the basal stem end. We investigated the possible role of peroxidase and catechol oxidase in the blockage in cv. van Zijverden flowers, which

  14. Antioxidant Activity of Some Plant Extracts Towards Xanthine Oxidase, Lipoxygenase and Tyrosinase

    Directory of Open Access Journals (Sweden)

    Pi-Yu Chen

    2009-08-01

    Full Text Available Natural products have the potential to be developed into new drugs for the treatment of various diseases. The aim of the present study was to screen the antioxidant activities of some common edible fruits, garden plants and medicinal plants indigenous to Taiwan. This was performed by assessing the activities of lipoxygenase, xanthine oxidase and tyrosinase following incubation with extracts from these plants. A further aim was to use HPLC-DAD and tyrosinase to chromatographically identify the antioxidative constituents obtained from an extract exhibiting strong antioxidative properties. The acetone extracts of 27 cultivated plant species from Taiwan were tested for antioxidant activities towards xanthine oxidase, tyrosinase and lipoxygenase using spectrophotometric assays. Koelreuteria henryi, Prunus campanulata, and Rhodiola rosea showed the highest xanthine oxidase inhibitory activities. Camellia sinensis, Rhodiola rosea, and Koelreuteria henryi exhibited good tyrosinase inhibitory activities and potent anti-lipoxygenase activities. As Koelreuteria henryi had notable significant inhibitory activities towards xanthine oxidase, tyrosinase, and lipoxygenase, it was further tested with tyrosinase and HPLC-DAD. The results from this part of the study revealed that the more powerful the antioxidant capability of the extracted component, the greater the decrease in peak height obtained after reacting with tyrosinase. Additional studies are warranted to further characterize the compounds responsible for the antioxidant properties of the examined extracts.

  15. Season-controlled changes in biochemical constituents and oxidase enzyme activities in tomato (Lycopersicon esculentum Mill.).

    Science.gov (United States)

    Sen, Supatra; Mukherji, S

    2009-07-01

    Season-controlled changes in biochemical constituents viz. carotenoids (carotene and xanthophyll) and pectic substances along with IAA-oxidase and polyphenol oxidase (PPO) enzyme activities were estimated/assayed in leaves of Lycopersicon esculentum Mill. (tomato) in two developmental stages--pre-flowering (35 days after sowing) and post-flowering (75 days after sowing) in three different seasons--summer rainy and winter Carotenoid content along with pectic substances were highest in winter and declined significantly in summer followed by rainy i.e. winter > summer > rainy. Carotenoid content was significantly higher in the pre-flowering as compared to post-flowering in all three seasons while pectic substances increased in the post-flowering as compared to pre-flowering throughout the annual cycle. IAA oxidase and PPO enzyme activities were enhanced in rainy and decreased sharply in summer and winter i.e. rainy > summer > winter. Both the enzymes exhibited higher activity in the post-flowering stage as compared to pre-flowering in all three seasons. These results indicate winter to be the most favourable season for tomato plants while rainy season environmental conditions prove to be unfavourable (stressful) with diminished content of carotenoid and pectic substances and low activities of IAA oxidase and PPO, ultimately leading to poor growth and productivity.

  16. Engineering glucose oxidase to minimize the influence of oxygen on sensor response

    International Nuclear Information System (INIS)

    Horaguchi, Yohei; Saito, Shoko; Kojima, Katsuhiro; Tsugawa, Wakako; Ferri, Stefano; Sode, Koji

    2014-01-01

    Glucose oxidase (GOx) is an important industrial enzyme and is recognized as the gold standard for monitoring blood glucose. However, due to its inherent oxidase property, the presence of oxygen affects electrochemical measurements of venous blood glucose employing artificial electron mediators. We therefore attempted to engineer Penicillium amagasakiense-derived GOx into a dehydrogenase by focusing on the amino acid residues predicted to interact with oxygen. Our rational amino acid substitution approach resulted in the construction of the Ser114Ala/Phe355Leu mutant, which has an 11-fold decrease in oxidase activity and 2.8-fold increase in dehydrogenase activity compared with wild-type GOx. As a result, the dehydrogenase/oxidase activity ratio of the engineered enzyme was 32-fold greater than that of the wild-type enzyme. The enzyme sensor constructed with Ser114Ala/Phe355Leu was considerably less affected by oxygen than the wild-type GOx-based sensor at lower glucose concentrations

  17. Age-related ultrastructural and monoamine oxidase changes in the rat optic nerve.

    Science.gov (United States)

    Taurone, S; Ripandelli, G; Minni, A; Lattanzi, R; Miglietta, S; Pepe, N; Fumagalli, L; Micera, A; Pastore, F S; Artico, M

    2016-01-01

    The aim of this paper is to study the morphology and the distribution of the monoamine oxidase enzymatic system in the optic nerve of 4 month-old Wistar (young) and 28 month-old Wistar (old) rats. The optic nerve was harvested from 20 young and old rats. The segment of optic nerve was divided longitudinally into two pieces, each 0.1 mm in length. The first piece was used for transmission electron microscopy. The second piece was stained with histochemical reaction for monoamine oxidase. The agerelated changes in the optic nerve of rats include micro-anatomical details, ultrastructure and monoamine oxidase histochemical staining. A strong decrease of the thin nerve fibers and a swelling of the thick ones can be observed in optic nerve fibers of old rats. Increased monoamine oxidase histochemical staining of the optic nerve of aged rats is well demonstrated. The increase of meningeal shealth and the decrease of thin nerve fibers of the optic nerve in old rats are well documented. Morphological, ultrastructural and histochemical changes observed in optic nerve fibers of the old rats show a close relation with aging.

  18. Free radicals in hypoxic rat diaphragm contractility: no role for xanthine oxidase.

    NARCIS (Netherlands)

    Heunks, L.M.A.; Machiels, H.A.; Abreu, R.A. de; Zhu, X.; Heijden, E. van der; Dekhuijzen, P.N.R.

    2001-01-01

    Recent evidence indicates that hypoxia enhances the generation of oxidants. Little is known about the role of free radicals in contractility of the rat diaphragm during hypoxia. We hypothesized that antioxidants improve contractility of the hypoxic rat diaphragm and that xanthine oxidase (XO) is an

  19. Nitric oxide production and monoamine oxidase activity in cancer patients during interferon-a therapy

    NARCIS (Netherlands)

    D. Fekkes (Durk); A.R. van Gool (Arthur); M. Bannink (Marjolein); S. Sleijfer (Stefan); W.H.J. Kruit (Wim); B. van der Holt (Bronno); A.M.M. Eggermont (Alexander); M.W. Hengeveld (Michiel); G. Stoter (Gerrit)

    2009-01-01

    textabstractAbstract Both increased and decreased nitric oxide (NO) synthesis have been reported in patients treated with interferon-alpha (IFN-alpha). Animal studies showed that IFN-alpha administration results in increased levels of biogenic amines, subsequent activation of monoamine oxidases

  20. Tissue Printing to Visualize Polyphenol Oxidase and Peroxidase in Vegetables, Fruits, and Mushrooms

    Science.gov (United States)

    Melberg, Amanda R.; Flurkey, William H.; Inlow, Jennifer K.

    2009-01-01

    A simple tissue-printing procedure to determine the tissue location of the endogenous enzymes polyphenol oxidase and peroxidase in a variety of vegetables, fruits, and mushrooms is described. In tissue printing, cell contents from the surface of a cut section of the tissue are transferred to an adsorptive surface, commonly a nitrocellulose…

  1. Mapping the primary structure of copper/topaquinone-containing methylamine oxidase from Aspergillus niger.

    Science.gov (United States)

    Lenobel, R; Sebela, M; Frébort, I

    2005-01-01

    The amino acid sequence of methylamine oxidase (MeAO) from the fungus Aspergillus niger was analyzed using mass spectrometry (MS). First, MeAO was characterized by an accurate molar mass of 72.4 kDa of the monomer measured using MALDI-TOF-MS and by a pI value of 5.8 determined by isoelectric focusing. MALDI-TOF-MS revealed a clear peptide mass fingerprint after tryptic digestion, which did not provide any relevant hit when searched against a nonredundant protein database and was different from that of A. niger amine oxidase AO-I. Tandem mass spectrometry with electrospray ionization coupled to liquid chromatography allowed unambiguous reading of six peptide sequences (11-19 amino acids) and seven sequence tags (4-15 amino acids), which were used for MS BLAST homology searching. MeAO was found to be largely homologous to a hypothetical protein AN7641.2 (EMBL/GenBank protein-accession code EAA61827) from Aspergillus nidulans FGSC A4 with a theoretical molar mass of 76.46 kDa and pI 6.14, which belongs to the superfamily of copper amine oxidases. The protein AN7641.2 is only little homologous to the amine oxidase AO-I (32% identity, 49 % similarity).

  2. Salicylic Acid Regulation of Respiration in Higher Plants: Alternative Oxidase Expression.

    Science.gov (United States)

    Rhoads, DM; McIntosh, L

    1992-01-01

    Alternative respiratory pathway capacity increases during the development of the thermogenic appendix of a voodoo lily inflorescence. The levels of the alternative oxidase proteins increased dramatically between D-4 (4 days prior to the day of anthesis) and D-3 and continued to increase until the day of anthesis (D-day). The level of salicylic acid (SA) in the appendix is very low early on D-1, but increases to a high level in the evening of D-1. Thermogenesis occurs after a few hours of light on D-day. Therefore, the initial accumulation of the alternative oxidase proteins precedes the increase in SA by 3 days, indicating that other regulators may be involved. A 1.6-kb transcript encoding the alternative oxidase precursor protein accumulated to a high level in the appendix tissue by D-1. Application of SA to immature appendix tissue caused an increase in alternative pathway capacity and a dramatic accumulation of the alternative oxidase proteins and the 1.6-kb transcript. Time course experiments showed that the increase in capacity, protein levels, and transcript level corresponded precisely. The response to SA was blocked by cycloheximide or actinomycin D, indicating that de novo transcription and translation are required. However, nuclear, in vitro transcription assays indicated that the accumulation of the 1.6-kb transcript did not result from a simple increase in the rate of transcription of aox1. PMID:12297672

  3. Pyranose 2-oxidase from Phanerochaete chrysosporium--Expression in E.coli and Biochemical Characterization

    Czech Academy of Sciences Publication Activity Database

    Pisanelli, I.; Kujawa, M.; Spadiut, O.; Kittl, R.; Halada, Petr; Volc, Jindřich; Mozuch, M. D.; Kersten, P.; Haltrich, D.; Peterbauer, C.

    2009-01-01

    Roč. 142, č. 2 (2009), s. 97-106 ISSN 0168-1656 Institutional research plan: CEZ:AV0Z50200510 Keywords : Pyranose 2-oxidase * Phanerochaete chrysosporium * Lignocellulose degradation Subject RIV: CE - Biochemistry Impact factor: 2.881, year: 2009

  4. Reconstitution of apoglucose oxidase with FAD conjugates for biosensoring of progesterone

    NARCIS (Netherlands)

    Posthuma-Trumpie, G.A.; Berg, van den W.A.M.; Wiel, van de D.F.M.; Schaaper, W.M.M.; Korf, J.; Berkel, van W.J.H.

    2007-01-01

    The reconstitution of Aspergillus niger apoglucose oxidase (apoGOx) with FAD conjugates for biosensoring of progesterone was investigated. ApoGOx prepared by partial unfolding of the protein under acidic conditions consisted of reconstitutable monomers (50 ± 10%), reconstitutable dimers (20 ± 10%)

  5. A putative novel nuclear-encoded subunit of the cytochrome c oxidase complex in trypanosomatids

    Czech Academy of Sciences Publication Activity Database

    Maslov, D. A.; Zíková, Alena; Kyselová, Iveta; Lukeš, Julius

    2002-01-01

    Roč. 125, 1-2 (2002), s. 113-125 ISSN 0166-6851 R&D Projects: GA ČR GA204/00/1212 Grant - others:NIH(US) AI40634 Institutional research plan: CEZ:AV0Z6022909 Keywords : cytochrome c oxidase * mitochondrion * kinetoplast Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.911, year: 2002

  6. Galactose oxidase labeling of membrane proteins from human brain white matter

    International Nuclear Information System (INIS)

    Hukkanen, V.; Frey, H.; Salmi, A.

    1981-01-01

    Membrane proteins of human autopsy brain white matter were subjected to a galactose oxidase/NaB 3 H 4 labeling procedure and the membranes labeled by this method or by [ 3 H]acetic anhydride techniques were studied by lectin affinity chromatography using Lens culinaris phytohemagglutinin (lentil lectin) attached to Sepharose 4B beads. (Auth.)

  7. The caa(3) terminal oxidase of Bacillus stearothermophilus - Transient spectroscopy of electron transfer and ligand binding

    NARCIS (Netherlands)

    Giuffre, A; DItri, E; Giannini, S; Brunori, M; UbbinkKok, T; Konings, WN; Antonini, G

    1996-01-01

    The thermophilic bacterium Bacillus stearothermophilus possesses a caa(3)-type terminal oxidase, which was previously purified (De Vrij, W., Heyne, R. I. HL, and Konings, W. N. (1989) Ear. J. Biochem. 178, 763-770). We have carried out extensive kinetic experiments on the purified enzyme by

  8. Cytokinin oxidase/dehydrogenase genes in barley and wheat. Cloning and heterologous expression

    Czech Academy of Sciences Publication Activity Database

    Galuszka, P.; Frébortová, Jitka; Werner, T.; Yamada, M.; Strnad, Miroslav; Schmülling, T.; Frébort, I.

    2004-01-01

    Roč. 271, č. 20 (2004), s. 3990-4002 ISSN 0014-2956 Institutional research plan: CEZ:AV0Z5038910 Keywords : cereals * cloning * cytokinin oxidase/dehydrogenase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.260, year: 2004

  9. Pyruvate Oxidase Influences the Sugar Utilization Pattern and Capsule Production in Streptococcus pneumoniae

    NARCIS (Netherlands)

    Carvalho, Sandra M.; Farshchi Andisi, Vahid; Gradstedt, Henrik; Neef, Jolanda; Kuipers, Oscar P.; Neves, Ana R.; Bijlsma, Jetta J. E.

    2013-01-01

    Pyruvate oxidase is a key function in the metabolism and lifestyle of many lactic acid bacteria and its activity depends on the presence of environmental oxygen. In Streptococcus pneumoniae the protein has been suggested to play a major role in metabolism and has been implicated in virulence,

  10. Cloning and sequencing of phenol oxidase 1 (pox1) gene from ...

    African Journals Online (AJOL)

    The gene (pox1) encoding a phenol oxidase 1 from Pleurotus ostreatus was sequenced and the corresponding pox1-cDNA was also synthesized, cloned and sequenced. The isolated gene is flanked by an upstream region called the promoter (399 bp) prior to the start codon (ATG). The putative metalresponsive elements ...

  11. Galactose Oxidase from Fusarium oxysporum - Expression in E. coli and P. pastoris and Biochemical Characterization

    Czech Academy of Sciences Publication Activity Database

    Paukner, R.; Staudigl, P.; Choosri, W.; Sygmund, Ch.; Halada, Petr; Haltrich, D.; Leitner, CH.

    2014-01-01

    Roč. 9, č. 6 (2014) E-ISSN 1932-6203 Grant - others:Austrian Science Foundation(AT) L 504-B11 Institutional support: RVO:61388971 Keywords : galactose oxidase * gene * Fusarium * gene expression Subject RIV: CE - Biochemistry Impact factor: 3.234, year: 2014

  12. Application of NAD(P)H oxidase for cofactor regeneration in dehydrogenase catalyzed oxidations

    DEFF Research Database (Denmark)

    Rehn, Gustav; Pedersen, Asbjørn Toftgaard; Woodley, John

    2016-01-01

    alcohol dehydrogenases. However, their effective use requires an effective regeneration of the oxidized nicotinamide cofactor (NAD(P)+), which is critical for the economic feasibility of the process. NAD(P)H oxidase is an enzyme class of particular interest for this cofactor regeneration since it enables...

  13. Diurnal changes in polyamine content, arginine and ornithine decarboxylase, and diamine oxidase in tobacco leaves

    Czech Academy of Sciences Publication Activity Database

    Gemperlová, Lenka; Nováková, Marie; Vaňková, Radomíra; Eder, Josef; Cvikrová, Milena

    2006-01-01

    Roč. 57, č. 6 (2006), s. 1413-1421 ISSN 0022-0957 R&D Projects: GA ČR GA206/03/0369 Institutional research plan: CEZ:AV0Z50380511 Keywords : Arginine decarboxylase * diamine oxidase * ornithine decarboxylase Subject RIV: ED - Physiology Impact factor: 3.630, year: 2006

  14. Thermal and pH stabilities of partially purified polyphenol oxidase ...

    African Journals Online (AJOL)

    Dr. Paul Chidoka Chikezie

    2012-07-30

    Jul 30, 2012 ... indices of the two PPO extracts is summarized in Table. 1. At the end of the .... activity near neutral pH values (Jime´nez-Atie´nzar et al.,. 2004; Dogan and Dogan, 2004). ..... oxidase from white cherry fruit (Starks gold). GIDA.

  15. Potato and Mushroom Polyphenol Oxidase Activities Are Differently Modulated by Natural Plant Extracts

    NARCIS (Netherlands)

    Kuijpers, T.F.M.; Herk, van T.; Vincken, J.P.; Janssen, R.H.; Narh, D.L.; Berkel, van W.J.H.; Gruppen, H.

    2014-01-01

    Enzymatic browning is a major quality issue in fruit and vegetable processing and can be counteracted by different natural inhibitors. Often, model systems containing a single polyphenol oxidase (PPO) are used to screen for new inhibitors. To investigate the impact of the source of PPO on the

  16. Role of Lysyl oxidase-like 1 gene polymorphisms in Pakistani patients with pseudoexfoliative glaucoma

    NARCIS (Netherlands)

    Micheal, S.; Khan, M.I.; Akhtar, F.; Ali, M.; Ahmed, A.; Hollander, A.I. den; Qamar, R.

    2012-01-01

    PURPOSE: Single nucleotide polymorphisms (SNPs) rs1048661 (p.R141L) and rs3825942 (p.G153D) in the lysyl oxidase-like 1 (LOXL1) gene have been previously reported to be associated with pseudoexfoliation glaucoma (PEXG) in various Asian and European populations, but these SNPs have not yet been

  17. Regio- and Stereospecific Conversion of 4-Alkylphenols by the Covalent Flavoprotein Vanillyl-Alcohol Oxidase

    NARCIS (Netherlands)

    Heuvel, Robert H.H. van den; Fraaije, Marco W.; Laane, Colja; Berkel, Willem J.H. van

    1998-01-01

    The regio- and stereospecific conversion of prochiral 4-alkylphenols by the covalent flavoprotein vanillyl-alcohol oxidase was investigated. The enzyme was active, with 4-alkylphenols bearing aliphatic side chains of up to seven carbon atoms. Optimal catalytic efficiency occurred with 4-ethylphenol

  18. Cytochrome c oxidase loses catalytic activity and structural integrity during the aging process in Drosophila melanogaster

    International Nuclear Information System (INIS)

    Ren, Jian-Ching; Rebrin, Igor; Klichko, Vladimir; Orr, William C.; Sohal, Rajindar S.

    2010-01-01

    Research highlights: → Cytochrome c oxidase loses catalytic activity during the aging process. → Abundance of seven nuclear-encoded subunits of cytochrome c oxidase decreased with age in Drosophila. → Cytochrome c oxidase is specific intra-mitochondrial site of age-related deterioration. -- Abstract: The hypothesis, that structural deterioration of cytochrome c oxidase (CcO) is a causal factor in the age-related decline in mitochondrial respiratory activity and an increase in H 2 O 2 generation, was tested in Drosophila melanogaster. CcO activity and the levels of seven different nuclear DNA-encoded CcO subunits were determined at three different stages of adult life, namely, young-, middle-, and old-age. CcO activity declined progressively with age by 33%. Western blot analysis, using antibodies specific to Drosophila CcO subunits IV, Va, Vb, VIb, VIc, VIIc, and VIII, indicated that the abundance these polypeptides decreased, ranging from 11% to 40%, during aging. These and previous results suggest that CcO is a specific intra-mitochondrial site of age-related deterioration, which may have a broad impact on mitochondrial physiology.

  19. The role of double covalent flavin binding in chito-oligosaccharide oxidase from Fusarium graminearum

    NARCIS (Netherlands)

    Heuts, Dominic P. H. M.; Winter, Remko T.; Damsma, Gerke E.; Janssen, Dick B.; Fraaije, Marco W.

    2008-01-01

    ChitO (chito-oligosaccharide oxidase) from Fusarium graminearum catalyses the regioselective oxidation of N-acetylated oligosaccharides. The enzyme harbours an FAD cofactor that is covalently attached to His(94) and Cys(154). The functional role of this unusual bi-covalent flavin-protein linkage was

  20. Are colorimetric assays appropriate for measuring phenol oxidase activity in peat soils?

    Science.gov (United States)

    Magdalena M. Wiedermann; Evan S. Kane; Timothy J. Veverica; Erik A. Lilleskov

    2017-01-01

    The activity of extracellular phenol oxidases is believed to play a critical role in decomposition processes in peatlands. The water logged, acidic conditions, and recalcitrant litter from the peatland vegetation, lead to exceptionally high phenolics in the peat. In order to quantify the activity of oxidative enzymes involved in the modification and break down of...

  1. Peroxidasin-mediated crosslinking of collagen IV is independent of NADPH oxidases

    Directory of Open Access Journals (Sweden)

    Gábor Sirokmány

    2018-06-01

    Full Text Available Collagen IV is a major component of the basement membrane in epithelial tissues. The NC1 domains of collagen IV protomers are covalently linked together through sulfilimine bonds, the formation of which is catalyzed by peroxidasin. Although hydrogen peroxide is essential for this reaction, the exact source of the oxidant remains elusive. Members of the NOX/DUOX NADPH oxidase family are specifically devoted to the production of superoxide and hydrogen peroxide. Our aim in this study was to find out if NADPH oxidases contribute in vivo to the formation of collagen IV sulfilimine crosslinks. We used multiple genetically modified in vivo model systems to provide a detailed assessment of this question. Our data indicate that in various peroxidasin-expressing tissues sulfilimine crosslinks between the NC1 domains of collagen IV can be readily detected in the absence of functioning NADPH oxidases. We also analyzed how subatmospheric oxygen levels influence the collagen IV network in collagen-producing cultured cells with rapid matrix turnover. We showed that collagen IV crosslinks remain intact even under strongly hypoxic conditions. Our hypothesis is that during collagen IV network formation PXDN cooperates with a NOX/DUOX-independent H2O2 source that is functional also at very low ambient oxygen levels. Keywords: Peroxidasin, NADPH oxidase, Hydrogen peroxide, Collagen IV, Sulfilimine

  2. Pharmacological inhibition of NADPH oxidase protects against cisplatin induced nephrotoxicity in mice by two step mechanism.

    Science.gov (United States)

    Wang, Yimin; Luo, Xiao; Pan, Hao; Huang, Wei; Wang, Xueping; Wen, Huali; Shen, Kezhen; Jin, Baiye

    2015-09-01

    Cisplatin induced nephrotoxicity is primarily caused by ROS (Reactive Oxygen Species) induced proximal tubular cell death. NADPH oxidase is major source of ROS production by cisplatin. Here, we reported that pharmacological inhibition of NADPH oxidase by acetovanillone (obtained from medicinal herb Picrorhiza kurroa) led to reduced cisplatin nephrotoxicity in mice. In this study we used various molecular biology and biochemistry methods a clinically relevant model of nephropathy, induced by an important chemotherapeutic drug cisplatin. Cisplatin-induced nephrotoxicity was evident by histological damage from loss of the tubular structure. The damage was also marked by the increase in blood urea nitrogen, creatinine, protein nitration as well as cell death markers such as caspase 3/7 activity and DNA fragmentation. Tubular cell death by cisplatin led to pro-inflammatory response by production of TNFα and IL1β followed by leukocyte/neutrophil infiltration which resulted in new wave of ROS involving more NADPH oxidases. Cisplatin-induced markers of kidney damage such as oxidative stress, cell death, inflammatory cytokine production and nephrotoxicity were attenuated by acetovanillone. In addition to that, acetovanillone enhanced cancer cell killing efficacy of cisplatin. Thus, pharmacological inhibition of NADPH oxidase can be protective for cisplatin-induced nephrotoxicity in mice. Copyright © 2015. Published by Elsevier Ltd.

  3. The N-terminus of amine oxidase of Hansenula polymorpha contains a peroxisomal targeting signal

    NARCIS (Netherlands)

    Faber, Klaas Nico; Keizer-Gunnink, Ineke; Pluim, Dick; Harder, Willem; AB, Geert; Veenhuis, Marten

    1995-01-01

    Here we describe the identification of the targeting sequence of peroxisomal amine oxidase (AMO) of H. polymorpha. Deletion analysis revealed that essential targeting information is located within the extreme N-terminal 16 amino acids. Moreover, this sequence can direct a reporter protein to the

  4. The microglial NADPH oxidase complex as a source of oxidative stress in Alzheimer's disease

    Directory of Open Access Journals (Sweden)

    Landreth Gary E

    2006-11-01

    Full Text Available Abstract Alzheimer's disease is the most common cause of dementia in the elderly, and manifests as progressive cognitive decline and profound neuronal loss. The principal neuropathological hallmarks of Alzheimer's disease are the senile plaques and the neurofibrillary tangles. The senile plaques are surrounded by activated microglia, which are largely responsible for the proinflammatory environment within the diseased brain. Microglia are the resident innate immune cells in the brain. In response to contact with fibrillar beta-amyloid, microglia secrete a diverse array of proinflammatory molecules. Evidence suggests that oxidative stress emanating from activated microglia contribute to the neuronal loss characteristic of this disease. The source of fibrillar beta-amyloid induced reactive oxygen species is primarily the microglial nicotinamide adenine dinucleotide phosphate (NADPH oxidase. The NADPH oxidase is a multicomponent enzyme complex that, upon activation, produces the highly reactive free radical superoxide. The cascade of intracellular signaling events leading to NADPH oxidase assembly and the subsequent release of superoxide in fibrillar beta-amyloid stimulated microglia has recently been elucidated. The induction of reactive oxygen species, as well as nitric oxide, from activated microglia can enhance the production of more potent free radicals such as peroxynitrite. The formation of peroxynitrite causes protein oxidation, lipid peroxidation and DNA damage, which ultimately lead to neuronal cell death. The elimination of beta-amyloid-induced oxidative damage through the inhibition of the NADPH oxidase represents an attractive therapeutic target for the treatment of Alzheimer's disease.

  5. Electron transfer reactivity of the Arabidopsis thaliana sulfhydryl oxidase AtErv1

    DEFF Research Database (Denmark)

    Farver, Ole; Vitu, Elvira; Wherland, Scot

    2009-01-01

    The redox reactivity of the three disulfide bridges and the flavin present in each protomer of the wild-type Arabidopsis thaliana mitochondrial sulfhydryl oxidase (AtErv1) homodimer has been investigated. Pulse radiolytically produced CO2- radical ions were found to reduce the disulfide bridges...

  6. Copper radical oxidases and related extracellular oxidoreductases of wood-decay Agaricomycetes

    Science.gov (United States)

    Phil Kersten; Dan Cullen

    2014-01-01

    Extracellular peroxide generation, a key component of oxidative lignocellulose degradation, has been attributed to various enzymes including the copper radical oxidases. Encoded by a family of structurally related sequences, the genes are widely distributed among wood decay fungi including three recently completed polypore genomes. In all cases, core catalytic residues...

  7. Asp170 is crucial for the redox properties of vanillyl-alcohol oxidase

    NARCIS (Netherlands)

    Heuvel, van den R.H.H.; Fraaije, M.W.; Mattevi, A.; Berkel, van W.J.H.

    2000-01-01

    Vanillyl-alcohol oxidase is a flavoprotein containing a covalent flavin that catalyzes the oxidation of 4-(methoxymethyl)phenol to 4-hydroxybenzaldehyde. The reaction proceeds through the formation of a p-quinone methide intermediate, after which, water addition takes place. Asp-170, located near

  8. Asp-170 Is Crucial for the Redox Properties of Vanillyl-alcohol Oxidase

    NARCIS (Netherlands)

    Heuvel, Robert H.H. van den; Fraaije, Marco W.; Mattevi, Andrea; Berkel, Willem J.H. van

    2000-01-01

    Vanillyl-alcohol oxidase is a flavoprotein containing a covalent flavin that catalyzes the oxidation of 4-(methoxymethyl)phenol to 4-hydroxybenzaldehyde. The reaction proceeds through the formation of a p-quinone methide intermediate, after which, water addition takes place. Asp-170, located near

  9. Direction of the reactivity of vanillyl-alcohol oxidase with 4-alkylphenols

    NARCIS (Netherlands)

    van den Heuvel, RHH; Fraaije, MW; van Berkel, WJH; Heuvel, Robert H.H. van den; Berkel, Willem J.H. van

    2000-01-01

    The covalent flavoprotein vanillyl-alcohol oxidase (VAO) predominantly converts short-chain 4-alkylphenols, like 4-ethylphenol, to (R)-1-(4'-hydroxyphenyl)alcohols and medium-chain 4-alkylphenols, like 4-butylphenol, to 1-(4'-hydroxyphenyl)alkenes. Crystallographic studies have indicated that the

  10. Enzymatic halogenation and oxidation using an alcohol oxidase-vanadium chloroperoxidase cascade

    NARCIS (Netherlands)

    But, Andrada; Noord, Van Aster; Poletto, Francesca; Sanders, Johan P.M.; Franssen, Maurice C.R.; Scott, Elinor L.

    2017-01-01

    The chemo-enzymatic cascade which combines alcohol oxidase from Hansenula polymorpha (AOXHp) with vanadium chloroperoxidase (VCPO), for the production of biobased nitriles from amino acids was investigated. In the first reaction H2O2 (and acetaldehyde) are generated from ethanol and oxygen by AOXHp.

  11. Effect of high pressure on peanut allergens in the presence of polyphenol oxidase and caffeic acid

    Science.gov (United States)

    High pressure (HP) enhances enzymatic reactions. Because polyphenol oxidase (PPO) is an enzyme, and reduces IgE binding of peanut allergens in presence of caffeic acid (CA), we postulated that a further reduction in IgE binding can be achieved, using HP together with PPO and CA. Peanut extracts cont...

  12. Mechanism-Based Inhibitors of Cytokinin Oxidase/Dehydrogenase Attack FAD Cofactor

    Czech Academy of Sciences Publication Activity Database

    Kopečný, D.; Šebela, M.; Briozzo, P.; Spíchal, Lukáš; Houba-Hérin, N.; Mašek, V.; Joly, N.; Madzak, C.; Anzenbacher, P.; Laloue, M.

    2008-01-01

    Roč. 380, č. 5 (2008), s. 886-899 ISSN 0022-2836 R&D Projects: GA ČR(CZ) GP522/08/P113 Institutional research plan: CEZ:AV0Z50380511 Keywords : cytokinin oxidase/dehydrogenase * cytokinin signaling * protein structure Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.146, year: 2008

  13. The role of xanthine oxidase in ischemia/reperfusion damage of rat liver

    NARCIS (Netherlands)

    Frederiks, W. M.; Bosch, K. S.

    1995-01-01

    Oxygen radicals have been proposed to be involved in the induction of liver cell damage during reperfusion after ischemia. The role of xanthine oxidase in this process and the potential of the antioxidant system have been studied in a model of in vivo ischemia of rat liver followed by 1 h

  14. Evaluation of the Expression of Amine Oxidase Proteins in Breast Cancer

    Directory of Open Access Journals (Sweden)

    Woo Young Sun

    2017-12-01

    Full Text Available We aimed to evaluate the expression of amine oxidase proteins in breast cancer and their clinical implications. We performed immunohistochemical staining of amine oxidase proteins (LOX, lysyl oxidase, AOC3, amine oxidase, MAOA, monoamine oxidase A, MAOB, monoamine oxidase B. Based on their hormone receptors, such as estrogen receptor (ER and progesterone receptor (PR, human epidermal growth factor receptor 2 (HER-2, and Ki-67 immunohistochemical staining, breast cancer was divided into four molecular subtypes: luminal A, luminal B, HER-2 type, and triple-negative breast cancer (TNBC. Luminal A was observed in 380 cases (49.4%, luminal B in 224 (29.1%, HER-2 type in 68 (8.8%, and TNBC in 98 (12.7%. Stromal AOC3, MAO-A, and MAO-B expression varied according to molecular subtypes. Stromal AOC3 expression was high in luminal B and HER-2 type and MAO-A expression was high in luminal A and luminal B (p < 0.001. MAO-B expression was higher in TNBC than in other subtypes (p = 0.020. LOX positivity was associated with high histological grade (p < 0.001 and high Ki-67 labeling index (LI (p = 0.009, and stromal AOC3 positivity was associated with high histological grade (p = 0.001, high Ki-67 LI (p < 0.001, and HER-2 positivity (p = 0.002. MAO-A positivity was related to low histological grade (p < 0.001, ER positivity, PR positivity (p < 0.001, and low Ki-67 LI (p < 0.001. In univariate analysis, MAO-A positivity was related to short disease-free survival in HER-2 type (p = 0.013, AOC3 negativity was related to short disease-free survival and overall survival in ER-positive breast cancer, PR-positive breast cancer, HER-2-negative breast cancer, and lymph node metastasis. In conclusion, the expression of amine oxidase proteins varies depending on the molecular subtype of breast cancer. Stromal AOC3 expression was high in luminal B and HER-2 type, and MAO-A expression was high in luminal A and luminal B.

  15. Glycosylation site-targeted PEGylation of glucose oxidase retains native enzymatic activity.

    Science.gov (United States)

    Ritter, Dustin W; Roberts, Jason R; McShane, Michael J

    2013-04-10

    Targeted PEGylation of glucose oxidase at its glycosylation sites was investigated to determine the effect on enzymatic activity, as well as the bioconjugate's potential in an optical biosensing assay. Methoxy-poly(ethylene glycol)-hydrazide (4.5kDa) was covalently coupled to periodate-oxidized glycosylation sites of glucose oxidase from Aspergillus niger. The bioconjugate was characterized using gel electrophoresis, liquid chromatography, mass spectrometry, and dynamic light scattering. Gel electrophoresis data showed that the PEGylation protocol resulted in a drastic increase (ca. 100kDa) in the apparent molecular mass of the protein subunit, with complete conversion to the bioconjugate; liquid chromatography data corroborated this large increase in molecular size. Mass spectrometry data proved that the extent of PEGylation was six poly(ethylene glycol) chains per glucose oxidase dimer. Dynamic light scattering data indicated the absence of higher-order oligomers in the PEGylated GOx sample. To assess stability, enzymatic activity assays were performed in triplicate at multiple time points over the course of 29 days in the absence of glucose, as well as before and after exposure to 5% w/v glucose for 24h. At a confidence level of 95%, the bioconjugate's performance was statistically equivalent to native glucose oxidase in terms of activity retention over the 29 day time period, as well as following the 24h glucose exposure. Finally, the bioconjugate was entrapped within a poly(2-hydroxyethyl methacrylate) hydrogel containing an oxygen-sensitive phosphor, and the construct was shown to respond approximately linearly with a 220±73% signal change (n=4, 95% confidence interval) over the physiologically-relevant glucose range (i.e., 0-400mg/dL); to our knowledge, this represents the first demonstration of PEGylated glucose oxidase incorporated into an optical biosensing assay. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. Production of recombinant cholesterol oxidase containing covalently bound FAD in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Molla Gianluca

    2010-04-01

    Full Text Available Abstract Background Cholesterol oxidase is an alcohol dehydrogenase/oxidase flavoprotein that catalyzes the dehydrogenation of C(3-OH of cholesterol. It has two major biotechnological applications, i.e. in the determination of serum (and food cholesterol levels and as biocatalyst providing valuable intermediates for industrial steroid drug production. Cholesterol oxidases of type I are those containing the FAD cofactor tightly but not covalently bound to the protein moiety, whereas type II members contain covalently bound FAD. This is the first report on the over-expression in Escherichia coli of type II cholesterol oxidase from Brevibacterium sterolicum (BCO. Results Design of the plasmid construct encoding the mature BCO, optimization of medium composition and identification of the best cultivation/induction conditions for growing and expressing the active protein in recombinant E. coli cells, concurred to achieve a valuable improvement: BCO volumetric productivity was increased from ~500 up to ~25000 U/L and its crude extract specific activity from 0.5 up to 7.0 U/mg protein. Interestingly, under optimal expression conditions, nearly 55% of the soluble recombinant BCO is produced as covalently FAD bound form, whereas the protein containing non-covalently bound FAD is preferentially accumulated in insoluble inclusion bodies. Conclusions Comparison of our results with those published on non-covalent (type I COs expressed in recombinant form (either in E. coli or Streptomyces spp., shows that the fully active type II BCO can be produced in E. coli at valuable expression levels. The improved over-production of the FAD-bound cholesterol oxidase will support its development as a novel biotool to be exploited in biotechnological applications.

  17. Lower Growth Temperature Increases Alternative Pathway Capacity and Alternative Oxidase Protein in Tobacco 1

    Science.gov (United States)

    Vanlerberghe, Greg C.; McIntosh, Lee

    1992-01-01

    Suspension cells of NT1 tobacco (Nicotiana tabacum L. cv bright yellow) have been used to study the effect of growth temperature on the CN-resistant, salicylhydroxamic acid-sensitive alternative pathway of respiration. Mitochondria isolated from cells maintained at 30°C had a low capacity to oxidize succinate via the alternative pathway, whereas mitochondria isolated from cells 24 h after transfer to 18°C displayed, on average, a 5-fold increase in this capacity (from 7 to 32 nanoatoms oxygen per milligram protein per minute). This represented an increase in alternative pathway capacity from 18 to 45% of the total capacity of electron transport. This increased capacity was lost upon transfer of cells back to 30°C. A monoclonal antibody to the terminal oxidase of the alternative pathway (the alternative oxidase) from Sauromatum guttatum (T.E. Elthon, R.L. Nickels, L. McIntosh [1989] Plant Physiology 89: 1311-1317) recognized a 35-kilodalton mitochondrial protein in tobacco. There was an excellent correlation between the capacity of the alternative path in isolated tobacco mitochondria and the levels of this 35-kilodalton alternative oxidase protein. Cycloheximide could inhibit both the increased level of the 35-kilodalton alternative oxidase protein and the increased alternative pathway capacity normally seen upon transfer to 18°C. We conclude that transfer of tobacco cells to the lower temperature increases the capacity of the alternative pathway due, at least in part, to de novo synthesis of the 35-kilodalton alternative oxidase protein. Images Figure 3 Figure 4 PMID:16652932

  18. Interrupted reperfusion reduces the activation of NADPH oxidase after cerebral I/R injury.

    Science.gov (United States)

    Shen, Jia; Bai, Xiao-Yin; Qin, Yuan; Jin, Wei-Wei; Zhou, Jing-Yin; Zhou, Ji-Ping; Yan, Ying-Gang; Wang, Qiong; Bruce, Iain C; Chen, Jiang-Hua; Xia, Qiang

    2011-06-15

    Interrupted reperfusion reduces ischemia/reperfusion (I/R) injury. This study was designed to determine whether NADPH oxidase participates in the neural protection against global I/R injury after interrupted reperfusion. Mice were randomly divided into five groups: sham (sham-operated), I/R (20-min global I/R), RR (I/R+interrupted reperfusion), Apo (I/R+apocynin administration), and RR+Apo. Behavioral tests (pole test, beam walking, and Morris water maze) and Nissl staining were undertaken in all five groups; superoxide levels, expression of gp91(phox) and p47(phox), p47(phox) translocation, and Rac1 activation were measured in the sham, I/R, and RR groups. The motor coordination, bradykinesia, and spatial learning and memory, as well as the neuron survival rates, were better in the RR, Apo, and RR+Apo groups than in the I/R group. The NADPH oxidase-dependent superoxide levels, p47(phox) and gp91(phox) expression, p47(phox) translocation, and Rac1 activation were lower in the RR group than in the I/R group. In conclusion, the neural protective effect of interrupted reperfusion is at least partly mediated by decreasing the expression and assembly of NADPH oxidase and the levels of NADPH oxidase-derived superoxide. The most striking reduction Rac1-GTP in the RR group suggests that interrupted reperfusion also acts on the activation of assembled NADPH oxidase by reducing the availability of Rac1-GTP. Copyright © 2011 Elsevier Inc. All rights reserved.

  19. Critical role of NADPH oxidase in neuronal oxidative damage and microglia activation following traumatic brain injury.

    Directory of Open Access Journals (Sweden)

    Quan-Guang Zhang

    Full Text Available BACKGROUND: Oxidative stress is known to play an important role in the pathology of traumatic brain injury. Mitochondria are thought to be the major source of the damaging reactive oxygen species (ROS following TBI. However, recent work has revealed that the membrane, via the enzyme NADPH oxidase can also generate the superoxide radical (O(2(-, and thereby potentially contribute to the oxidative stress following TBI. The current study thus addressed the potential role of NADPH oxidase in TBI. METHODOLOGY/PRINCIPAL FINDINGS: The results revealed that NADPH oxidase activity in the cerebral cortex and hippocampal CA1 region increases rapidly following controlled cortical impact in male mice, with an early peak at 1 h, followed by a secondary peak from 24-96 h after TBI. In situ localization using oxidized hydroethidine and the neuronal marker, NeuN, revealed that the O(2(- induction occurred in neurons at 1 h after TBI. Pre- or post-treatment with the NADPH oxidase inhibitor, apocynin markedly inhibited microglial activation and oxidative stress damage. Apocynin also attenuated TBI-induction of the Alzheimer's disease proteins β-amyloid and amyloid precursor protein. Finally, both pre- and post-treatment of apocynin was also shown to induce significant neuroprotection against TBI. In addition, a NOX2-specific inhibitor, gp91ds-tat was also shown to exert neuroprotection against TBI. CONCLUSIONS/SIGNIFICANCE: As a whole, the study demonstrates that NADPH oxidase activity and superoxide production exhibit a biphasic elevation in the hippocampus and cortex following TBI, which contributes significantly to the pathology of TBI via mediation of oxidative stress damage, microglial activation, and AD protein induction in the brain following TBI.

  20. Extracellular oxidases and the transformation of solubilised low-rank coal by wood-rot fungi

    Energy Technology Data Exchange (ETDEWEB)

    Ralph, J.P. [Flinders Univ. of South Australia, Bedford Park (Australia). School of Biological Sciences; Graham, L.A. [Flinders Univ. of South Australia, Bedford Park (Australia). School of Biological Sciences; Catcheside, D.E.A. [Flinders Univ. of South Australia, Bedford Park (Australia). School of Biological Sciences

    1996-12-31

    The involvement of extracellular oxidases in biotransformation of low-rank coal was assessed by correlating the ability of nine white-rot and brown-rot fungi to alter macromolecular material in alkali-solubilised brown coal with the spectrum of oxidases they produce when grown on low-nitrogen medium. The coal fraction used was that soluble at 3.0{<=}pH{<=}6.0 (SWC6 coal). In 15-ml cultures, Gloeophyllum trabeum, Lentinus lepideus and Trametes versicolor produced little or no lignin peroxidase, manganese (Mn) peroxidase or laccase activity and caused no change to SWC6 coal. Ganoderma applanatum and Pycnoporus cinnabarinus also produced no detectable lignin or Mn peroxidases or laccase yet increased the absorbance at 400 nm of SWC6 coal. G. applanatum, which produced veratryl alcohol oxidase, also increased the modal apparent molecular mass. SWC6 coal exposed to Merulius tremellosus and Perenniporia tephropora, which secreted Mn peroxidases and laccase and Phanerochaete chrysosporium, which produced Mn and lignin peroxidases was polymerised but had unchanged or decreased absorbance. In the case of both P. chrysosporium and M. tremellosus, polymerisation of SWC6 coal was most extensive, leading to the formation of a complex insoluble in 100 mM NaOH. Rigidoporus ulmarius, which produced only laccase, both polymerised and reduced the A{sub 400} of SWC6 coal. P. chrysosporium, M. tremellosus and P. tephropora grown in 10-ml cultures produced a spectrum of oxidases similar to that in 15-ml cultures but, in each case, caused more extensive loss of A{sub 400}, and P. chrysosporium depolymerised SWC6 coal. It is concluded that the extracellular oxidases of white-rot fungi can transform low-rank coal macromolecules and that increased oxygen availability in the shallower 10-ml cultures favours catabolism over polymerisation. (orig.)

  1. Mediation of glycosylated and partially-deglycosylated glucose oxidase of Aspergillus niger by a ferrocene-derivatised detergent.

    Science.gov (United States)

    Fraser, D M; Zakeeruddin, S M; Grätzel, M

    1992-01-30

    A ferrocene-derivatised detergent, (11-ferrocenylundecyl) trimethylammonium bromide (FTMAB), when oxidised to the corresponding ferricinium ion, was found by electrochemical studies to be an effective electron acceptor for reduced glucose oxidase of Aspergillus niger (EC 1.13.4) and thus acts as a electron-transfer mediator between glucose oxidase and a working electrode held at a potential sufficiently positive to reoxidise reduced FTMAB. An increase in mediating activity was produced when FTMAB was present in concentrations above its critical micelle concentration. An 'enzyme electrode' was formed by adsorption of glucose oxidase and FTMAB surfactant on a graphite rod. The electrode functioned as an amperometric biosensor for glucose in phosphate-buffered saline solution. A mixed micelle of glucose oxidase and FTMAB, probably adsorbed on the electrode surface, appears to be advantageous for the amperometric determination of glucose. Additionally, glucose oxidase was treated with alpha-mannosidase. When this partially-deglycosylated glucose oxidase was incorporated in an enzyme electrode, a 100-fold increase in the second-order rate constant (k) for electron transfer between the enzyme and FTMAB was observed, together with increased current densities, with respect to the equivalent values for FTMAB and commercial glucose oxidase. The use of deglycosylated enzymes in biosensors is suggested.

  2. Oxoferryl-Porphyrin Radical Catalytic Intermediate in Cytochrome bd Oxidases Protects Cells from Formation of Reactive Oxygen Species*

    Science.gov (United States)

    Paulus, Angela; Rossius, Sebastiaan Gijsbertus Hendrik; Dijk, Madelon; de Vries, Simon

    2012-01-01

    The quinol-linked cytochrome bd oxidases are terminal oxidases in respiration. These oxidases harbor a low spin heme b558 that donates electrons to a binuclear heme b595/heme d center. The reaction with O2 and subsequent catalytic steps of the Escherichia coli cytochrome bd-I oxidase were investigated by means of ultra-fast freeze-quench trapping followed by EPR and UV-visible spectroscopy. After the initial binding of O2, the O–O bond is heterolytically cleaved to yield a kinetically competent heme d oxoferryl porphyrin π-cation radical intermediate (compound I) magnetically interacting with heme b595. Compound I accumulates to 0.75–0.85 per enzyme in agreement with its much higher rate of formation (∼20,000 s−1) compared with its rate of decay (∼1,900 s−1). Compound I is next converted to a short lived heme d oxoferryl intermediate (compound II) in a phase kinetically matched to the oxidation of heme b558 before completion of the reaction. The results indicate that cytochrome bd oxidases like the heme-copper oxidases break the O–O bond in a single four-electron transfer without a peroxide intermediate. However, in cytochrome bd oxidases, the fourth electron is donated by the porphyrin moiety rather than by a nearby amino acid. The production of reactive oxygen species by the cytochrome bd oxidase was below the detection level of 1 per 1000 turnovers. We propose that the two classes of terminal oxidases have mechanistically converged to enzymes in which the O–O bond is broken in a single four-electron transfer reaction to safeguard the cell from the formation of reactive oxygen species. PMID:22287551

  3. Paraquat and maneb co-exposure induces noradrenergic locus coeruleus neurodegeneration through NADPH oxidase-mediated microglial activation

    International Nuclear Information System (INIS)

    Hou, Liyan; Zhang, Cong; Wang, Ke; Liu, Xiaofang; Wang, Hongwei; Che, Yuning; Sun, Fuqiang; Zhou, Xueying; Zhao, Xiulan; Wang, Qingshan

    2017-01-01

    Highlights: • Microglial activation induced by paraquat and maneb precedes noradrenergic neurodegeneration in locus coeruleus. • NADPH oxidase activation contributes to microglia-mediated neuroinflammation and related noradrenergic neurodegeneration. • Inhibition of NADPH oxidase by apocynin protects noradrenergic neurons against paraquat and maneb-induced toxicity. - Abstract: Co-exposure to paraquat (PQ) and maneb (Mb) has been shown to increase the risk of Parkinson’s disease (PD) and dopaminergic (DA) neurodegeneration in the substantia nigra pars compacta (SNpc) is observed in PQ and Mb-treated experimental animals. The loss of noradrenergic locus coeruleus (LC/NE) neurons in brainstem is a common feature shared by multiple neurodegenerative diseases, including PD. However, whether PQ and Mb is able to damage LC/NE neurons remains undefined. In this study, mice treated with combined PQ and Mb displayed progressive LC/NE neurodegeneration. Time course studies revealed that the activation of microglia preceded LC/NE neurodegeneration. Mechanistically, the activation of NADPH oxidase contributed to microglial activation and subsequent LC/NE neurodegeneration. We found that PQ and Mb co-exposure induced activation of NADPH oxidase as shown by increased superoxide production and membrane translocation of p47 phox , a cytosolic subunit of NADPH oxidase. Inhibition of NADPH oxidase by apocynin, a widely used NADPH oxidase inhibitor, suppressed microglial activation and gene expressions of proinflammatory factors. Furthermore, reduced activation of nuclear factor-κB (NF-κB) pathway was observed in apocynin-treated mice. More importantly, inhibition of NADPH oxidase by apocynin afforded LC/NE neuroprotection against PQ and Mb-induced neurotoxicity. Thus, our findings revealed the critical role NADPH oxidase-mediated microglial activation in driving LC/NE neurodegeneration induced by PQ and Mb, providing new insights into the pathogenesis of environmental

  4. The Role of Aldehyde Oxidase and Xanthine Oxidase in the Biotransformation of a Novel Negative Allosteric Modulator of Metabotropic Glutamate Receptor Subtype 5

    Science.gov (United States)

    Morrison, Ryan D.; Blobaum, Anna L.; Byers, Frank W.; Santomango, Tammy S.; Bridges, Thomas M.; Stec, Donald; Brewer, Katrina A.; Sanchez-Ponce, Raymundo; Corlew, Melany M.; Rush, Roger; Felts, Andrew S.; Manka, Jason; Bates, Brittney S.; Venable, Daryl F.; Rodriguez, Alice L.; Jones, Carrie K.; Niswender, Colleen M.; Conn, P. Jeffrey; Lindsley, Craig W.; Emmitte, Kyle A.

    2012-01-01

    Negative allosteric modulation (NAM) of metabotropic glutamate receptor subtype 5 (mGlu5) represents a therapeutic strategy for the treatment of childhood developmental disorders, such as fragile X syndrome and autism. VU0409106 emerged as a lead compound within a biaryl ether series, displaying potent and selective inhibition of mGlu5. Despite its high clearance and short half-life, VU0409106 demonstrated efficacy in rodent models of anxiety after extravascular administration. However, lack of a consistent correlation in rat between in vitro hepatic clearance and in vivo plasma clearance for the biaryl ether series prompted an investigation into the biotransformation of VU0409106 using hepatic subcellular fractions. An in vitro appraisal in rat, monkey, and human liver S9 fractions indicated that the principal pathway was NADPH-independent oxidation to metabolite M1 (+16 Da). Both raloxifene (aldehyde oxidase inhibitor) and allopurinol (xanthine oxidase inhibitor) attenuated the formation of M1, thus implicating the contribution of both molybdenum hydroxylases in the biotransformation of VU0409106. The use of 18O-labeled water in the S9 experiments confirmed the hydroxylase mechanism proposed, because 18O was incorporated into M1 (+18 Da) as well as in a secondary metabolite (M2; +36 Da), the formation of which was exclusively xanthine oxidase-mediated. This unusual dual and sequential hydroxylase metabolism was confirmed in liver S9 and hepatocytes of multiple species and correlated with in vivo data because M1 and M2 were the principal metabolites detected in rats administered VU0409106. An in vitro-in vivo correlation of predicted hepatic and plasma clearance was subsequently established for VU0409106 in rats and nonhuman primates. PMID:22711749

  5. Superoxide production and expression of NAD(P)H oxidases by transformed and primary human colonic epithelial cells

    DEFF Research Database (Denmark)

    Perner, A; Andresen, Lars; Pedersen, G

    2003-01-01

    Superoxide (O(2)(-)) generation through the activity of reduced nicotinamide dinucleotide (NADH) or reduced nicotinamide dinucleotide phosphate (NADPH) oxidases has been demonstrated in a variety of cell types, but not in human colonic epithelial cells....

  6. Direct electrochemistry and electrocatalysis of a glucose oxidase-functionalized bioconjugate as a trace label for ultrasensitive detection of thrombin.

    Science.gov (United States)

    Bai, Lijuan; Yuan, Ruo; Chai, Yaqin; Yuan, Yali; Wang, Yan; Xie, Shunbi

    2012-11-18

    For the first time, a glucose oxidase-functionalized bioconjugate was prepared and served as a new trace label through its direct electrochemistry and electrocatalysis in a sandwich-type electrochemical aptasensor for ultrasensitive detection of thrombin.

  7. Carbon fiber microelectrodes modified with carbon nanotubes as a new support for immobilization of glucose oxidase

    International Nuclear Information System (INIS)

    Wen, H.; Nallathambi, V.; Chakraborty, D.; Barton, S.C.

    2011-01-01

    Carboxylated carbon nanotubes were coated onto carbon microfiber electrodes to create a micron-scale bioelectrode. This material has a high surface area and can serve as a support for immobilization of enzymes such as glucose oxidase. A typical carbon nanotube loading of 13 μg cm -1 yields a coating thickness of 17 μm and a 2000-fold increase in surface capacitance. The modified electrode was further coated with a biocatalytic hydrogel composed of a conductive redox polymer, glucose oxidase, and a crosslinker to create a glucose bioelectrode. The current density on oxidation of glucose is 16.6 mA cm-2 at 0.5 V (vs. Ag/AgCl) in oxygen-free glucose solution. We consider this approach to be useful for designing and characterizing surface treatments for carbon mats and papers by mimicking their local microenvironment. (author)

  8. Gold Nanoparticles Like A Matrix For Covalent Immobilization Of Cholesterol Oxidase – Application For Biosensing

    Directory of Open Access Journals (Sweden)

    Wojnarowska R.

    2015-09-01

    Full Text Available Gold nanoparticles are emerging as promising agents for various areas of material science as well as nanotechnology, electronics and medicine. The interest in this material is provided due to its unique optical, electronic and molecular-recognition properties. This paper presents results of preparation, characterization and biofunctionalization of gold nanoparticles. Nanoparticles have been conjugated with the cholesterol oxidase enzyme in order to prepare the active element for biosensors. Cholesterol oxidase is one of the most important analytical enzyme, used for cholesterol assay in clinical diagnostics, and there is still a necessity in improvement of existing analytical techniques, including bio-nanotechnological approaches based on modern nanosystems. The prepared bio-nanosystem was characterized by the enzyme activity test. Obtained results showed a stable binding of the enzyme with nanoparticles and preserved the bioactivity approves which gives possibility to use the prepared bio-nanosystems for analytical purposes.

  9. Efficacy, safety, and patient preference of monoamine oxidase B inhibitors in the treatment of Parkinson's disease

    Directory of Open Access Journals (Sweden)

    Bradley J Robottom

    2011-01-01

    Full Text Available Bradley J RobottomDepartment of Neurology, University of Maryland School of Medicine, Baltimore, MD, USAAbstract: Parkinson's disease (PD is the second most common neurodegenerative disease and the most treatable. Treatment of PD is symptomatic and generally focuses on the replacement or augmentation of levodopa. A number of options are available for treatment, both in monotherapy of early PD and to treat complications of advanced PD. This review focuses on rasagiline and selegiline, two medications that belong to a class of antiparkinsonian drugs called monoamine oxidase B (MAO-B inhibitors. Topics covered in the review include mechanism of action, efficacy in early and advanced PD, effects on disability, the controversy regarding disease modification, safety, and patient preference for MAO-B inhibitors.Keywords: monoamine oxidase inhibitors, rasagiline, selegiline, Parkinson's disease, efficacy, safety

  10. Fabrication and optimisation of optical biosensor using alcohol oxidase enzyme to evaluate detection of formaldehyde

    Science.gov (United States)

    Rachim, A.; Sari, A. P.; Nurlely, Fauzia, V.

    2017-07-01

    In this study, a new and simple biosensor base on alcohol oxidase (AOX)-enzyme for detecting formaldehyde in aqueous solutions has been successfully fabricated. The alcohol oxidase (AOX) enzyme was immobilized on poly-n-butyl acrylic-co-N-acryloxysuccinimide (nBA-NAS) membrane containing chromoionophore. The chemical reaction between AOX and formaldehyde generates a colour change of chromoionophore detected by optical absorbance measured in UV Vis. This paper focuses on the concentration optimization of buffer phosphate solution, response time, the quantity of enzyme and the measurement of the detection range of biosensors. The result shows that the optimum concentration and pH of buffer phosphate solution is 0.05 M and pH 7, respectively. The optimum response time is 3 min, the optimum unit of enzyme for biosensor is 1 unit/sample and the detection range of biosensor is 0.264 mM with R2 = 0.9421.

  11. Lysyl oxidase: properties, specificity, and biological roles inside and outside of the cell.

    Science.gov (United States)

    Kagan, Herbert M; Li, Wande

    2003-03-01

    Lysyl oxidase (LO) plays a critical role in the formation and repair of the extracellular matrix (ECM) by oxidizing lysine residues in elastin and collagen, thereby initiating the formation of covalent crosslinkages which stabilize these fibrous proteins. Its catalytic activity depends upon both its copper cofactor and a unique carbonyl cofactor and has been shown to extend to a variety of basic globular proteins, including histone H1. Although the three-dimensional structure of LO has yet to be determined, the present treatise offers hypotheses based upon its primary sequence, which may underlie the prominent electrostatic component of its unusual substrate specificity as well as the catalysis-suppressing function of the propeptide domain of prolysyl oxidase. Recent studies have demonstrated that LO appears to function within the cell in a manner, which strongly modifies cellular activity. Newly discovered LO-like proteins also likely play unique roles in biology. Copyright 2002 Wiley-Liss, Inc.

  12. Ag-doped CdO nanocatalysts: Preparation, characterization and catechol oxidase activity

    Science.gov (United States)

    El-Kemary, Maged; El-Mehasseb, Ibrahim; El-Shamy, Hany

    2018-06-01

    Silver doped cadmium oxide (Ag/CdO) nanoparticles with an average size of 41 nm have been successfully synthesized via thermal decomposition and liquid impregnation technique. The structural characterization has been performed by using several spectroscopic techniques, e.g., X-ray diffraction (XRD), scanning electron microscopy (SEM) and fourier-transform infrared (FT-IR). The catechol oxidase has been studied by UV-visible absorption spectroscopy and fourier-transform infrared as well as the mechanism has been assured by cyclic voltammetry and fluorescence spectroscopy. The results indicate that the oxidation does not occur in the presence of unsupported cadmium oxide particles by silver and in the same time, the catechol oxidase activity of silver doped CdO nanoparticles were improved by about three orders of magnitude than silver ions.

  13. [Substrate-inhibitory analysis of monoamine oxidase from hepatopancreas of the octopus Bathypolypus arcticus].

    Science.gov (United States)

    Basova, I N; Iagodina, O V

    2012-01-01

    Study of the substrate-inhibitory specificity of mitochondrial monoamine oxidase (MAO) of hepatopancreas of the octopus Bathypolypus arcticus revealed distinctive peculiarities of catalytic properties of this enzyme. The studied enzyme, on one hand, like the classic MAO of homoiothermal animals, is able to deaminate tyramine, serotonin, benzylamine, tryptamine, beta-phenylethylamine, while, on the other hand, deaminates histamine and does not deaminate putrescine--classic substrates of diamine oxidase (DAO). Results of the substrate-inhibitory analysis with use of chlorgiline and deprenyl are indirect proofs of the existence in the octopus hepatopancreas of one molecular MAO form. Semicarbazide and pyronine G turned out to be weak irreversible inhibitors, four derivatives of acridine--irreversible inhibitors of the intermediate effectiveness with respect to the octopus hepatopancreas MAO; specificity of action of inhibitors at deamination of different substrates was equal.

  14. Immobilized glucose oxidase--catalase and their deactivation in a differential-bed loop reactor.

    Science.gov (United States)

    Prenosil, J E

    1979-01-01

    Glucose oxidase containing catalase was immobilized with a copolymer of phenylenediamine and glutaraldehyde on pumice and titania carrier to study the enzymatic oxidation of glucose in a differential-bed loop reactor. The reaction rate was found to be first order with respect to the concentration of limiting oxygen substrate, suggesting a strong external mass-transfer resistance for all the flow rates used. The partial pressure of oxygen was varied from 21.3 up to 202.6 kPa. The use of a differential-bed loop reactor for the determination of the active enzyme concentration in the catalyst with negligible internal pore diffusion resistance is shown. Catalyst deactivation was studied, especially with respect to the presence of catalase. It is believed that the hydrogen peroxide formed in the oxidation reaction deactivates catalase first; if an excess of catalase is present, the deactivation of glucose oxidase remains small. The mathematical model subsequently developed adequately describes the experimental results.

  15. Valencene oxidase CYP706M1 from Alaska cedar (Callitropsis nootkatensis).

    Science.gov (United States)

    Cankar, Katarina; van Houwelingen, Adèle; Goedbloed, Miriam; Renirie, Rokus; de Jong, René M; Bouwmeester, Harro; Bosch, Dirk; Sonke, Theo; Beekwilder, Jules

    2014-03-18

    (+)-Nootkatone is a natural sesquiterpene ketone used in grapefruit and citrus flavour compositions. It occurs in small amounts in grapefruit and is a major component of Alaska cedar (Callitropsis nootkatensis) heartwood essential oil. Upon co-expression of candidate cytochrome P450 enzymes from Alaska cedar in yeast with a valencene synthase, a C. nootkatensis valencene oxidase (CnVO) was identified to produce trans-nootkatol and (+)-nootkatone. Formation of (+)-nootkatone was detected at 144±10μg/L yeast culture. CnVO belongs to a new subfamily of the CYP706 family of cytochrome P450 oxidases. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  16. Urate oxidase for the prevention and treatment of tumour lysis syndrome in children with cancer.

    Science.gov (United States)

    Cheuk, Daniel Kl; Chiang, Alan Ks; Chan, Godfrey Cf; Ha, Shau Yin

    2017-03-08

    Tumour lysis syndrome (TLS) is a serious complication of malignancies and can result in renal failure or death. Previous reviews did not find clear evidence of benefit of urate oxidase in children with cancer. This review is the second update of a previously published Cochrane review. To assess the effects and safety of urate oxidase for the prevention and treatment of TLS in children with malignancies. In March 2016 we searched CENTRAL, MEDLINE, Embase, and CINAHL. In addition, we searched the reference lists of all identified relevant papers, trials registers and other databases. We also screened conference proceedings and we contacted experts in the field and the manufacturer of rasburicase, Sanofi-aventis. Randomised controlled trials (RCT) and controlled clinical trials (CCT) of urate oxidase for the prevention or treatment of TLS in children under 18 years with any malignancy. Two review authors independently extracted trial data and assessed individual trial quality. We used risk ratios (RR) for dichotomous data and mean difference (MD) for continuous data. We included seven trials, involving 471 participants in the treatment groups and 603 participants in the control groups. No new studies were identified in the update. One RCT and five CCTs compared urate oxidase and allopurinol. Three trials tested Uricozyme, and three trials tested rasburicase for the prevention of TLS.The RCT did not evaluate the primary outcome (incidence of clinical TLS). It showed no clear evidence of a difference in mortality (both all-cause mortality (Fisher's exact test P = 0.23) and mortality due to TLS (no deaths in either group)), renal failure (Fisher's exact test P = 0.46), and adverse effects between the treatment and the control groups (Fisher's exact test P = 1.0). The frequency of normalisation of uric acid at four hours (10 out of 10 participants in the treatment group versus zero out of nine participants in the control group, Fisher's exact test P oxidase (RR 9.10, 95

  17. Cytochrome oxidase subunit II gene in mitochondria of Oenothera has no intron

    Science.gov (United States)

    Hiesel, Rudolf; Brennicke, Axel

    1983-01-01

    The cytochrome oxidase subunit II gene has been localized in the mitochondrial genome of Oenothera berteriana and the nucleotide sequence has been determined. The coding sequence contains 777 bp and, unlike the corresponding gene in Zea mays, is not interrupted by an intron. No TGA codon is found within the open reading frame. The codon CGG, as in the maize gene, is used in place of tryptophan codons of corresponding genes in other organisms. At position 742 in the Oenothera sequence the TGG of maize is changed into a CGG codon, where Trp is conserved as the amino acid in other organisms. Homologous sequences occur more than once in the mitochondrial genome as several mitochondrial DNA species hybridize with DNA probes of the cytochrome oxidase subunit II gene. ImagesFig. 5. PMID:16453484

  18. Immobilization of the Enzyme Glucose Oxidase on Both Bulk and Porous SiO2 Surfaces

    Directory of Open Access Journals (Sweden)

    Fulvia Sinatra

    2008-09-01

    Full Text Available Silicon dioxide surfaces, both bulk and porous, were used to anchor the enzyme glucose oxidase. The immobilization protocol was optimized and the samples characterized using X-ray Photoelectron Spectroscopy, Energy Dispersive X-rays coupled to scanning electron microscopy and enzymatic activity measurements. We show that a uniform layer was obtained by activating the oxide before immobilization. X-ray Photoelectron Spectroscopy measurements carried out on bulk oxide showed that the silicon substrate signal was fully screened after the enzyme deposition showing the absence of uncovered surface regions. The enzyme presence was detected monitoring both the C 1s and N 1s signals. Finally, enzymatic activity measurements confirmed that the glucose oxidase activity was preserved after immobilization and maintained after three months of shelf life if the sample was properly stored. The importance of using porous silicon oxide to maximize the surface area was also evidenced.

  19. In vitro analysis of the role of glucose oxidase from Talaromyces flavus in biocontrol of the plant pathogen Verticillium dahliae.

    OpenAIRE

    Stosz, S K; Fravel, D R; Roberts, D P

    1996-01-01

    Culture filtrates from Talaromyces flavus grown on glucose contained high levels of glucose oxidase activity, while culture filtrates from T. flavus grown on xylan contained negligible glucose oxidase activity. Culture filtrates from T-flavus grown on both media contained complex protein profiles. However, only culture filtrates from T. flavus grown on glucose inhibited germination of microsclerotia of Verticillium dahliae in in vitro inhibition assays. A polyclonal antiserum preparation, pAB...

  20. Optimization of Glucose oxidase towards oxygen independency and high mediator activity for amperometric glucose determination in diabetes analytics

    OpenAIRE

    Arango Gutierrez, Erik Uwe

    2015-01-01

    Glucose oxidase is an oxidoreductase exhibiting a high β-D-glucose specificity and high stability which renders glucose oxidase well-suited for applications in diabetes care. Nevertheless, GOx activity is highly oxygen dependent which can lead to inaccuracies in amperometric β-D-glucose determinations. Therefore a directed evolution campaign with two rounds of random mutagenesis (SeSaM followed by epPCR), site saturation mutagenesis studies, and one simultaneous site saturation library (OmniC...

  1. Biocatalytic Properties and Structural Analysis of Eugenol Oxidase from Rhodococcus jostii RHA1: A Versatile Oxidative Biocatalyst

    OpenAIRE

    Nguyen, Quoc-Thai; de Gonzalo, Gonzalo; Binda, Claudia; Rioz-Martínez, Ana; Mattevi, Andrea; Fraaije, Marco W.

    2016-01-01

    Abstract Eugenol oxidase (EUGO) from Rhodococcus jostii RHA1 had previously been shown to convert only a limited set of phenolic compounds. In this study, we have explored the biocatalytic potential of this flavoprotein oxidase, resulting in a broadened substrate scope and a deeper insight into its structural properties. In addition to the oxidation of vanillyl alcohol and the hydroxylation of eugenol, EUGO can efficiently catalyze the dehydrogenation of various phenolic ketones and the selec...

  2. Size-selective QD@MOF core-shell nanocomposites for the highly sensitive monitoring of oxidase activities.

    Science.gov (United States)

    Wang, Ke; Li, Nan; Zhang, Jing; Zhang, Zhiqi; Dang, Fuquan

    2017-01-15

    In this work, we proposed a novel and facile method to monitor oxidase activities based on size-selective fluorescent quantum dot (QD)@metal-organic framework (MOF) core-shell nanocomposites (CSNCPs). The CSNCPs were synthesized from ZIF-8 and CdTe QDs in aqueous solution in 40min at room temperature with stirring. The prepared CdTe@ZIF-8 CSNCPs , which have excellent water dispersibility and stability, displays distinct fluorescence responses to hole scavengers of different molecular sizes (e.g., H 2 O 2 , substrate, and oxidase) due to the aperture limitation of the ZIF-8 shell. H 2 O 2 can efficiently quench the fluorescence of CdTe@ZIF-8 CSNCPs over a linearity range of 1-100nM with a detection limit of 0.29nM, whereas large molecules such as substrate and oxidase have very little effect on its fluorescence. Therefore, the highly sensitive detection of oxidase activities was achieved by monitoring the fluorescence quenching of CdTe@ZIF-8 CSNCPs by H 2 O 2 produced in the presence of substrate and oxidase, which is proportional to the oxidase activities. The linearity ranges of the uricase and glucose oxidase activity are 0.1-50U/L and 1-100U/L, respectively, and their detection limits are 0.024U/L and 0.26U/L, respectively. Therefore, the current QD@MOF CSNCPs based sensing system is a promising, widely applicable means of monitoring oxidase activities in biochemical research. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Activation of NADPH oxidase mediates increased endoplasmic reticulum stress and left ventricular remodeling after myocardial infarction in rabbits.

    Science.gov (United States)

    Li, Bao; Tian, Jing; Sun, Yi; Xu, Tao-Rui; Chi, Rui-Fang; Zhang, Xiao-Li; Hu, Xin-Ling; Zhang, Yue-An; Qin, Fu-Zhong; Zhang, Wei-Fang

    2015-05-01

    Nicotinamide adenine dinucleotide 3-phosphate (NADPH) oxidase activity and endoplasmic reticulum (ER) stress are increased after myocardial infarction (MI). In this study, we proposed to test whether activation of the NADPH oxidase in the remote non-infarcted myocardium mediates ER stress and left ventricular (LV) remodeling after MI. Rabbits with MI or sham operation were randomly assigned to orally receive an NADPH oxidase inhibitor apocynin or placebo for 30 days. The agents were administered beginning at 1 week after surgery. MI rabbits exhibited decreases in LV fractional shortening, LV ejection fraction and the first derivative of the LV pressure rise, which were abolished by apocynin treatment. NADPH oxidase Nox2 protein and mRNA expressions were increased in the remote non-infarcted myocardium after MI. Immunolabeling further revealed that Nox2 was increased in cardiac myocytes in the remote myocardium. The apocynin treatment prevented increases in the Nox2 expression, NADPH oxidase activity, oxidative stress, myocyte apoptosis and GRP78, CHOP and cleaved caspase 12 protein expression in the remote myocardium. The apocynin treatment also attenuated increases in myocyte diameter and cardiac fibrosis. In cultured H9C2 cardiomyocytes exposed to angiotensin II, an important stimulus for post-MI remodeling, Nox2 knockdown with siRNA significantly inhibited angiotensin II-induced NADPH oxidase activation, reactive oxygen species and GRP78 and CHOP protein expression. We conclude that NADPH oxidase inhibition attenuates increased ER stress in the remote non-infarcted myocardium and LV remodeling late after MI in rabbits. These findings suggest that the activation of NADPH oxidase in the remote non-infarcted myocardium mediates increased ER stress, contributing to myocyte apoptosis and LV remodeling after MI. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Cyanobacterial Lactate Oxidases Serve as Essential Partners in N2 Fixation and Evolved into Photorespiratory Glycolate Oxidases in Plants[w

    Science.gov (United States)

    Hackenberg, Claudia; Kern, Ramona; Hüge, Jan; Stal, Lucas J.; Tsuji, Yoshinori; Kopka, Joachim; Shiraiwa, Yoshihiro; Bauwe, Hermann; Hagemann, Martin

    2011-01-01

    Glycolate oxidase (GOX) is an essential enzyme involved in photorespiratory metabolism in plants. In cyanobacteria and green algae, the corresponding reaction is catalyzed by glycolate dehydrogenases (GlcD). The genomes of N2-fixing cyanobacteria, such as Nostoc PCC 7120 and green algae, appear to harbor genes for both GlcD and GOX proteins. The GOX-like proteins from Nostoc (No-LOX) and from Chlamydomonas reinhardtii showed high l-lactate oxidase (LOX) and low GOX activities, whereas glycolate was the preferred substrate of the phylogenetically related At-GOX2 from Arabidopsis thaliana. Changing the active site of No-LOX to that of At-GOX2 by site-specific mutagenesis reversed the LOX/GOX activity ratio of No-LOX. Despite its low GOX activity, No-LOX overexpression decreased the accumulation of toxic glycolate in a cyanobacterial photorespiratory mutant and restored its ability to grow in air. A LOX-deficient Nostoc mutant grew normally in nitrate-containing medium but died under N2-fixing conditions. Cultivation under low oxygen rescued this lethal phenotype, indicating that N2 fixation was more sensitive to O2 in the Δlox Nostoc mutant than in the wild type. We propose that LOX primarily serves as an O2-scavenging enzyme to protect nitrogenase in extant N2-fixing cyanobacteria, whereas in plants it has evolved into GOX, responsible for glycolate oxidation during photorespiration. PMID:21828292

  5. Auxin-activated NADH oxidase activity of soybean plasma membranes is distinct from the constitutive plasma membrane NADH oxidase and exhibits prion-like properties

    Science.gov (United States)

    Morre, D. James; Morre, Dorothy M.; Ternes, Philipp

    2003-01-01

    The hormone-stimulated and growth-related cell surface hydroquinone (NADH) oxidase activity of etiolated hypocotyls of soybeans oscillates with a period of about 24 min or 60 times per 24-h day. Plasma membranes of soybean hypocotyls contain two such NADH oxidase activities that have been resolved by purification on concanavalin A columns. One in the apparent molecular weight range of 14-17 kDa is stimulated by the auxin herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The other is larger and unaffected by 2,4-D. The 2,4-D-stimulated activity absolutely requires 2,4-D for activity and exhibits a period length of about 24 min. Also exhibiting 24-min oscillations is the rate of cell enlargement induced by the addition of 2,4-D or the natural auxin indole-3-acetic acid (IAA). Immediately following 2,4-D or IAA addition, a very complex pattern of oscillations is frequently observed. However, after several hours a dominant 24-min period emerges at the expense of the constitutive activity. A recruitment process analogous to that exhibited by prions is postulated to explain this behavior.

  6. Glucose Oxidase Directly Immobilized onto Highly Porous Gold Electrodes for Sensing and Fuel Cell applications

    International Nuclear Information System (INIS)

    Toit, Hendrik du; Di Lorenzo, Mirella

    2014-01-01

    Highlights: • Electrochemical adsorption of glucose oxidase (GOx) on highly porous gold (hPG); • Rapid one-step immobilisation protocol with no use of expensive and/or harsh reagents; • Linear response to glucose in the range 50 μM -10 mM; • Lower detection limit, stable over 5 days: 25 μM. • The use of the GOx-hPG in a fuel cell lead to the peak power density of 6 μW cm −2 . - Abstract: The successful implementation of redox-enzyme electrodes in biosensors and enzymatic biofuel cells has been the subject of extensive research. For high sensitivity and high energy-conversion efficiency, the effective electron transfer at the protein-electrode interface has a key role. This is difficult to achieve in the case of glucose oxidase, due to the fact that for this enzyme the redox centre is buried inside the structure, far from any feasible electrode binding sites. This study reports, a simple and rapid methodology for the direct immobilisation of glucose oxidase into highly porous gold electrodes. When the resulting electrode was tested as glucose sensor, a Michaelis-Menten kinetic trend was observed, with a detection limit of 25 μM. The bioelectrode sensitivity, calculated against the superficial surface area of the bioelectrode, was of 22.7 ± 0.1 μA mM −1 cm −2 . This glucose oxidase electrode was also tested as an anode in a glucose/O 2 enzymatic biofuel cell, leading to a peak power density of 6 μW cm −2 at a potential of 0.2 V

  7. Glucose oxidase-functionalized fluorescent gold nanoclusters as probes for glucose

    Energy Technology Data Exchange (ETDEWEB)

    Xia, Xiaodong [College of Chemistry and Chemical Engineering, Central South University, Changsha 410083 (China); School of Chemistry and Chemical Engineering, Hunan University of Science and Technology, Xiangtan 411201 (China); Long, Yunfei, E-mail: l_yunfei927@163.com [School of Chemistry and Chemical Engineering, Hunan University of Science and Technology, Xiangtan 411201 (China); Wang, Jianxiu, E-mail: jxiuwang@csu.edu.cn [College of Chemistry and Chemical Engineering, Central South University, Changsha 410083 (China)

    2013-04-15

    Highlights: ► A glucose oxidase/gold nanocluster conjugates formed by etching chemistry. ► Integration of the bioactivities and fluorescence properties within a single unit. ► These conjugates serve as novel fluorescent probe for glucose. -- Abstract: Creation and application of noble metal nanoclusters have received continuous attention. By integrating enzyme activity and fluorescence for potential applications, enzyme-capped metal clusters are more desirable. This work demonstrated a glucose oxidase (an enzyme for glucose)-functionalized gold cluster as probe for glucose. Under physiological conditions, such bioconjugate was successfully prepared by an etching reaction, where tetrakis (hydroxylmethyl) phosphonium-protected gold nanoparticle and thioctic acid-modified glucose oxidase were used as precursor and etchant, respectively. These bioconjugates showed unique fluorescence spectra (λ{sub em} {sub max} = 650 nm, λ{sub ex} {sub max} = 507 nm) with an acceptable quantum yield (ca. 7%). Moreover, the conjugated glucose oxidase remained active and catalyzed reaction of glucose and dissolved O{sub 2} to produce H{sub 2}O{sub 2}, which quenched quantitatively the fluorescence of gold clusters and laid a foundation of glucose detection. A linear range of 2.0 × 10{sup −6}–140 × 10{sup −6} M and a detection limit of 0.7 × 10{sup −6} M (S/N = 3) were obtained. Also, another horseradish peroxidase/gold cluster bioconjugate was produced by such general synthesis method. Such enzyme/metal cluster bioconjugates represented a promising class of biosensors for biologically important targets in organelles or cells.

  8. Adaptive evolution of cytochrome c oxidase: Infrastructure for a carnivorous plant radiation

    OpenAIRE

    Jobson, Richard W.; Nielsen, Rasmus; Laakkonen, Liisa; Wikström, Mårten; Albert, Victor A.

    2004-01-01

    Much recent attention in the study of adaptation of organismal form has centered on developmental regulation. As such, the highly conserved respiratory machinery of eukaryotic cells might seem an unlikely target for selection supporting novel morphologies. We demonstrate that a dramatic molecular evolutionary rate increase in subunit I of cytochrome c oxidase (COX) from an active-trapping lineage of carnivorous plants is caused by positive Darwinian selection. Bladderworts (Utricularia) trap ...

  9. Kinetic isotope effect studies on milk xanthine oxidase and on chicken liver xanthine dehydrogenase

    International Nuclear Information System (INIS)

    D'Ardenne, S.C.; Edmondson, D.E.

    1990-01-01

    The effect of isotopic substitution of the 8-H of xanthine (with 2 H and 3 H) on the rate of oxidation by bovine xanthine oxidase and by chicken xanthine dehydrogenase has been measured. V/K isotope effects were determined from competition experiments. No difference in H/T (V/K) values was observed between xanthine oxidase and xanthine dehydrogenase. Xanthine dehydrogenase exhibited a larger T/D (V/K) value than that observed for xanthine oxidase. Observed H/T (V/K) values for either enzyme are less than those H/T (V/K) values calculated with D/T (V/K) data. These discrepancies are suggested to arise from the presence of a rate-limiting step(s) prior to the irreversible C-H bond cleavage step in the mechanistic pathways of both enzymes. These kinetic complexities preclude examination of whether tunneling contributes to the reaction coordinate for the H-transfer step in each enzyme. No observable exchange of tritium with solvent is observed during the anaerobic incubation of [8- 3 H]xanthine with either enzyme, which suggests the reverse commitment to catalysis (C r ) is essentially zero. With the assumption of adherence to reduced mass relationships, the intrinsic deuterium isotope effect ( D k) for xanthine oxidation is calculated. By the use of these values and steady-state kinetic data, the minimal rate for the hydrogen-transfer step is calculated to be ∼75-fold faster than k cat for xanthine oxidase and ∼10-fold faster than k cat for xanthine dehydrogenase. Values calculated for each enzyme were found to be identical within experimental uncertainty

  10. Improving Glyphosate Oxidation Activity of Glycine Oxidase from Bacillus cereus by Directed Evolution

    Science.gov (United States)

    Zhan, Tao; Zhang, Kai; Chen, Yangyan; Lin, Yongjun; Wu, Gaobing; Zhang, Lili; Yao, Pei; Shao, Zongze; Liu, Ziduo

    2013-01-01

    Glyphosate, a broad spectrum herbicide widely used in agriculture all over the world, inhibits 5-enolpyruvylshikimate-3-phosphate synthase in the shikimate pathway, and glycine oxidase (GO) has been reported to be able to catalyze the oxidative deamination of various amines and cleave the C-N bond in glyphosate. Here, in an effort to improve the catalytic activity of the glycine oxidase that was cloned from a glyphosate-degrading marine strain of Bacillus cereus (BceGO), we used a bacteriophage T7 lysis-based method for high-throughput screening of oxidase activity and engineered the gene encoding BceGO by directed evolution. Six mutants exhibiting enhanced activity toward glyphosate were screened from two rounds of error-prone PCR combined with site directed mutagenesis, and the beneficial mutations of the six evolved variants were recombined by DNA shuffling. Four recombinants were generated and, when compared with the wild-type BceGO, the most active mutant B3S1 showed the highest activity, exhibiting a 160-fold increase in substrate affinity, a 326-fold enhancement in catalytic efficiency against glyphosate, with little difference between their pH and temperature stabilities. The role of these mutations was explored through structure modeling and molecular docking, revealing that the Arg51 mutation is near the active site and could be an important residue contributing to the stabilization of glyphosate binding, while the role of the remaining mutations is unclear. These results provide insight into the application of directed evolution in optimizing glycine oxidase function and have laid a foundation for the development of glyphosate-tolerant crops. PMID:24223901

  11. Lysyl oxidase enzymatic function increases stiffness to drive colorectal cancer progression through FAK

    DEFF Research Database (Denmark)

    Baker, A-M; Bird, D; Lang, G

    2013-01-01

    The extracellular, matrix-modifying enzyme lysyl oxidase (LOX) has recently been linked to colorectal cancer (CRC) progression, in particular to the stages of invasion and metastasis. In this report, we use cell lines expressing a catalytically inactive mutant form of LOX to show that catalytic a...... for patients with metastatic CRC.Oncogene advance online publication, 28 May 2012; doi:10.1038/onc.2012.202....

  12. Serum xanthine oxidase profile in stressed Marwari sheep from arid tracts in India

    Directory of Open Access Journals (Sweden)

    Maan R.

    2012-08-01

    Full Text Available The present investigation was aimed to determine serum xanthine oxidase profile in stressed Marwari breed of sheep belonging to arid tracts in Rajasthan, India. Extreme hot and cold ambiences were considered as stress conditions to the animals. Blood samples were collected to obtain sera during moderate, extreme hot and cold ambiences. The mean value of serum xanthine oxidase during moderate ambience was 93.33±1.11 mU L-1.The mean value of serum xanthine oxidase was significantly (p≤0.05higher during hot and significantly (p≤0.05 lower during cold ambiences as compared to moderate mean value serving as control. The sex and age effects were significant (p≤0.05 in all ambiences. The mean values were significantly (p≤0.05 higher in males than females. In each ambience the age effect showed a significant (p≤0.05 increase in the mean values being highest in the animals of 2.5-4.5 years of age. The effects of extreme ambiences were observed on the male and female animals of all age groups as revealed by various interactions studied viz. ambience X age; ambience X sex and age X sex (p≤0.01. Further sex effect was present in the animals of each age group. It can be concluded that serum xanthine oxidase can be used as an effective marker to assess oxidative stress in these animals. Mean values obtained from large number of animals during moderate ambience will help in providing physiological reference values for future research and clinical interpretations.

  13. Effects of trace elements and mono- and dithiols on mitochondrial monoamine oxidase of rats

    Energy Technology Data Exchange (ETDEWEB)

    Revis, N.; Horton, C.

    1978-01-01

    The effects of several trace elements on mitochondrial monoamine oxidase (MAO) were studied. Elements were studied at a concentration of 1 mM; only mercury, cadmium, and copper were significantly effective in reducing the activity of this enzyme. Of several thiols tested, only dithiothreitol could reverse the inhibition of MAO by these elements. Evidence is also presented in this report to show that cysteine, homocysteine, and reduced glutathione inhibit this MAO, whereas dithiothreitol or dithioerythritol evoke stimulatory responses.

  14. Tissue- and species-specific differences in cytochrome c oxidase assembly induced by SURF1 defects

    Czech Academy of Sciences Publication Activity Database

    Kovářová, Nikola; Pecina, Petr; Nůsková, Hana; Vrbacký, Marek; Zeviani, M.; Mráček, Tomáš; Viscomi, C.; Houštěk, Josef

    2016-01-01

    Roč. 1862, č. 4 (2016), s. 705-715 ISSN 0925-4439 R&D Projects: GA ČR(CZ) GB14-36804G; GA MŠk(CZ) LL1204; GA MZd(CZ) NT12370 Institutional support: RVO:67985823 Keywords : cytochrome c oxidase * respiratory supercomplexes * leigh syndrome * SURF1−/− mouse knockout * doxycycline * pulse-chase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.476, year: 2016

  15. Evolutionary origin and function of NOX4-art, an arthropod specific NADPH oxidase

    OpenAIRE

    Gandara, Ana Caroline Paiva; Torres, Andr?; Bahia, Ana Cristina; Oliveira, Pedro L.; Schama, Renata

    2017-01-01

    Background NADPH oxidases (NOX) are ROS producing enzymes that perform essential roles in cell physiology, including cell signaling and antimicrobial defense. This gene family is present in most eukaryotes, suggesting a common ancestor. To date, only a limited number of phylogenetic studies of metazoan NOXes have been performed, with few arthropod genes. In arthropods, only NOX5 and DUOX genes have been found and a gene called NOXm was found in mosquitoes but its origin and function has not b...

  16. Cyanide inhibition and pyruvate-induced recovery of cytochrome c oxidase

    Czech Academy of Sciences Publication Activity Database

    Nůsková, Hana; Vrbacký, Marek; Drahota, Zdeněk; Houštěk, Josef

    2010-01-01

    Roč. 42, č. 5 (2010), s. 395-403 ISSN 0145-479X R&D Projects: GA ČR(CZ) GA303/07/0781; GA MŠk(CZ) 1M0520; GA MŠk OC08017 Institutional research plan: CEZ:AV0Z50110509 Keywords : cytochrom c oxidase * cyanide * oxygen affinity Subject RIV: CE - Biochemistry Impact factor: 3.637, year: 2010

  17. Adaptation of respiratory chain biogenesis to cytochrome c oxidase deficiency caused by SURF1 gene mutations

    Czech Academy of Sciences Publication Activity Database

    Kovářová, Nikola; Vrbacká-Čížková, Alena; Pecina, Petr; Stránecký, V.; Pronicka, E.; Kmoch, S.; Houštěk, Josef

    2012-01-01

    Roč. 1822, č. 7 (2012), s. 1114-1124 ISSN 0925-4439 R&D Projects: GA MZd(CZ) NS9759; GA MZd(CZ) NT12370; GA ČR(CZ) GD305/08/H037 Institutional research plan: CEZ:AV0Z50110509 Institutional support: RVO:67985823 Keywords : mitochondrial disorder * SURF1 gene * Leigh syndrome * gene expression * oxidative phosphorylation * cytochrome c oxidase Subject RIV: FG - Pediatrics Impact factor: 4.910, year: 2012

  18. Oxidized low-density lipoproteins upregulate proline oxidase to initiate ROS-dependent autophagy

    OpenAIRE

    Zabirnyk, Olga; Liu, Wei; Khalil, Shadi; Sharma, Anit; Phang, James M.

    2009-01-01

    Epidemiological studies showed that high levels of oxidized low-density lipoproteins (oxLDLs) are associated with increased cancer risk. We examined the direct effect of physiologic concentrations oxLDL on cancer cells. OxLDLs were cytotoxic and activate both apoptosis and autophagy. OxLDLs have ligands for peroxisome proliferator-activated receptor gamma and upregulated proline oxidase (POX) through this nuclear receptor. We identified 7-ketocholesterol (7KC) as a main component responsible ...

  19. Enhanced Production of Glucose Oxidase Using Penicillium notatum and Rice Polish

    OpenAIRE

    Shazia Sabir; Haq Nawaz Bhatti; Muhammad Anjum Zia; Munir Ahmad Sheikh

    2007-01-01

    Glucose oxidase (GOD) is an important enzyme that finds a wide range of applications in food and pharmaceutical industry. In this investigation the feasibility of using rice polish as a substrate for the production of GOD by Penicillium notatum in submerged fermentation (SmF) has been evaluated. The intention was to enhance total GOD activity by the selection of economical substrate, microorganism and consecutive optimization of various cultural conditions. Maximum GOD activity of (112±5) U/m...

  20. Evaluation of Antimicrobial Activity of Glucose Oxidase from Aspergillus niger EBL-A and Penicillium notatum

    OpenAIRE

    Zia, Muhammad Anjum; Riaz, Ayesha; Rasul, Samreen; Abbas, Rao Zahid

    2013-01-01

    This work aimed to study the production and purification of glucose oxidase by Aspergillus niger and Penicillium notatum using corn steep liquor as the substrate and evaluate its antimicrobial activity for use in pharmaceutical and food industries. The enzyme was purified by ammonium sulfate precipitation (60-85%), DEAE-cellulose ion exchange and Sephadex G-200 size exclusion chromatography. The crude enzyme extracts of A. niger and P. notatum showed 2.32 and 5.53 U mg-1 specific activities, ...

  1. Cytokinin metabolism of pathogenic fungus Leptosphaeria maculans involves isopentenyltransferase, adenosine kinase and cytokinin oxidase/dehydrogenase

    Czech Academy of Sciences Publication Activity Database

    Trdá, Lucie; Barešová, Monika; Šašek, Vladimír; Nováková, Miroslava; Zahajská, Lenka; Dobrev, Petre; Motyka, Václav; Burketová, Lenka

    2017-01-01

    Roč. 8, JUL 21 (2017), č. článku 1374. ISSN 1664-302X R&D Projects: GA ČR GA13-26798S; GA ČR(CZ) GA16-14649S Institutional support: RVO:61389030 Keywords : Adenosine kinase * Cytokinin * Cytokinin oxidase/dehydrogenase * Isopentenyltransferase * Leptosphaeria maculans * Zeatin cis/trans isomerase Subject RIV: GF - Plant Pathology, Vermin, Weed, Plant Protection OBOR OECD: Microbiology Impact factor: 4.076, year: 2016

  2. Optimum pH and pH Stability of Crude Polyphenol Oxidase (PPO ...

    African Journals Online (AJOL)

    The effect of pH on the activity and stability of crude polyphenol oxidase (PPO) extracted from garden egg (Solanum aethiopicum), pawpaw (Carica papaya), pumpkin ... Optimum pH values were found to be 6.0,6.5,6.0, 4.5 and 4.0/or 8.0 for the enzyme extracted from Solanum aethiopicum, Carica papaya, Cucurbita pepo, ...

  3. Inhibitors of NADPH oxidase decrease endotoxin mediated induction of inducible nitric oxide expression in mouse macrophages

    Czech Academy of Sciences Publication Activity Database

    Krejčová, Daniela; Okénková, Kateřina; Konopka, Roman; Lojek, Antonín; Kubala, Lukáš

    2007-01-01

    Roč. 101, č. 14 (2007), s203-s204 E-ISSN 1213-7103. [Mezioborová česko-slovenská toxikologická konference /12./. Praha, 11.06.2007-13.06.2007] R&D Projects: GA ČR(CZ) GA524/06/1197 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : lipopolysaccharide * inhibitors of NADPH oxidase * macrophage s Subject RIV: BO - Biophysics

  4. Disparate phenotypic effects from the knockdown of various Trypanosoma brucei cytochrome c oxidase subunits

    Czech Academy of Sciences Publication Activity Database

    Gnipová, Anna; Panicucci, Brian; Paris, Zdeněk; Verner, Zdeněk; Horváth, A.; Lukeš, Julius; Zíková, Alena

    2012-01-01

    Roč. 184, č. 2 (2012), s. 90-98 ISSN 0166-6851 R&D Projects: GA AV ČR KJB500960901; GA ČR GA204/09/1667 Institutional research plan: CEZ:AV0Z60220518 Keywords : Trypanosoma * RNA interference * Mitochondrion * Respiratory complexes * Cytochrome c oxidase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.734, year: 2012 http://www.sciencedirect.com/science/article/pii/S0166685112001065#

  5. A highly sensitive electrochemical glucose sensor structuring with nickel hydroxide and enzyme glucose oxidase

    International Nuclear Information System (INIS)

    Mathew, Manjusha; Sandhyarani, N.

    2013-01-01

    Graphical abstract: A combination of Ni 2+ /Ni 3+ redox couple and glucose oxidase has successfully been exploited for the realization of a highly sensitive glucose sensor for the first time. -- Highlights: • A multilayered glucose biosensor with enhanced sensitivity was fabricated. • Combination of Ni 2+ /Ni 3+ redox couple and glucose oxidase has been exploited for the first time. • Exhibits a lower detection limit of 100 nM with a high sensitivity of 16,840 μA mM −1 cm −2 . • The surface shows a low Michaelis–Menten constant value of 2.4 μM. • Detailed mechanism of sensing was proposed and justified. -- Abstract: A multilayered glucose biosensor with enhanced electron transport was fabricated via the sequential electrodeposition of chitosan gold nanocomposite (CGNC) and nickel hydroxide (Ni(OH) 2 ) on a bare gold electrode and subsequent immobilization of glucose oxidase. A thin film of Ni(OH) 2 deposited on CGNC modified gold electrode serves as an electrochemical redox probe as well as a matrix for the immobilization of glucose oxidase retaining its activity. Electron transport property of CGNC has been exploited to enhance the electron transport between the analyte and electrode. Electrochemical characteristics of the biosensor were studied by cyclic voltammetry and chronoamperometry. Under optimal conditions the biosensor exhibits a linear range from 1 μM to 100 μM with a limit of detection (lod) down to 100 nM. The sensor shows a low Michaelis-Menten constant value of 2.4 μM indicates the high affinity of enzyme to the analyte points to the retained activity of enzyme after immobilization. The present glucose sensor with the high selectivity, sensitivity and stability is promising for practical clinical applications

  6. Growth of non-Campylobacter, oxidase-positive bacteria on selective Campylobacter agar.

    OpenAIRE

    Moskowitz, L B; Chester, B

    1982-01-01

    A total of 67 oxidase-positive, gram-negative bacteria were tested for growth on selective Campylobacter agar (Blaser formulation, BBL Microbiology Systems, Cockeysville, Md.) at 42 degrees C under microaerophilic conditions. Although the growth of most of these bacteria was prevented, all strains of Achromobacter xylosoxidans, Pseudomonas aeruginosa, Pseudomonas putrefaciens, Pseudomonas alcaligenes, and Pseudomonas pseudoalcaligenes grew as well as Campylobacter fetus subsp. jejuni.

  7. Improving glyphosate oxidation activity of glycine oxidase from Bacillus cereus by directed evolution.

    Directory of Open Access Journals (Sweden)

    Tao Zhan

    Full Text Available Glyphosate, a broad spectrum herbicide widely used in agriculture all over the world, inhibits 5-enolpyruvylshikimate-3-phosphate synthase in the shikimate pathway, and glycine oxidase (GO has been reported to be able to catalyze the oxidative deamination of various amines and cleave the C-N bond in glyphosate. Here, in an effort to improve the catalytic activity of the glycine oxidase that was cloned from a glyphosate-degrading marine strain of Bacillus cereus (BceGO, we used a bacteriophage T7 lysis-based method for high-throughput screening of oxidase activity and engineered the gene encoding BceGO by directed evolution. Six mutants exhibiting enhanced activity toward glyphosate were screened from two rounds of error-prone PCR combined with site directed mutagenesis, and the beneficial mutations of the six evolved variants were recombined by DNA shuffling. Four recombinants were generated and, when compared with the wild-type BceGO, the most active mutant B3S1 showed the highest activity, exhibiting a 160-fold increase in substrate affinity, a 326-fold enhancement in catalytic efficiency against glyphosate, with little difference between their pH and temperature stabilities. The role of these mutations was explored through structure modeling and molecular docking, revealing that the Arg(51 mutation is near the active site and could be an important residue contributing to the stabilization of glyphosate binding, while the role of the remaining mutations is unclear. These results provide insight into the application of directed evolution in optimizing glycine oxidase function and have laid a foundation for the development of glyphosate-tolerant crops.

  8. Effect of gamma irradiation on aspergillus niger for enhanced production of glucose oxidase

    International Nuclear Information System (INIS)

    Zia, M.A.; Rasul, S.

    2012-01-01

    Developing countries have a high prevalence of diabetes and their populations are suffering from associated adverse factors. Such a frequency requires more effective diagnosis, mostly achieved by glucose diagnostic kits. Although high priced kits are available in market but local production of such kits can be highly cost effective and may confer the decline in incidence of the disease. Glucose oxidase is the key enzyme for the determination of glucose in such analytical tools. Enhanced production of glucose oxidase was performed by mutagenesis of Aspergillus niger by gamma irradiation. A dose of 80 krad was found as optimum for derivation of positive mutant strains. Following the screening by triton X-100 and 2-deoxy-D-glucose, the selected strains A. niger G-80-A, A. niger G-80-B and A. niger G-80-C showed 27.5, 23.20 and 20.55 UmL/sub -1/ glucose oxidase activity in enzyme diffusion zone test; which is much higher to parental strain (7.5 UmL/sup -1/). A. niger G-80-A was subjected to submerged fermentation and obtained highest yields after 36 h, at CSL 2%, pH 6.5, 30 degree C, KH/sub 2/PO/sub 4/ 0.8% and urea 0.3%. Partial purification by ammonium sulfate resulted in 175 UmL/sup -1/ of glucose oxidase activity after dialysis. Kinetic parameters like optimum pH, temperature, K/sub m/ and V/sub max/ were found to be 6.0 (180 +- 2 UmL/sup -1/), 30 degree C (185 +- 0.5 UmL/sup -1/), 5.26 mM and 400 U mL/sup -1/, respectively. Active inhibition of the enzyme by increasing concentration of PLP in reaction mixture confirmed the presence of functional lysyl residue on the active site of enzyme. (author)

  9. Phenyl- and benzylurea cytokinins as competitive inhibitors of cytokinin oxidase/dehydrogenase: a structural study

    Czech Academy of Sciences Publication Activity Database

    Kopečný, D.; Briozzo, P.; Popelková, H.; Šebela, M.; Končitíková, R.; Spíchal, Lukáš; Nisler, Jaroslav; Madzak, C.; Frébort, Ivo; Laloue, M.; Houba-Herin, N.

    2010-01-01

    Roč. 92, č. 8 (2010), s. 1052-1062 ISSN 0300-9084 R&D Projects: GA ČR GA522/08/0555; GA ČR GA301/08/1649 Institutional research plan: CEZ:AV0Z50380511 Keywords : benzylurea * crystal structure * cytokinin oxidase/dehydrogenase Subject RIV: CE - Biochemistry Impact factor: 3.787, year: 2010

  10. Immobilization of the enzyme polyphenol oxidase on dendrispheres: In partial fulfilment of the degree Magister Scientiae

    CSIR Research Space (South Africa)

    Bannister, M

    2011-04-01

    Full Text Available OF THE ENZYME POLYPHENOL OXIDASE ON DENDRISPHERES IN PARTIAL FULFILMENT OF THE DEGREE MAGISTER SCIENTIAE Magdalien Bannister 26112664 April 2011 TEA (Camellia sinensis plant) ? CSIR 2011 Slide 2 Second most consumed beverage in the world Grouped into...: ?Green tea Non-fermented ?Oolong tea Partially fermented ?Black tea Fermented Catechins: ?Major component present in green tea leaves ?(-)-Epigallocatechin gallate (EGCG), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG) and (-)-epicatechin...

  11. A Xylenol Orange-Based Screening Assay for the Substrate Specificity of Flavin-Dependent para-Phenol Oxidases

    Directory of Open Access Journals (Sweden)

    Tom A. Ewing

    2018-01-01

    Full Text Available Vanillyl alcohol oxidase (VAO and eugenol oxidase (EUGO are flavin-dependent enzymes that catalyse the oxidation of para-substituted phenols. This makes them potentially interesting biocatalysts for the conversion of lignin-derived aromatic monomers to value-added compounds. To facilitate their biocatalytic exploitation, it is important to develop methods by which variants of the enzymes can be rapidly screened for increased activity towards substrates of interest. Here, we present the development of a screening assay for the substrate specificity of para-phenol oxidases based on the detection of hydrogen peroxide using the ferric-xylenol orange complex method. The assay was used to screen the activity of VAO and EUGO towards a set of twenty-four potential substrates. This led to the identification of 4-cyclopentylphenol as a new substrate of VAO and EUGO and 4-cyclohexylphenol as a new substrate of VAO. Screening of a small library of VAO and EUGO active-site variants for alterations in their substrate specificity led to the identification of a VAO variant (T457Q with increased activity towards vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol and a EUGO variant (V436I with increased activity towards chavicol (4-allylphenol and 4-cyclopentylphenol. This assay provides a quick and efficient method to screen the substrate specificity of para-phenol oxidases, facilitating the enzyme engineering of known para-phenol oxidases and the evaluation of the substrate specificity of novel para-phenol oxidases.

  12. A Xylenol Orange-Based Screening Assay for the Substrate Specificity of Flavin-Dependent para-Phenol Oxidases.

    Science.gov (United States)

    Ewing, Tom A; van Noord, Aster; Paul, Caroline E; van Berkel, Willem J H

    2018-01-14

    Vanillyl alcohol oxidase (VAO) and eugenol oxidase (EUGO) are flavin-dependent enzymes that catalyse the oxidation of para -substituted phenols. This makes them potentially interesting biocatalysts for the conversion of lignin-derived aromatic monomers to value-added compounds. To facilitate their biocatalytic exploitation, it is important to develop methods by which variants of the enzymes can be rapidly screened for increased activity towards substrates of interest. Here, we present the development of a screening assay for the substrate specificity of para -phenol oxidases based on the detection of hydrogen peroxide using the ferric-xylenol orange complex method. The assay was used to screen the activity of VAO and EUGO towards a set of twenty-four potential substrates. This led to the identification of 4-cyclopentylphenol as a new substrate of VAO and EUGO and 4-cyclohexylphenol as a new substrate of VAO. Screening of a small library of VAO and EUGO active-site variants for alterations in their substrate specificity led to the identification of a VAO variant (T457Q) with increased activity towards vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) and a EUGO variant (V436I) with increased activity towards chavicol (4-allylphenol) and 4-cyclopentylphenol. This assay provides a quick and efficient method to screen the substrate specificity of para -phenol oxidases, facilitating the enzyme engineering of known para- phenol oxidases and the evaluation of the substrate specificity of novel para -phenol oxidases.

  13. Comparison of brain mitochondrial cytochrome c oxidase activity with cyanide LD(50) yields insight into the efficacy of prophylactics.

    Science.gov (United States)

    Marziaz, Mandy L; Frazier, Kathryn; Guidry, Paul B; Ruiz, Robyn A; Petrikovics, Ilona; Haines, Donovan C

    2013-01-01

    Cyanide inhibits cytochrome c oxidase, the terminal oxidase of the mitochondrial respiratory pathway, therefore inhibiting the cell oxygen utilization and resulting in the condition of histotoxic anoxia. The enzyme rhodanese detoxifies cyanide by utilizing sulfur donors to convert cyanide to thiocyanate, and new and improved sulfur donors are actively sought as researchers seek to improve cyanide prophylactics. We have determined brain cytochrome c oxidase activity as a marker for cyanide exposure for mice pre-treated with various cyanide poisoning prophylactics, including sulfur donors thiosulfate (TS) and thiotaurine (TT3). Brain mitochondria were isolated by differential centrifugation, the outer mitochondrial membrane was disrupted by a maltoside detergent, and the decrease in absorbance at 550 nm as horse heart ferrocytochrome c (generated by the dithiothreitol reduction of ferricytochrome c) was oxidized was monitored. Overall, the TS control prophylactic treatment provided significant protection of the cytochrome c oxidase activity. The TT3-treated mice showed reduced cytochrome c oxidase activity even in the absence of cyanide. In both treatment series, addition of exogenous Rh did not significantly enhance the prevention of cytochrome c oxidase inhibition, but the addition of sodium nitrite did. These findings can lead to a better understanding of the protection mechanism by various cyanide antidotal systems. Copyright © 2011 John Wiley & Sons, Ltd.

  14. Electrochemical L-lactic acid sensor based on immobilized ZnO nanorods with lactate oxidase.

    Science.gov (United States)

    Ibupoto, Zafar Hussain; Shah, Syed Muhammad Usman Ali; Khun, Kimleang; Willander, Magnus

    2012-01-01

    In this work, fabrication of gold coated glass substrate, growth of ZnO nanorods and potentiometric response of lactic acid are explained. The biosensor was developed by immobilizing the lactate oxidase on the ZnO nanorods in combination with glutaraldehyde as a cross linker for lactate oxidase enzyme. The potentiometric technique was applied for the measuring the output (EMF) response of l-lactic acid biosensor. We noticed that the present biosensor has wide linear detection range of concentration from 1 × 10(-4)-1 × 10(0) mM with acceptable sensitivity about 41.33 ± 1.58 mV/decade. In addition, the proposed biosensor showed fast response time less than 10 s, a good selectivity towards l-lactic acid in presence of common interfering substances such as ascorbic acid, urea, glucose, galactose, magnesium ions and calcium ions. The present biosensor based on immobilized ZnO nanorods with lactate oxidase sustained its stability for more than three weeks.

  15. Development of Galactose Biosensor Based on Functionalized ZnO Nanorods with Galactose Oxidase

    Directory of Open Access Journals (Sweden)

    K. Khun

    2012-01-01

    Full Text Available The fabrication of galactose biosensor based on functionalised ZnO nanorods is described. The galactose biosensor was developed by immobilizing galactose oxidase on ZnO nanorods in conjunction with glutaraldehyde as a cross-linker molecule. The IRAS study provided evidence for the interaction of galactose oxidase with the surface of ZnO nanorods. The electromotive force (EMF response of the galactose biosensor was measured by potentiometric method. We observed that the proposed biosensor has a linear detection range over a concentration range from 10 mM to 200 mM with good sensitivity of 89.10±1.23 mV/decade. In addition, the proposed biosensor has shown fast time response of less than 10 s and a good selectivity towards galactose in the presence of common interferents such as ascorbic acid, uric acid, glucose, and magnesium ions. The galactose biosensor based on galactose oxidase immobilized ZnO nanorods has a shelf life more than four weeks.

  16. Characterization of three bioenergetically active respiratory terminal oxidases in the cyanobacterium Synechocystis sp. strain PCC 6803.

    Science.gov (United States)

    Pils, D; Schmetterer, G

    2001-09-25

    Synechocystis sp. PCC 6803 contains three respiratory terminal oxidases (RTOs): cytochrome c oxidase (Cox), quinol oxidase (Cyd), and alternate RTO (ARTO). Mutants lacking combinations of the RTOs were used to characterize these key enzymes of respiration. Pentachlorophenol and 2-heptyl-4-hydroxy-quinoline-N-oxide inhibited Cyd completely, but had little effect on electron transport to the other RTOs. KCN inhibited all three RTOs but the in vivo K(I) for Cox and Cyd was quite different (7 vs. 27 microM), as was their affinity for oxygen (K(M) 1.0 vs. 0.35 microM). ARTO has a very low respiratory activity. However, when uptake of 3-O-methylglucose, an active H+ co-transport, was used to monitor energization of the cytoplasmic membrane, ARTO was similarly effective as the other RTOs. As removal of the gene for cytochrome c(553) had the same effects as removal of ARTO genes, we propose that the ARTO might be a second Cox. The possible functions, localization and regulation of the RTOs are discussed.

  17. Expression and Characterization of Glucose Oxidase from Aspergillus niger in Yarrowia lipolytica.

    Science.gov (United States)

    Khadivi Derakshan, Fatemeh; Darvishi, Farshad; Dezfulian, Mehrouz; Madzak, Catherine

    2017-08-01

    Glucose oxidase (GOX) is currently used in clinical, pharmaceutical, food and chemical industries. The aim of this study was expression and characterization of Aspergillus niger glucose oxidase gene in the yeast Yarrowia lipolytica. For the first time, the GOX gene of A. niger was successfully expressed in Y. lipolytica using a mono-integrative vector containing strong hybrid promoter and secretion signal. The highest total glucose oxidase activity was 370 U/L after 7 days of cultivation. An innovative method was used to cell wall disruption in current study, and it could be recommended to use for efficiently cell wall disruption of Y. lipolytica. Optimum pH and temperature for recombinant GOX activity were 5.5 and 37 °C, respectively. A single band with a molecular weight of 80 kDa similar to the native and pure form of A. niger GOX was observed for the recombinant GOX in SDS-PAGE analysis. Y. lipolytica is a suitable and efficient eukaryotic expression system to production of recombinant GOX in compered with other yeast expression systems and could be used to production of pure form of GOX for industrial applications.

  18. Biological Effects of Potato Plants Transformation with Glucose Oxidase Gene and their Resistance to Hyperthermia

    Directory of Open Access Journals (Sweden)

    O.I. Grabelnych

    2017-02-01

    Full Text Available It is known that regulation of plant tolerance to adverse environmental factors is connected with short term increase of the concentration of endogenous reactive oxygen species (ROS, which are signalling molecules for the induction of protective mechanisms. Introduction and expression of heterologous gox gene, which encodes glucose oxidase enzyme in plant genome, induce constantly higher content of hydrogen peroxide in plant tissues. It is not known how the introduction of native or modified gox gene affects the plant resistance to high-temperature stress, one of the most commonly used model for the study of stress response and thermal tolerance. In this study, we investigated biological effects of transformation and evaluated the resistance to temperature stress of potato plants with altered levels of glucose oxidase expression. Transformation of potato plants by gox gene led to the more early coming out from tuber dormancy of transformed plants and slower growth rate. Transformants containing the glucose oxidase gene were more sensitive to lethal thermal shock (50 °C, 90 min than the transformant with the empty vector (pBI or untransformed plants (CK. Pre-heating of plants at 37 °C significantly weakened the damaging effect of lethal thermal shock. This attenuation was more significant in the non-transformed plants.

  19. Immobilization of glucose oxidase on sepharose by UV-initiated graft copolymerization

    International Nuclear Information System (INIS)

    D'Angiuro, L.; Cremonesi, P.

    1982-01-01

    The performance of a new method of enzyme immobilization based on photochemically initiated direct graft copolymerization was recently investigated. The immobilization reaction can be carried out in a simple way and by carefully selecting the reaction conditions, the enzyme-graft copolymer can be obtained as the main reaction product. Coupling efficiency of glucose oxidase has been found to depend only on the amount of photocatalyst (FeCl 3 ) fixed on Sepharose used as polysaccharide support. Small quantities of glycidylmethacrylate (GMA) (0.25 g/g dry Sepharose) are sufficient but necessary to achieve the best enzyme coupling efficiency (20-40%). Enzyme immobilization occurs very rapidly and the entire reaction occurs within 60 min. Reaction patterns and physicochemical characteristics of the obtained enzyme-graft copolymers exclude the glucose oxidase entrapment: therefore a covalent attachment mechanism may be proposed. The kinetic parameters of immobilized glucose oxidase (K/sub m/' = 2.0 x 10 -2 M) are quite similar to those of free enzyme (K/sub m/ = 1.93 x 10 -2 M), and no diffusion limitation phenomena are evidenced in samples having different enzyme or polymer content. Lyophilization, thermostability, and long-term continuous operation also have been investigated. The advantages of this method over that using vinylenzyme copolymerization are discussed

  20. Feasibility of converting lactic acid to ethanol in food waste fermentation by immobilized lactate oxidase

    International Nuclear Information System (INIS)

    Ma, Hong-zhi; Xing, Yi; Yu, Miao; Wang, Qunhui

    2014-01-01

    Highlights: • Residue lactic acid in food waste could be converted to pyruvic acid. • Calcium alginate immobilized the lactate oxidase with high pH and thermal stability. • Immobilized enzyme could convert 70% lactic acid to pyruvic acid. • Ethanol yield could be increased by 20% with lactate oxidase added. - Abstract: Adoption of lactic acid bacteria (LAB) into ethanol fermentation from food waste can replace the sterilization process. However, LAB inoculation will convert part of the substrate into lactic acid (LA), not ethanol. This study adopted lactate oxidase to convert the produced LA to pyruvate, and then ethanol fermentation was carried out. The immobilization enzyme was utilized, and corresponding optimum conditions were determined. Results showed that calcium alginate could successfully immobilize the enzyme and improve pH and thermal stability. The optimum pH and temperature were 6.2 and 55 °C, respectively. The utilization of immobilized enzyme with catalytic time of 5 h could convert 70% LA to pyruvate, and the addition of enzyme increased the ethanol yield by 20% more than that of the control. The process could be applied in food waste storage and can help in reducing carbon source consumption

  1. Nitric oxide partitioning into mitochondrial membranes and the control of respiration at cytochrome c oxidase

    Science.gov (United States)

    Shiva, Sruti; Brookes, Paul S.; Patel, Rakesh P.; Anderson, Peter G.; Darley-Usmar, Victor M.

    2001-06-01

    An emerging and important site of action for nitric oxide (NO) within cells is the mitochondrial inner membrane, where NO binds to and inhibits members of the electron transport chain, complex III and cytochrome c oxidase. Although it is known that inhibition of cytochrome c oxidase by NO is competitive with O2, the mechanisms that underlie this phenomenon remain unclear, and the impact of both NO and O2 partitioning into biological membranes has not been considered. These properties are particularly interesting because physiological O2 tensions can vary widely, with NO having a greater inhibitory effect at low O2 tensions (mitochondrial membranes in the absence of substrate, in a nonsaturable process that is O2 dependent. This consumption modulates inhibition of cytochrome c oxidase by NO and is enhanced by the addition of exogenous membranes. From these data, it is evident that the partition of NO into mitochondrial membranes has a major impact on the ability of NO to control mitochondrial respiration. The implications of this conclusion are discussed in the context of mitochondrial lipid:protein ratios and the importance of NO as a regulator of respiration in pathophysiology.

  2. Molecular Modeling of Peroxidase and Polyphenol Oxidase: Substrate Specificity and Active Site Comparison

    Directory of Open Access Journals (Sweden)

    Lalida Shank

    2010-09-01

    Full Text Available Peroxidases (POD and polyphenol oxidase (PPO are enzymes that are well known to be involved in the enzymatic browning reaction of fruits and vegetables with different catalytic mechanisms. Both enzymes have some common substrates, but each also has its specific substrates. In our computational study, the amino acid sequence of grape peroxidase (ABX was used for the construction of models employing homology modeling method based on the X-ray structure of cytosolic ascorbate peroxidase from pea (PDB ID:1APX, whereas the model of grape polyphenol oxidase was obtained directly from the available X-ray structure (PDB ID:2P3X. Molecular docking of common substrates of these two enzymes was subsequently studied. It was found that epicatechin and catechin exhibited high affinity with both enzymes, even though POD and PPO have different binding pockets regarding the size and the key amino acids involved in binding. Predicted binding modes of substrates with both enzymes were also compared. The calculated docking interaction energy of trihydroxybenzoic acid related compounds shows high affinity, suggesting specificity and potential use as common inhibitor to grape ascorbate peroxidase and polyphenol oxidase.

  3. Conformational lock and dissociative thermal inactivation of lentil seedling amine oxidase.

    Science.gov (United States)

    Moosavi-Nejad, S Zahra; Moosavi-Movahedi, Ali-Akbar; Rezaei-Tavirani, Mostafa; Floris, Giovanni; Medda, Rosaria

    2003-03-31

    The kinetics of thermal inactivation of copper-containing amine oxidase from lentil seedlings were studied in a 100 mM potassium phosphate buffer, pH 7, using putrescine as the substrate. The temperature range was between 47-60 degrees C. The thermal inactivation curves were not linear at 52 and 57 degrees C; three linear phases were shown. The first phase gave some information about the number of dimeric forms of the enzyme that were induced by the higher temperatures using the "conformational lock" pertaining theory to oligomeric enzyme. The "conformational lock" caused two additional dimeric forms of the enzyme when the temperature increased to 57 degrees C. The second and third phases were interpreted according to a dissociative thermal inactivation model. These phases showed that lentil amine oxidase was reversibly-dissociated before the irreversible thermal inactivation. Although lentil amine oxidase is not a thermostable enzyme, its dimeric structure can form "conformational lock," conferring a structural tolerance to the enzyme against heat stress.

  4. Electrochemical L-Lactic Acid Sensor Based on Immobilized ZnO Nanorods with Lactate Oxidase

    Directory of Open Access Journals (Sweden)

    Kimleang Khun

    2012-02-01

    Full Text Available In this work, fabrication of gold coated glass substrate, growth of ZnO nanorods and potentiometric response of lactic acid are explained. The biosensor was developed by immobilizing the lactate oxidase on the ZnO nanorods in combination with glutaraldehyde as a cross linker for lactate oxidase enzyme. The potentiometric technique was applied for the measuring the output (EMF response of L-lactic acid biosensor. We noticed that the present biosensor has wide linear detection range of concentration from 1 × 10−4–1 × 100 mM with acceptable sensitivity about 41.33 ± 1.58 mV/decade. In addition, the proposed biosensor showed fast response time less than 10 s, a good selectivity towards L-lactic acid in presence of common interfering substances such as ascorbic acid, urea, glucose, galactose, magnesium ions and calcium ions. The present biosensor based on immobilized ZnO nanorods with lactate oxidase sustained its stability for more than three weeks.

  5. Improvement of exopolysaccharide production in Lactobacillus casei LC2W by overexpression of NADH oxidase gene.

    Science.gov (United States)

    Li, Nan; Wang, Yuanlong; Zhu, Ping; Liu, Zhenmin; Guo, Benheng; Ren, Jing

    2015-02-01

    Lactobacillus casei LC2W is an exopolysaccharide (EPS)-producing strain with probiotic effects. To investigate the regulation mechanism of EPS biosynthesis and to improve EPS production through cofactor engineering, a H₂O-forming NADH oxidase gene was cloned from Streptococcus mutans and overexpressed in L. casei LC2W under the control of constitutive promoter P₂₃. The recombinant strain LC-nox exhibited 0.854 U/mL of NADH oxidase activity, which was elevated by almost 20-fold in comparison with that of wild-type strain. As a result, overexpression of NADH oxidase resulted in a reduction in growth rate. In addition, lactate production was decreased by 22% in recombinant strain. It was proposed that more carbon source was saved and used for the biosynthesis of EPS, the production of which was reached at 219.4 mg/L, increased by 46% compared to that of wild-type strain. This work provided a novel and convenient genetic approach to manipulate metabolic flux and to increase EPS production. To the best of our knowledge, this is the first report which correlates cofactor engineering with EPS production. Copyright © 2015 Elsevier GmbH. All rights reserved.

  6. Thermal Characterization of Purified Glucose Oxidase from A Newly Isolated Aspergillus Niger UAF-1

    Science.gov (United States)

    Anjum Zia, Muhammad; Khalil-ur-Rahman; K. Saeed, Muhammad; Andaleeb, Fozia; I. Rajoka, Muhammad; A. Sheikh, Munir; A. Khan, Iftikhar; I. Khan, Azeem

    2007-01-01

    An intracellular glucose oxidase was isolated from the mycelium extract of a locally isolated strain of Aspergillus niger UAF-1. The enzyme was purified to a yield of 28.43% and specific activity of 135 U mg−1 through ammonium sulfate precipitation, anion exchange and gel filtration chromatography. The enzyme showed high affinity for D-glucose with a Km value of 2.56 mM. The enzyme exhibited optimum catalytic activity at pH 5.5. Temperature optimum for glucose oxidase, catalyzed D-glucose oxidation was 40°C. The enzyme showed a high thermostability having a half-life 30 min, enthalpy of denaturation 99.66 kJ mol−1 and free energy of denaturation 103.63 kJ mol−1. These characteristics suggest the use of glucose oxidase from Aspergillus niger UAF-1 as an analytical reagent and in the design of biosensors for clinical, biochemical and diagnostic assays. PMID:18193107

  7. Evaluation of Antimicrobial Activity of Glucose Oxidase from Aspergillus niger EBL-A and Penicillium notatum

    Directory of Open Access Journals (Sweden)

    Muhammad Anjum Zia

    2013-12-01

    Full Text Available This work aimed to study the production and purification of glucose oxidase by Aspergillus niger and Penicillium notatum using corn steep liquor as the substrate and evaluate its antimicrobial activity for use in pharmaceutical and food industries. The enzyme was purified by ammonium sulfate precipitation (60-85%, DEAE-cellulose ion exchange and Sephadex G-200 size exclusion chromatography. The crude enzyme extracts of A. niger and P. notatum showed 2.32 and 5.53 U mg-1 specific activities, respectively, which after desalting was 15.52 and 12.05 U mg-1, and after ion exchange and gel filtration chromatography was 29.09 - 62 and 25.72 - 59.37 U mg-1 for A. niger and P. notatum, respectively. The antimicrobial activity was determined by disc diffusion method against selected microbial strains where glucose oxidase from A. niger showed anti-bacterial activity, while no fungicidal effects were shown by both A. niger and P. notatum glucose oxidases.

  8. Ozone affects pollen viability and NAD(P)H oxidase release from Ambrosia artemisiifolia pollen.

    Science.gov (United States)

    Pasqualini, Stefania; Tedeschini, Emma; Frenguelli, Giuseppe; Wopfner, Nicole; Ferreira, Fatima; D'Amato, Gennaro; Ederli, Luisa

    2011-10-01

    Air pollution is frequently proposed as a cause of the increased incidence of allergy in industrialised countries. We investigated the impact of ozone (O(3)) on reactive oxygen species (ROS) and allergen content of ragweed pollen (Ambrosia artemisiifolia). Pollen was exposed to acute O(3) fumigation, with analysis of pollen viability, ROS and nitric oxide (NO) content, activity of nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase, and expression of major allergens. There was decreased pollen viability after O(3) fumigation, which indicates damage to the pollen membrane system, although the ROS and NO contents were not changed or were only slightly induced, respectively. Ozone exposure induced a significant enhancement of the ROS-generating enzyme NAD(P)H oxidase. The expression of the allergen Amb a 1 was not affected by O(3), determined from the mRNA levels of the major allergens. We conclude that O(3) can increase ragweed pollen allergenicity through stimulation of ROS-generating NAD(P)H oxidase. Copyright © 2011 Elsevier Ltd. All rights reserved.

  9. A Plastid Terminal Oxidase Associated with Carotenoid Desaturation during Chromoplast Differentiation1

    Science.gov (United States)

    Josse, Eve-Marie; Simkin, Andrew J.; Gaffé, Joël; Labouré, Anne-Marie; Kuntz, Marcel; Carol, Pierre

    2000-01-01

    The Arabidopsis IMMUTANS gene encodes a plastid homolog of the mitochondrial alternative oxidase, which is associated with phytoene desaturation. Upon expression in Escherichia coli, this protein confers a detectable cyanide-resistant electron transport to isolated membranes. In this assay this activity is sensitive to n-propyl-gallate, an inhibitor of the alternative oxidase. This protein appears to be a plastid terminal oxidase (PTOX) that is functionally equivalent to a quinol:oxygen oxidoreductase. This protein was immunodetected in achlorophyllous pepper (Capsicum annuum) chromoplast membranes, and a corresponding cDNA was cloned from pepper and tomato (Lycopersicum esculentum) fruits. Genomic analysis suggests the presence of a single gene in these organisms, the expression of which parallels phytoene desaturase and ζ-carotene desaturase gene expression during fruit ripening. Furthermore, this PTOX gene is impaired in the tomato ghost mutant, which accumulates phytoene in leaves and fruits. These data show that PTOX also participates in carotenoid desaturation in chromoplasts in addition to its role during early chloroplast development. PMID:10938359

  10. Bioconversion of Airborne Methylamine by Immobilized Recombinant Amine Oxidase from the Thermotolerant Yeast Hansenula polymorpha

    Directory of Open Access Journals (Sweden)

    Sasi Sigawi

    2014-01-01

    Full Text Available Aliphatic amines, including methylamine, are air-pollutants, due to their intensive use in industry and the natural degradation of proteins, amino acids, and other nitrogen-containing compounds in biological samples. It is necessary to develop systems for removal of methylamine from the air, since airborne methylamine has a negative effect on human health. The primary amine oxidase (primary amine : oxygen oxidoreductase (deaminating or amine oxidase, AMO; EC 1.4.3.21, a copper-containing enzyme from the thermotolerant yeast Hansenula polymorpha which was overexpressed in baker’s yeast Saccharomyces cerevisiae, was tested for its ability to oxidize airborne methylamine. A continuous fluidized bed bioreactor (CFBR was designed to enable bioconversion of airborne methylamine by AMO immobilized in calcium alginate (CA beads. The results demonstrated that the bioreactor with immobilized AMO eliminates nearly 97% of the airborne methylamine. However, the enzymatic activity of AMO causes formation of formaldehyde. A two-step bioconversion process was therefore proposed. In the first step, airborne methylamine was fed into a CFBR which contained immobilized AMO. In the second step, the gas flow was passed through another CFBR, with alcohol oxidase from the yeast H. polymorpha immobilized in CA, in order to decompose the formaldehyde formed in the first step. The proposed system provided almost total elimination of the airborne methylamine and the formaldehyde.

  11. Cell Wall Amine Oxidases: New Players in Root Xylem Differentiation under Stress Conditions

    Directory of Open Access Journals (Sweden)

    Sandip A. Ghuge

    2015-07-01

    Full Text Available Polyamines (PAs are aliphatic polycations present in all living organisms. A growing body of evidence reveals their involvement as regulators in a variety of physiological and pathological events. They are oxidatively deaminated by amine oxidases (AOs, including copper amine oxidases (CuAOs and flavin adenine dinucleotide (FAD-dependent polyamine oxidases (PAOs. The biologically-active hydrogen peroxide (H2O2 is a shared compound in all of the AO-catalyzed reactions, and it has been reported to play important roles in PA-mediated developmental and stress-induced processes. In particular, the AO-driven H2O2 biosynthesis in the cell wall is well known to be involved in plant wound healing and pathogen attack responses by both triggering peroxidase-mediated wall-stiffening events and signaling modulation of defense gene expression. Extensive investigation by a variety of methodological approaches revealed high levels of expression of cell wall-localized AOs in root xylem tissues and vascular parenchyma of different plant species. Here, the recent progresses in understanding the role of cell wall-localized AOs as mediators of root xylem differentiation during development and/or under stress conditions are reviewed. A number of experimental pieces of evidence supports the involvement of apoplastic H2O2 derived from PA oxidation in xylem tissue maturation under stress-simulated conditions.

  12. Molecular characterisation and expression analysis of acc oxidase gene from guzmania ruiz and pav

    International Nuclear Information System (INIS)

    Jianxin, L.; Huaqiao, D.; Weiyong, W.; Danqing, T.

    2017-01-01

    ACC oxidase is the last key enzyme of ethylene synthesis pathway, while ethylene is a key factor affecting flowering in ornamental bromeliad. To understand ACC oxidase gene's characteristics and its effect on ornamental bromeliad flowering, we cloned 1504bp full-length cDNA sequence (GenBank: JX972145) and 2546bp corresponding genomic sequence (GenBank: JX972146)of GoACO1 (ACC oxidase gene) from Guzmania variety: Ostara. Prokaryotic expression study showed that expression of GoACO1 can produced a 41 KD protein precipitation in Escherichia coli DE3(BL-21); Real-time quantitative analysis showed that GoACO1 can express in all tested tissues including floral organ, bract, leaf and scape, and expression quantity in bract was the highest. Through constructing plant overexpression vector, transforming into Arabidopsis thaliana, and investigating blossom character of T2 generation seeds, we found that first flowering time of the goal Arabidopsis thaliana was 1.5 days earlier, and their peak flowering time(the number of flowering more than 50%) was 1.8 days earlier, compared with wild type one. Taken together, our results suggested that GoACO1can express in all kinds of tissues and seems to promote Arabidopsis thaliana flowering earlier. (author)

  13. Structure of choline oxidase in complex with the reaction product glycine betaine.

    Science.gov (United States)

    Salvi, Francesca; Wang, Yuan-Fang; Weber, Irene T; Gadda, Giovanni

    2014-02-01

    Choline oxidase from Arthrobacter globiformis, which is involved in the biosynthesis of glycine betaine from choline, has been extensively characterized in its mechanistic and structural properties. Despite the knowledge gained on the enzyme, the details of substrate access to the active site are not fully understood. The `loop-and-lid' mechanism described for the glucose-methanol-choline enzyme superfamily has not been confirmed for choline oxidase. Instead, a hydrophobic cluster on the solvent-accessible surface of the enzyme has been proposed by molecular dynamics to control substrate access to the active site. Here, the crystal structure of the enzyme was solved in complex with glycine betaine at pH 6.0 at 1.95 Å resolution, allowing a structural description of the ligand-enzyme interactions in the active site. This structure is the first of choline oxidase in complex with a physiologically relevant ligand. The protein structures with and without ligand are virtually identical, with the exception of a loop at the dimer interface, which assumes two distinct conformations. The different conformations of loop 250-255 define different accessibilities of the proposed active-site entrance delimited by the hydrophobic cluster on the other subunit of the dimer, suggesting a role in regulating substrate access to the active site.

  14. Crystallization and preliminary X-ray analysis of Aspergillus oryzae catechol oxidase

    International Nuclear Information System (INIS)

    Kaljunen, Heidi; Gasparetti, Chiara; Kruus, Kristiina; Rouvinen, Juha; Hakulinen, Nina

    2011-01-01

    Catechol oxidase from A. oryzae was crystallized by the hanging-drop vapour-diffusion method. Catechol oxidase is an enzyme that catalyzes the oxidation of o-diphenols to the corresponding o-quinones. It is a copper-containing enzyme with a binuclear copper active site. Here, the crystallization and multiple-wavelength anomalous dispersion data collection of catechol oxidase from the mould fungus Aspergillus oryzae are described. During the purification, three forms of the enzyme (39.3, 40.5 and 44.3 kDa) were obtained. A mixture of these three forms was initially crystallized and gave crystals that diffracted to 2.5 Å resolution and belonged to space group P3 2 21, with unit-cell parameters a = b = 118.9, c = 84.5 Å, α = β = 90, γ = 120°. A preparation containing only the shorter form (39.3 kDa) produced crystals that diffracted to 2.9 Å resolution and belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 51.8, b = 95.3, c = 139.5 Å, α = β = γ = 90°

  15. Study of allosteric communications in chimeric two-domain proteins

    Czech Academy of Sciences Publication Activity Database

    Boušová, Kristýna

    2017-01-01

    Roč. 26, S1 (2017), s. 74 ISSN 0961-8368. [Annual Symposium of the Protein Society /31./. 24.07.2017-27.07.2017, Montreal] Institutional support: RVO:61388963 Keywords : protein domains * chimeric structures Subject RIV: CE - Biochemistry

  16. Characterization of the cydAB-Encoded Cytochrome bd Oxidase from Mycobacterium smegmatis

    Science.gov (United States)

    Kana, Bavesh D.; Weinstein, Edward A.; Avarbock, David; Dawes, Stephanie S.; Rubin, Harvey; Mizrahi, Valerie

    2001-01-01

    The cydAB genes from Mycobacterium smegmatis have been cloned and characterized. The cydA and cydB genes encode the two subunits of a cytochrome bd oxidase belonging to the widely distributed family of quinol oxidases found in prokaryotes. The cydD and cydC genes located immediately downstream of cydB encode a putative ATP-binding cassette-type transporter. At room temperature, reduced minus oxidized difference spectra of membranes purified from wild-type M. smegmatis displayed spectral features that are characteristic of the γ-proteobacterial type cytochrome bd oxidase. Inactivation of cydA or cydB by insertion of a kanamycin resistance marker resulted in loss of d-heme absorbance at 631 nm. The d-heme could be restored by transformation of the M. smegmatis cyd mutants with a replicating plasmid carrying the highly homologous cydABDC gene cluster from Mycobacterium tuberculosis. Inactivation of cydA had no effect on the ability of M. smegmatis to exit from stationary phase at 37 or 42°C. The growth rate of the cydA mutant was tested under oxystatic conditions. Although no discernible growth defect was observed under moderately aerobic conditions (9.2 to 37.5 × 102 Pa of pO2 or 5 to 21% air saturation), the mutant displayed a significant growth disadvantage when cocultured with the wild type under extreme microaerophilia (0.8 to 1.7 × 102 Pa of pO2 or 0.5 to 1% air saturation). These observations were in accordance with the two- to threefold increase in cydAB gene expression observed upon reduction of the pO2 of the growth medium from 21 to 0.5% air saturation and with the concomitant increase in d-heme absorbance in spectra of membranes isolated from wild-type M. smegmatis cultured at 1% air saturation. Finally, the cydA mutant displayed a competitive growth disadvantage in the presence of the terminal oxidase inhibitor, cyanide, when cocultured with wild type at 21% air saturation in an oxystat. In conjunction with these findings, our results suggest that

  17. RhoA/ROCK downregulates FPR2-mediated NADPH oxidase activation in mouse bone marrow granulocytes.

    Science.gov (United States)

    Filina, Julia V; Gabdoulkhakova, Aida G; Safronova, Valentina G

    2014-10-01

    Polymorphonuclear neutrophils (PMNs) express the high and low affinity receptors to formylated peptides (mFPR1 and mFPR2 in mice, accordingly). RhoA/ROCK (Rho activated kinase) pathway is crucial for cell motility and oxidase activity regulated via FPRs. There are contradictory data on RhoA-mediated regulation of NADPH oxidase activity in phagocytes. We have shown divergent Rho GTPases signaling via mFPR1 and mFPR2 to NADPH oxidase in PMNs from inflammatory site. The present study was aimed to find out the role of RhoA/ROCK in the respiratory burst activated via mFPR1 and mFPR2 in the bone marrow PMNs. Different kinetics of RhoA activation were detected with 0.1μM fMLF and 1μM WKYMVM operating via mFPR1 and mFPR2, accordingly. RhoA was translocated in fMLF-activated cells towards the cell center and juxtamembrane space versus uniform allocation in the resting cells. Specific inhibition of RhoA by CT04, Rho inhibitor I, weakly depressed the respiratory burst induced via mFPR1, but significantly increased the one induced via mFPR2. Inhibition of ROCK, the main effector of RhoA, by Y27632 led to the same effect on the respiratory burst. Regulation of mFPR2-induced respiratory response by ROCK was impossible under the cytoskeleton disruption by cytochalasin D, whereas it persisted in the case of mFPR1 activation. Thus we suggest RhoA to be one of the regulatory and signal transduction components in the respiratory burst through FPRs in the mouse bone marrow PMNs. Both mFPR1 and mFPR2 binding with a ligand trigger the activation of RhoA. FPR1 signaling through RhoA/ROCK increases NADPH-oxidase activity. But in FPR2 action RhoA/ROCK together with cytoskeleton-linked systems down-regulates NADPH-oxidase. This mechanism could restrain the reactive oxygen species dependent damage of own tissues during the chemotaxis of PMNs and in the resting cells. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Nox1 oxidase suppresses influenza a virus-induced lung inflammation and oxidative stress.

    Directory of Open Access Journals (Sweden)

    Stavros Selemidis

    Full Text Available Influenza A virus infection is an ongoing clinical problem and thus, there is an urgent need to understand the mechanisms that regulate the lung inflammation in order to unravel novel generic pharmacological strategies. Evidence indicates that the Nox2-containing NADPH oxidase enzyme promotes influenza A virus-induced lung oxidative stress, inflammation and dysfunction via ROS generation. In addition, lung epithelial and endothelial cells express the Nox1 isoform of NADPH oxidase, placing this enzyme at key sites to regulate influenza A virus-induced lung inflammation. The aim of this study was to investigate whether Nox1 oxidase regulates the inflammatory response and the oxidative stress to influenza infection in vivo in mice. Male WT and Nox1-deficient (Nox1(-/y mice were infected with the moderately pathogenic HkX-31 (H3N2, 1×10(4 PFU influenza A virus for analysis of bodyweight, airways inflammation, oxidative stress, viral titre, lung histopathology, and cytokine/chemokine expression at 3 and 7 days post infection. HkX-31 virus infection of Nox1(-/y mice resulted in significantly greater: loss of bodyweight (Day 3; BALF neutrophilia, peri-bronchial, peri-vascular and alveolar inflammation; Nox2-dependent inflammatory cell ROS production and peri-bronchial, epithelial and endothelial oxidative stress. The expression of pro-inflammatory cytokines including CCL2, CCL3, CXCL2, IL-1β, IL-6, GM-CSF and TNF-α was higher in Nox1(-/y lungs compared to WT mice at Day 3, however, the expression of CCL2, CCL3, CXCL2, IFN-γ and the anti-inflammatory cytokine IL-10 were lower in lungs of Nox1(-/y mice vs. WT mice at Day 7. Lung viral titre, and airways infiltration of active CD8(+ and CD4(+ T lymphocytes, and of Tregs were similar between WT and Nox1(-/y mice. In conclusion, Nox1 oxidase suppresses influenza A virus induced lung inflammation and oxidative stress in mice particularly at the early phases of the infection. Nox1 and Nox2 oxidases appear

  19. Unique role of NADPH oxidase 5 in oxidative stress in human renal proximal tubule cells

    Directory of Open Access Journals (Sweden)

    Peiying Yu

    2014-01-01

    Full Text Available NADPH oxidases are the major sources of reactive oxygen species in cardiovascular, neural, and kidney cells. The NADPH oxidase 5 (NOX5 gene is present in humans but not rodents. Because Nox isoforms in renal proximal tubules (RPTs are involved in the pathogenesis of hypertension, we tested the hypothesis that NOX5 is differentially expressed in RPT cells from normotensive (NT and hypertensive subjects (HT. We found that NOX5 mRNA, total NOX5 protein, and apical membrane NOX5 protein were 4.2±0.7-fold, 5.2±0.7-fold, and 2.8±0.5-fold greater in HT than NT. Basal total NADPH oxidase activity was 4.5±0.2-fold and basal NOX5 activity in NOX5 immunoprecipitates was 6.2±0.2-fold greater in HT than NT (P=<0.001, n=6–14/group. Ionomycin increased total NOX and NOX5 activities in RPT cells from HT (P<0.01, n=4, ANOVA, effects that were abrogated by pre-treatment of the RPT cells with diphenylene-iodonium or superoxide dismutase. Silencing NOX5 using NOX5-siRNA decreased NADPH oxidase activity (−45.1±3.2% vs. mock-siRNA, n=6–8 in HT. D1-like receptor stimulation decreased NADPH oxidase activity to a greater extent in NT (−32.5±1.8% than HT (−14.8±1.8. In contrast to the marked increase in expression and activity of NOX5 in HT, NOX1 mRNA and protein were minimally increased in HT, relative to NT; total NOX2 and NOX4 proteins were not different between HT and NT, while the increase in apical RPT cell membrane NOX1, NOX2, and NOX4 proteins in HT, relative to NT, was much less than those observed with NOX5. Thus, we demonstrate, for the first time, that NOX5 is expressed in human RPT cells and to greater extent than the other Nox isoforms in HT than NT. We suggest that the increased expression of NOX5, which may be responsible for the increased oxidative stress in RPT cells in human essential hypertension, is caused, in part, by a defective renal dopaminergic system.

  20. NADPH oxidase is involved in regulation of gene expression and ROS overproduction in soybean (Glycine max L. seedlings exposed to cadmium

    Directory of Open Access Journals (Sweden)

    Jagna Chmielowska-Bąk

    2017-06-01

    Full Text Available Cadmium-induced oxidative burst is partially mediated by NADPH oxidase. The aim of the present research was to evaluate the role of NADPH oxidase in soybeans’ response to short-term cadmium stress. The application of an NADPH oxidase inhibitor, diphenyleneiodonium chloride (DPI, affected expression of two Cd-inducible genes, encoding DOF1 and MYBZ2 transcription factors. This effect was observed after 3 h of treatment. Interestingly, Cd-dependent increases in NADPH oxidase activity occurred only after a period of time ranging from 6 and 24 h of stress. Stimulation of the enzyme correlated in time with a significant accumulation of reactive oxygen species (ROS. Further analysis revealed that pharmacological inhibition of NADPH oxidase activity during 24 h of Cd stress does not affect Cd uptake, seedling growth, or the level of lipid peroxidation. The role of NADPH oxidase in the response of soybean seedlings to short-term Cd exposure is discussed.

  1. Isolation and Characterization of a Catabolite Repression-Insensitive Mutant of a Methanol Yeast, Candida boidinii A5, Producing Alcohol Oxidase in Glucose-Containing Medium

    OpenAIRE

    Sakai, Yasuyoshi; Sawai, Tohru; Tani, Yoshiki

    1987-01-01

    Mutants exhibiting alcohol oxidase (EC 1.1.3.13) activity when grown on glucose in the presence of methanol were found among 2-deoxyglucose-resistant mutants derived from a methanol yeast, Candida boidinii A5. One of these mutants, strain ADU-15, showed the highest alcohol oxidase activity in glucose-containing medium. The growth characteristics and also the induction and degradation of alcohol oxidase were compared with the parent strain and mutant strain ADU-15. In the parent strain, initia...

  2. Substrate specificity of flavin-dependent vanillyl-alcohol oxidase from Penicillium simplicissimum.Evidence for the production of 4-hydroxycinnamyl alcohols from 4-allylphenols

    OpenAIRE

    Fraaije, Marco W.; Veeger, Cees; Berkel, Willem J.H. van

    1995-01-01

    The substrate specificity of the flavoprotein vanillyl-alcohol oxidase from Penicillium simplicissimum was investigated. Vanillyl-alcohol oxidase catalyzes besides the oxidation of 4-hydroxybenzyl alcohols, the oxidative deamination of 4-hydroxybenzylamines and the oxidative demethylation of 4-(methoxymethyl)phenols. During the conversion of vanillylamine to vanillin, a transient intermediate, most probably vanillylimine, is observed. Vanillyl-alcohol oxidase weakly interacts with 4-hydroxyph...

  3. Myeloperoxidase amplified high glucose-induced endothelial dysfunction in vasculature: Role of NADPH oxidase and hypochlorous acid.

    Science.gov (United States)

    Tian, Rong; Ding, Yun; Peng, Yi-Yuan; Lu, Naihao

    2017-03-11

    Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived reactive oxygen species (ROS) such as superoxide and hydrogen peroxide (H 2 O 2 ), have emerged as important molecules in the pathogenesis of diabetic endothelial dysfunction. Additionally, neutrophils-derived myeloperoxidase (MPO) and MPO-catalyzed hypochlorous acid (HOCl) play important roles in the vascular injury. However, it is unknown whether MPO can use vascular-derived ROS to induce diabetic endothelial dysfunction. In the present study, we demonstrated that NADPH oxidase was the main source of ROS formation in high glucose-cultured human umbilical vein endothelial cells (HUVECs), and played a critical role in high glucose-induced endothelial dysfunction such as cell apoptosis, loss of cell viability and reduction of nitric oxide (NO). However, the addition of MPO could amplify the high glucose-induced endothelial dysfunction which was inhibited by the presence of apocynin (NADPH oxidase inhibitor), catalase (H 2 O 2 scavenger), or methionine (HOCl scavenger), demonstrating the contribution of NADPH oxidase-H 2 O 2 -MPO-HOCl pathway in the MPO/high glucose-induced vascular injury. In high glucose-incubated rat aortas, MPO also exacerbated the NADPH oxidase-induced impairment of endothelium-dependent relaxation. Consistent with these in vitro data, in diabetic rat aortas, both MPO expresion and NADPH oxidase activity were increased while the endothelial function was simultaneously impaired. The results suggested that vascular-bound MPO could amplify high glucose-induced vascular injury in diabetes. MPO-NADPH oxidase-HOCl may represent an important pathogenic pathway in diabetic vascular diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Resveratrol protects vascular endothelial cells from high glucose-induced apoptosis through inhibition of NADPH oxidase activation-driven oxidative stress.

    Science.gov (United States)

    Chen, Feng; Qian, Li-Hua; Deng, Bo; Liu, Zhi-Min; Zhao, Ying; Le, Ying-Ying

    2013-09-01

    Hyperglycemia-induced oxidative stress has been implicated in diabetic vascular complications in which NADPH oxidase is a major source of reactive oxygen species (ROS) generation. Resveratrol is a naturally occurring polyphenol, which has vasoprotective effects in diabetic animal models and inhibits high glucose (HG)-induced oxidative stress in endothelial cells. We aimed to examine whether HG-induced NADPH oxidase activation and ROS production contribute to glucotoxicity to endothelial cells and the effect of resveratrol on glucotoxicity. Using a murine brain microvascular endothelial cell line bEnd3, we found that NADPH oxidase inhibitor (apocynin) and resveratrol both inhibited HG-induced endothelial cell apoptosis. HG-induced elevation of NADPH oxidase activity and production of ROS were inhibited by apocynin, suggesting that HG induces endothelial cell apoptosis through NADPH oxidase-mediated ROS production. Mechanistic studies revealed that HG upregulated NADPH oxidase subunit Nox1 but not Nox2, Nox4, and p22(phox) expression through NF-κB activation, which resulted in elevation of NADPH oxidase activity and consequent ROS production. Resveratrol prevented HG-induced endothelial cell apoptosis through inhibiting HG-induced NF-κB activation, NADPH oxidase activity elevation, and ROS production. HG induces endothelial cell apoptosis through NF-κB/NADPH oxidase/ROS pathway, which was inhibited by resveratrol. Our findings provide new potential therapeutic targets against brain vascular complications of diabetes. © 2013 John Wiley & Sons Ltd.

  5. Combined effect of loss of the caa3 oxidase and Crp regulation drives Shewanella to thrive in redox-stratified environments.

    Science.gov (United States)

    Zhou, Guangqi; Yin, Jianhua; Chen, Haijiang; Hua, Yijie; Sun, Linlin; Gao, Haichun

    2013-09-01

    Shewanella species are a group of facultative Gram-negative microorganisms with remarkable respiration abilities that allow the use of a diverse array of terminal electron acceptors (EA). Like most bacteria, S. oneidensis possesses multiple terminal oxidases, including two heme-copper oxidases (caa3- and cbb3-type) and a bd-type quinol oxidase. As aerobic respiration is energetically favored, mechanisms underlying the fact that these microorganisms thrive in redox-stratified environments remain vastly unexplored. In this work, we discovered that the cbb3-type oxidase is the predominant system for respiration of oxygen (O2), especially when O2 is abundant. Under microaerobic conditions, the bd-type quinol oxidase has a significant role in addition to the cbb3-type oxidase. In contrast, multiple lines of evidence suggest that under test conditions the caa3-type oxidase, an analog to the mitochondrial enzyme, has no physiological significance, likely because of its extremely low expression. In addition, expression of both cbb3- and bd-type oxidases is under direct control of Crp (cAMP receptor protein) but not the well-established redox regulator Fnr (fumarate nitrate regulator) of canonical systems typified in Escherichia coli. These data, collectively, suggest that adaptation of S. oneidensis to redox-stratified environments is likely due to functional loss of the caa3-type oxidase and switch of the regulatory system for respiration.

  6. Evaluation of different disinfectants on the performance of an on-meter dosed amperometric glucose-oxidase-based glucose meter.

    Science.gov (United States)

    Sarmaga, Don; Dubois, Jeffrey A; Lyon, Martha E

    2011-11-01

    Off-meter dosed photometric glucose-oxidase-based glucose meters have been reported to be susceptible to interference by hydrogen-peroxide-based disinfecting agents. The objective of this study was to determine if a single application of hydrogen-peroxide-containing Accel® wipe to disinfect an on-meter dosed amperometric glucose-oxidase-based glucose meter will influence its performance. The performance of five on-meter dosed amperometric glucose-oxidase-based glucose meters was determined before and after disinfecting the devices with a single application of either CaviWipes® (14.3% isopropanol and 0.23% diisobutyl-phenoxy-ethoxyethyl dimethyl benzyl ammonium chloride) or Accel (0.5% hydrogen peroxide) wipes. Replicate glucose measurements were conducted before disinfecting the devices, immediately after disinfecting, and then 1 and 2 min postdisinfecting, with measurements in triplicate. Analysis was sequentially completed for five different meters. Results were analyzed by a two-way analysis of variance (Analyze-it software). No clinical ( .05) in glucose concentration were detected when the on-meter dosed amperometric glucose-oxidase-based glucose meters were disinfected with either CaviWipes or Accel wipes and measured immediately or 1 or 2 min postdisinfecting. No clinically significant difference in glucose concentration was detected between meters (glucose oxidase amperometric-based glucose meters are not analytically susceptible to interference by a single application of hydrogen-peroxide-containing Accel disinfectant wipes. © 2011 Diabetes Technology Society.

  7. Acupuncture elicits neuroprotective effect by inhibiting NAPDH oxidase-mediated reactive oxygen species production in cerebral ischaemia.

    Science.gov (United States)

    Shi, Guang-Xia; Wang, Xue-Rui; Yan, Chao-Qun; He, Tian; Yang, Jing-Wen; Zeng, Xiang-Hong; Xu, Qian; Zhu, Wen; Du, Si-Qi; Liu, Cun-Zhi

    2015-12-10

    In the current study, we aimed to investigate whether NADPH oxidase, a major ROS-producing enzyme, was involved in the antioxidant effect of acupuncture on cognitive impairment after cerebral ischaemia. The cognitive function, infract size, neuron cell loss, level of superoxide anion and expression of NADPH oxidase subunit in hippocampus of two-vessel occlusion (2VO) rats were determined after 2-week acupuncture. Furthermore, the cognitive function and production of O2(-) were determined in the presence and absence of NADPH oxidase agonist (TBCA) and antagonist (Apocynin). The effect of acupuncture on cognitive function after cerebral ischaemia in gp91phox-KO mice was evaluated by Morris water maze. Acupuncture reduced infarct size, attenuated overproduction of O2(-), and reversed consequential cognitive impairment and neuron cell loss in 2VO rats. The elevations of gp91phox and p47phox after 2VO were significantly decreased after acupuncture treatment. However, no differences of gp91phox mRNA were found among any experimental groups. Furthermore, these beneficial effects were reversed by TBCA, whereas apocynin mimicked the effect of acupuncture by improving cognitive function and decreasing O2(-) generation. Acupuncture failed to improve the memory impairment in gp91phox KO mice. Full function of the NADPH oxidase enzyme plays an important role in neuroprotective effects against cognitive impairment via inhibition of NAPDH oxidase-mediated oxidative stress.

  8. Intermittent hypoxia-induced cognitive deficits are mediated by NADPH oxidase activity in a murine model of sleep apnea.

    Directory of Open Access Journals (Sweden)

    Deepti Nair

    Full Text Available In rodents, exposure to intermittent hypoxia (IH, a hallmark of obstructive sleep apnea (OSA, is associated with neurobehavioral impairments, increased apoptosis in the hippocampus and cortex, as well as increased oxidant stress and inflammation. Excessive NADPH oxidase activity may play a role in IH-induced CNS dysfunction.The effect of IH during light period on two forms of spatial learning in the water maze and well as markers of oxidative stress was assessed in mice lacking NADPH oxidase activity (gp91phox(_/Y and wild-type littermates. On a standard place training task, gp91phox(_/Y displayed normal learning, and were protected from the spatial learning deficits observed in wild-type littermates exposed to IH. Moreover, anxiety levels were increased in wild-type mice exposed to IH as compared to room air (RA controls, while no changes emerged in gp91phox(_/Y mice. Additionally, wild-type mice, but not gp91phox(_/Y mice had significantly elevated levels of NADPH oxidase expression and activity, as well as MDA and 8-OHDG in cortical and hippocampal lysates following IH exposures.The oxidative stress responses and neurobehavioral impairments induced by IH during sleep are mediated, at least in part, by excessive NADPH oxidase activity, and thus pharmacological agents targeting NADPH oxidase may provide a therapeutic strategy in sleep-disordered breathing.

  9. Dynamic 1-aminocyclopropane-1-carboxylate-synthase and -oxidase transcript accumulation patterns during pollen tube growth in tobacco styles.

    Science.gov (United States)

    Weterings, Koen; Pezzotti, Mario; Cornelissen, Marc; Mariani, Celestina

    2002-11-01

    In flowering plants, pollination of the stigma sets off a cascade of responses in the distal flower organs. Ethylene and its biosynthetic precursor 1-aminocyclopropane-1-carboxylate (ACC) play an important role in regulating these responses. Because exogenous application of ethylene or ACC does not invoke the full postpollination syndrome, the pollination signal probably consists of a more complex set of stimuli. We set out to study how and when the pollination signal moves through the style of tobacco (Nicotiana tabacum) by analyzing the expression patterns of pistil-expressed ACC-synthase and -oxidase genes. Results from this analysis showed that pollination induces high ACC-oxidase transcript levels in all cells of the transmitting tissue. ACC-synthase mRNA accumulated only in a subset of transmitting tract cells and to lower levels as compared with ACC-oxidase. More significantly, we found that although ACC-oxidase transcripts accumulate to uniform high levels, the ACC-synthase transcripts accumulate in a wave-like pattern in which the peak coincides with the front of the ingrowing pollen tube tips. This wave of ACC-synthase expression can also be induced by incongruous pollination and (partially) by wounding. This indicates that wounding-like features of pollen tube invasion might be part of the stimuli evoking the postpollination response and that these stimuli are interpreted differently by the regulatory mechanisms of the ACC-synthase and -oxidase genes.

  10. Interference of aldehyde metabolizing enzyme with diamine oxidase/histaminase/activity as determined by /sup 14/C putrescine method

    Energy Technology Data Exchange (ETDEWEB)

    Fogel, W A [Polish Academy of Sciences, Cracow (Poland). Inst. of Pharmacology; Bieganski, T; Wozniak, J; Maslinski, C

    1978-01-01

    The ..delta../sup 1/ pyrroline formation, as an indicator of diamine oxidase activity according to Okuyama and Kobayashi /sup 14/C putrescine test (1961, Archs Biochem. Biophys., vol.95, 242), has been investigated in several tissue homogenates. When guinea pig liver homogenate was used as a source of enzyme in the presence of aldehyde dehydrogenase inhibitors chlorate hydrate and acetaldehyde the level of formation ..delta../sup 1/ pyrroline was strongly increased in a dose-dependent manner. Also inhibition of aldehyde reductase by phenobarbital enhanced ..delta../sup 1/ pyrroline formation, but to a lesser degree. In other tissues, with very high initial diamine oxidase activity (rat intestine, dog kidney) or with very low diamine oxidase activity (guinea pig skin, dog liver) the influence of these inhibitors was only slight. Pyrazole, an inhibitor of alcohol dehydrogenase exerted only a small effect on ..delta../sup 1/ pyrroline formation. All aldehyde-metabolizing enzymes inhibitors, except pyrazole, were without effect on purified pea seddling and hog kidney diamine oxidases. The use of aldehyde-metabolizing enzymes inhibitors may help to reveal the real values of diamine oxidase activity, when tissues homogenates are used as a source of enzyme.

  11. Interference of aldehyde metabolizing enzyme with diamine oxidase/histaminase/activity as determined by 14C putrescine method

    International Nuclear Information System (INIS)

    Fogel, W.A.; Bieganski, T.; Wozniak, J.; Maslinski, C.

    1978-01-01

    The Δ 1 pyrroline formation, as an indicator of diamine oxidase activity according to Okuyama and Kobayashi 14 C putrescine test (1961, Archs Biochem. Biophys., vol.95, 242), has been investigated in several tissue homogenates. When guinea pig liver homogenate was used as a source of enzyme in the presence of aldehyde dehydrogenase inhibitors chlorate hydrate and acetaldehyde the level of formation Δ 1 pyrroline was strongly increased in a dose-dependent manner. Also inhibition of aldehyde reductase by phenobarbital enhanced Δ 1 pyrroline formation, but to a lesser degree. In other tissues, with very high initial diamine oxidase activity (rat intestine, dog kidney) or with very low diamine oxidase activity (guinea pig skin, dog liver) the influence of these inhibitors was only slight. Pyrazole, an inhibitor of alcohol dehydrogenase exerted only a small effect on Δ 1 pyrroline formation. All aldehyde-metabolizing enzymes inhibitors, except pyrazole, were without effect on purified pea seddling and hog kidney diamine oxidases. The use of aldehyde-metabolizing enzymes inhibitors may help to reveal the real values of diamine oxidase activity, when tissues homogenates are used as a source of enzyme. (author)

  12. Distinguishing the Roles of Thylakoid Respiratory Terminal Oxidases in the Cyanobacterium Synechocystis sp. PCC 6803.

    Science.gov (United States)

    Ermakova, Maria; Huokko, Tuomas; Richaud, Pierre; Bersanini, Luca; Howe, Christopher J; Lea-Smith, David J; Peltier, Gilles; Allahverdiyeva, Yagut

    2016-06-01

    Various oxygen-utilizing electron sinks, including the soluble flavodiiron proteins (Flv1/3), and the membrane-localized respiratory terminal oxidases (RTOs), cytochrome c oxidase (Cox) and cytochrome bd quinol oxidase (Cyd), are present in the photosynthetic electron transfer chain of Synechocystis sp. PCC 6803. However, the role of individual RTOs and their relative importance compared with other electron sinks are poorly understood, particularly under light. Via membrane inlet mass spectrometry gas exchange, chlorophyll a fluorescence, P700 analysis, and inhibitor treatment of the wild type and various mutants deficient in RTOs, Flv1/3, and photosystem I, we investigated the contribution of these complexes to the alleviation of excess electrons in the photosynthetic chain. To our knowledge, for the first time, we demonstrated the activity of Cyd in oxygen uptake under light, although it was detected only upon inhibition of electron transfer at the cytochrome b6f site and in ∆flv1/3 under fluctuating light conditions, where linear electron transfer was drastically inhibited due to impaired photosystem I activity. Cox is mostly responsible for dark respiration and competes with P700 for electrons under high light. Only the ∆cox/cyd double mutant, but not single mutants, demonstrated a highly reduced plastoquinone pool in darkness and impaired gross oxygen evolution under light, indicating that thylakoid-based RTOs are able to compensate partially for each other. Thus, both electron sinks contribute to the alleviation of excess electrons under illumination: RTOs continue to function under light, operating on slower time ranges and on a limited scale, whereas Flv1/3 responds rapidly as a light-induced component and has greater capacity. © 2016 American Society of Plant Biologists. All Rights Reserved.

  13. Davallialactone reduces inflammation and repairs dentinogenesis on glucose oxidase-induced stress in dental pulp cells.

    Science.gov (United States)

    Lee, Young-Hee; Kim, Go-Eun; Song, Yong-Beom; Paudel, Usha; Lee, Nan-Hee; Yun, Bong-Sik; Yu, Mi-Kyung; Yi, Ho-Keun

    2013-11-01

    The chronic nature of diabetes mellitus (DM) raises the risk of oral complication diseases. In general, DM causes oxidative stress to organs. This study aimed to evaluate the cellular change of dental pulp cells against glucose oxidative stress by glucose oxidase with a high glucose state. The purpose of this study was to test the antioxidant character of davallialactone and to reduce the pathogenesis of dental pulp cells against glucose oxidative stress. The glucose oxidase with a high glucose concentration was tested for hydroxy peroxide (H2O2) production, cellular toxicity, reactive oxygen species (ROS) formation, induction of inflammatory molecules and disturbance of dentin mineralization in human dental pulp cells. The anti-oxidant effect of Davallilactone was investigated to restore dental pulp cells' vitality and dentin mineralization via reduction of H2O2 production, cellular toxicity, ROS formation and inflammatory molecules. The treatment of glucose oxidase with a high glucose concentration increased H2O2 production, cellular toxicity, and inflammatory molecules and disturbed dentin mineralization by reducing pulp cell activity. However, davallialactone reduced H2O2 production, cellular toxicity, ROS formation, inflammatory molecules, and dentin mineralization disturbances even with a long-term glucose oxidative stress state. The results of this study imply that the development of oral complications is related to the irreversible damage of dental pulp cells by DM-induced oxidative stress. Davallialactone, a natural antioxidant, may be useful to treat complicated oral disease, representing an improvement for pulp vital therapy. Copyright © 2013 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  14. The amine oxidase inhibitor phenelzine limits lipogenesis in adipocytes without inhibiting insulin action on glucose uptake.

    Science.gov (United States)

    Carpéné, Christian; Grès, Sandra; Rascalou, Simon

    2013-06-01

    The antidepressant phenelzine is a monoamine oxidase inhibitor known to inhibit various other enzymes, among them semicarbazide-sensitive amine oxidase (currently named primary amine oxidase: SSAO/PrAO), absent from neurones but abundant in adipocytes. It has been reported that phenelzine inhibits adipocyte differentiation of cultured preadipocytes. To further explore the involved mechanisms, our aim was to study in vitro the acute effects of phenelzine on de novo lipogenesis in mature fat cells. Therefore, glucose uptake and incorporation into lipid were measured in mouse adipocytes in response to phenelzine, other hydrazine-based SSAO/PrAO-inhibitors, and reference agents. None of the inhibitors was able to impair the sevenfold activation of 2-deoxyglucose uptake induced by insulin. Phenelzine did not hamper the effect of lower doses of insulin. However, insulin-stimulated glucose incorporation into lipids was dose-dependently inhibited by phenelzine and pentamidine, but not by semicarbazide or BTT2052. In contrast, all these SSAO/PrAO inhibitors abolished the transport and lipogenesis stimulation induced by benzylamine. These data indicate that phenelzine does not inhibit glucose transport, the first step of lipogenesis, but inhibits at 100 μM the intracellular triacylglycerol assembly, consistently with its long-term anti-adipogenic effect and such rapid action was not found with all the hydrazine derivatives tested. Therefore, the alterations of body weight control consecutive to the use of this antidepressant drug might be not only related to central effects on food intake/energy expenditure, but could also depend on its direct action in adipocytes. Nonetheless, phenelzine antilipogenic action is not merely dependent on SSAO/PrAO inhibition.

  15. Stability of glucose oxidase and catalase adsorbed on variously activated 13X zeolite.

    Science.gov (United States)

    Pifferi, P G; Vaccari, A; Ricci, G; Poli, G; Ruggeri, O

    1982-10-01

    The use of 13X zeolite (0.1-0.4-mm granules), treated with 2N and 0.01N HCI, 0.01M citric acid, 0.1M citric-phosphate buffer (pH 3.6), and in untreated form to adsorb glucose oxidase of fungal origin and microbial catalase was examined. Physicochemical analysis of the support demonstrated that its crystalline structure, greatly altered by the HCl and buffer, could be partially maintained with citric acid. The specific adsorption of the enzymes increased with decreasing pH and proved to be considerable for all the supports. The stability with storage at 25 degrees C is strictly correlated with the titrable acidity of the activated zeolite expressed as meq NaOH/g and with pH value of the activation solution. It proved to be lower than 55 h for both enzymes if adsorbed on zeolite treated with 2N HCl, and 15-fold and 30-fold higher for glucose oxidase and catalase adsorbed, respectively, on zeolite treated with the 0.1M citric-phosphate buffer and 0.01M citric acid. The specific adsorption of glucose oxidase and catalase was, respectively, 1840 U/g at pH 3.0 and 6910 U/g at pH 5.0. Their half-life at 25 degrees C with storage at pH 3.5 for the former and at pH 5.0 for the latter was 800 and 1560 h vs. 40 and 110 h for the corresponding free enzymes.

  16. NADPH Oxidase-Mediated ROS Production Determines Insulin's Action on the Retinal Microvasculature.

    Science.gov (United States)

    Kida, Teruyo; Oku, Hidehiro; Horie, Taeko; Matsuo, Junko; Kobayashi, Takatoshi; Fukumoto, Masanori; Ikeda, Tsunehiko

    2015-10-01

    To determine whether insulin induces nitric oxide (NO) formation in retinal microvessels and to examine the effects of high glucose on the formation of NO. Freshly isolated rat retinal microvessels were incubated in normal (5.5 mM) or high (20 mM) glucose with or without insulin (100 nM). The levels of insulin-induced NO and reactive oxygen species (ROS) in the retinal microvessels were determined semiquantitatively using fluorescent probes, 4,5-diaminofluorescein diacetate, and hydroethidine, respectively, and a laser scanning confocal microscope. The insulin-induced changes of NO in rat retinal endothelial cells and pericytes cultured at different glucose concentrations (5.5 and 25 mM) were determined using flow cytometry. Nitric oxide synthase (NOS) protein levels were determined by Western blot analysis; intracellular levels of ROS were determined using fluorescence-activated cell sorting (FACS) analysis of ethidium fluorescence; and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase RNA expression was quantified using real-time PCR. Exposure of microvessels to insulin under normal glucose conditions led to a significant increase in NO levels; however, this increase was significantly suppressed when the microvessels were incubated under high glucose conditions. Intracellular levels of ROS were significantly increased in both retinal microvessels and cultured microvascular cells under high glucose conditions. The expression of NOS and NADPH oxidase were significantly increased in endothelial cells and pericytes under high glucose conditions. The increased formation of NO by insulin and its suppression by high glucose conditions suggests that ROS production mediated by NADPH oxidase is important by insulin's effect on the retinal microvasculature.

  17. Mitochondrial terminal alternative oxidase and its enhancement by thermal stress in the coral symbiont Symbiodinium

    Science.gov (United States)

    Oakley, Clinton A.; Hopkinson, Brian M.; Schmidt, Gregory W.

    2014-06-01

    A terminal electron acceptor alternative to mitochondrial cytochrome c oxidase (COX), mitochondrial alternative oxidase (AOX), is ubiquitous in higher plants and represented in nearly every algal taxon but is poorly documented in dinoflagellates. AOX competes for electrons with the conventional COX and has been hypothesized to function as a means of reducing oxidative stress in mitochondria, as well as a potential mechanism for ameliorating thermal and other physiological stressors. Here, the presence of an active AOX in cultured Symbiodinium was assayed by the response of oxygen consumption to the AOX inhibitor salicylhydroxamic acid (SHAM) and the COX inhibitor cyanide (CN). CN-insensitive, SHAM-sensitive oxygen consumption was found to account for a large portion (26 %) of Symbiodinium dark respiration and is consistent with high levels of AOX activity. This experimental evidence of the existence of a previously unreported terminal oxidase was further corroborated by analysis of publicly available Symbiodinium transcriptome data. The potential for enhanced AOX expression to play a compensatory role in mediating thermal stress was supported by inhibitor assays of cultured Symbiodinium at low (18 °C), moderate (26 °C), and high (32 °C) temperature conditions. Maximum capacity of the putative AOX pathway as a proportion of total dark oxygen consumption was found to increase from 26 % at 26 °C to 45 % and 53 % at 18 °C and 32 °C, respectively, when cells were acclimated to the treatment temperatures. Cells assayed at 18 and 32 °C without acclimation exhibited either the same or lower AOX capacity as controls, suggesting that the AOX protein is upregulated under temperature stress. The physiological implications for the presence of AOX in the coral/algal symbiosis and its potential role in response to many forms of biotic and abiotic stress, particularly oxidative stress, are discussed.

  18. fMLP-Induced IL-8 Release Is Dependent on NADPH Oxidase in Human Neutrophils

    Directory of Open Access Journals (Sweden)

    María A. Hidalgo

    2015-01-01

    Full Text Available N-Formyl-methionyl-leucyl-phenylalanine (fMLP and platelet-activating factor (PAF induce similar intracellular signalling profiles; but only fMLP induces interleukin-8 (IL-8 release and nicotinamide adenine dinucleotide phosphate reduced (NADPH oxidase activity in neutrophils. Because the role of ROS on IL-8 release in neutrophils is until now controversial, we assessed if NADPH oxidase is involved in the IL-8 secretions and PI3K/Akt, MAPK, and NF-κB pathways activity induced by fMLP. Neutrophils were obtained from healthy volunteers. IL-8 was measured by ELISA, IL-8 mRNA by qPCR, and ROS production by luminol-amplified chemiluminescence, reduction of ferricytochrome c, and FACS. Intracellular pH changes were detected by spectrofluorescence. ERK1/2, p38 MAPK, and Akt phosphorylation were analysed by immunoblotting and NF-κB was analysed by immunocytochemistry. Hydroxy-3-methoxyaceto-phenone (HMAP, diphenyleneiodonium (DPI, and siRNA Nox2 reduced the ROS and IL-8 release in neutrophils treated with fMLP. HMAP, DPI, and amiloride (a Na+/H+ exchanger inhibitor inhibited the Akt phosphorylation and did not affect the p38 MAPK and ERK1/2 activity. DPI and HMAP reduced NF-κB translocation induced by fMLP. We showed that IL-8 release induced by fMLP is dependent on NADPH oxidase, and ROS could play a redundant role in cell signalling, ultimately activating the PI3K/Akt and NF-κB pathways in neutrophils.

  19. Urotensin II inhibits skeletal muscle glucose transport signaling pathways via the NADPH oxidase pathway.

    Directory of Open Access Journals (Sweden)

    Hong-Xia Wang

    Full Text Available Our previous studies have demonstrated that the urotensin (UII and its receptor are up-regulated in the skeletal muscle of mice with type II diabetes mellitus (T2DM, but the significance of UII in skeletal muscle insulin resistance remains unknown. The purpose of this study was to investigate the effect of UII on NADPH oxidase and glucose transport signaling pathways in the skeletal muscle of mice with T2DM and in C2C12 mouse myotube cells. KK/upj-AY/J mice (KK mice were divided into the following groups: KK group, with saline treatment for 2 weeks; KK+ urantide group, with daily 30 µg/kg body weight injections over the same time period of urantide, a potent urotensin II antagonist peptide; Non-diabetic C57BL/6J mice were used as normal controls. After urantide treatment, mice were subjected to an intraperitoneal glucose tolerance test, in addition to measurements of the levels of ROS, NADPH oxidase and the phosphorylated AKT, PKC and ERK. C2C12 cells were incubated with serum-free DMEM for 24 hours before conducting the experiments, and then administrated with 100 nM UII for 2 hours or 24 hours. Urantide treatment improved glucose tolerance, decreased the translocation of the NADPH subunits p40-phox and p47-phox, and increased levels of the phosphorylated PKC, AKT and ERK. In contrast, UII treatment increased ROS production and p47-phox and p67-phox translocation, and decreased the phosphorylated AKT, ERK1/2 and p38MAPK; Apocynin abrogated this effect. In conclusion, UII increased ROS production by NADPH oxidase, leading to the inhibition of signaling pathways involving glucose transport, such as AKT/PKC/ERK. Our data imply a role for UII at the molecular level in glucose homeostasis, and possibly in skeletal muscle insulin resistance in T2DM.

  20. Isolated cytochrome c oxidase deficiency in G93A SOD1 mice overexpressing CCS protein.

    Science.gov (United States)

    Son, Marjatta; Leary, Scot C; Romain, Nadine; Pierrel, Fabien; Winge, Dennis R; Haller, Ronald G; Elliott, Jeffrey L

    2008-05-02

    G93A SOD1 transgenic mice overexpressing CCS protein develop an accelerated disease course that is associated with enhanced mitochondrial pathology and increased mitochondrial localization of mutant SOD1. Because these results suggest an effect of mutant SOD1 on mitochondrial function, we assessed the enzymatic activities of mitochondrial respiratory chain complexes in the spinal cords of CCS/G93A SOD1 and control mice. CCS/G93A SOD1 mouse spinal cord demonstrates a 55% loss of complex IV (cytochrome c oxidase) activity compared with spinal cord from age-matched non-transgenic or G93A SOD1 mice. In contrast, CCS/G93A SOD1 spinal cord shows no reduction in the activities of complex I, II, or III. Blue native gel analysis further demonstrates a marked reduction in the levels of complex IV but not of complex I, II, III, or V in spinal cords of CCS/G93A SOD1 mice compared with non-transgenic, G93A SOD1, or CCS/WT SOD1 controls. With SDS-PAGE analysis, spinal cords from CCS/G93A SOD1 mice showed significant decreases in the levels of two structural subunits of cytochrome c oxidase, COX1 and COX5b, relative to controls. In contrast, CCS/G93A SOD1 mouse spinal cord showed no reduction in levels of selected subunits from complexes I, II, III, or V. Heme A analyses of spinal cord further support the existence of cytochrome c oxidase deficiency in CCS/G93A SOD1 mice. Collectively, these results establish that CCS/G93A SOD1 mice manifest an isolated complex IV deficiency which may underlie a substantial part of mutant SOD1-induced mitochondrial cytopathy.

  1. NADPH Oxidase Contributes to Resistance against Aggregatibacter actinomycetemcomitans-Induced Periodontitis in Mice.

    Science.gov (United States)

    Bast, Antje; Kubis, Helen; Holtfreter, Birte; Ribback, Silvia; Martin, Heiner; Schreiner, Helen C; Dominik, Malte J; Breitbach, Katrin; Dombrowski, Frank; Kocher, Thomas; Steinmetz, Ivo

    2017-02-01

    Aggregatibacter actinomycetemcomitans is a Gram-negative commensal bacterium of the oral cavity which has been associated with the pathogenesis of periodontitis with severe alveolar bone destruction. The role of host factors such as reactive oxygen and nitrogen intermediates in periodontal A. actinomycetemcomitans infection and progression to periodontitis is still ill-defined. Therefore, this study aimed to analyze the role of NADPH oxidase and inducible nitric oxide synthase (iNOS) in a murine model of A. actinomycetemcomitans-induced periodontitis. NADPH oxidase-deficient (gp91 phox knockout [KO]), iNOS-deficient (iNOS KO), and C57BL/6 wild-type mice were orally infected with A. actinomycetemcomitans and analyzed for bacterial colonization at various time points. Alveolar bone mineral density and alveolar bone volume were quantified by three-dimensional micro-computed tomography, and the degree of tissue inflammation was calculated by histological analyses. At 5 weeks after infection, A. actinomycetemcomitans persisted at significantly higher levels in the murine oral cavities of infected gp91 phox KO mice than in those of iNOS KO and C57BL/6 mice. Concomitantly, alveolar bone mineral density was significantly lower in all three infected groups than in uninfected controls, but with the highest loss of bone density in infected gp91 phox KO mice. Only infected gp91 phox KO mice revealed significant loss of alveolar bone volume and enhanced inflammatory cell infiltration, as well as an increased number of osteoclasts. Our results indicate that NADPH oxidase is important to control A. actinomycetemcomitans infection in the murine oral cavity and to prevent subsequent alveolar bone destruction and osteoclastogenesis. Copyright © 2017 American Society for Microbiology.

  2. A preliminary neutron diffraction study of rasburicase, a recombinant urate oxidase enzyme, complexed with 8-azaxanthin

    Energy Technology Data Exchange (ETDEWEB)

    Budayova-Spano, Monika, E-mail: spano@embl-grenoble.fr [European Molecular Biology Laboratory Grenoble Outstation, 6 Rue Jules Horowitz, 38042 Grenoble (France); Institut Laue-Langevin, 6 Rue Jules Horowitz, BP 156, 38042 Grenoble (France); Bonneté, Françoise; Ferté, Natalie [Centre de Recherche en Matière Condensée et Nanosciences, Campus de Luminy, Case 913, 13288 Marseille (France); El Hajji, Mohamed [Sanofi-Aventis, 371 Rue du Professeur Blayac, 34184 Montpellier (France); Meilleur, Flora [Institut Laue-Langevin, 6 Rue Jules Horowitz, BP 156, 38042 Grenoble (France); Blakeley, Matthew Paul [European Molecular Biology Laboratory Grenoble Outstation, 6 Rue Jules Horowitz, 38042 Grenoble (France); Castro, Bertrand [Sanofi-Aventis, 371 Rue du Professeur Blayac, 34184 Montpellier (France); European Molecular Biology Laboratory Grenoble Outstation, 6 Rue Jules Horowitz, 38042 Grenoble (France)

    2006-03-01

    Neutron diffraction data of hydrogenated recombinant urate oxidase enzyme (Rasburicase), complexed with a purine-type inhibitor 8-azaxanthin, was collected to 2.1 Å resolution from a crystal grown in D{sub 2}O by careful control and optimization of crystallization conditions via knowledge of the phase diagram. Deuterium atoms were clearly seen in the neutron-scattering density map. Crystallization and preliminary neutron diffraction measurements of rasburicase, a recombinant urate oxidase enzyme expressed by a genetically modified Saccharomyces cerevisiae strain, complexed with a purine-type inhibitor (8-azaxanthin) are reported. Neutron Laue diffraction data were collected to 2.1 Å resolution using the LADI instrument from a crystal (grown in D{sub 2}O) with volume 1.8 mm{sup 3}. The aim of this neutron diffraction study is to determine the protonation states of the inhibitor and residues within the active site. This will lead to improved comprehension of the enzymatic mechanism of this important enzyme, which is used as a protein drug to reduce toxic uric acid accumulation during chemotherapy. This paper illustrates the high quality of the neutron diffraction data collected, which are suitable for high-resolution structural analysis. In comparison with other neutron protein crystallography studies to date in which a hydrogenated protein has been used, the volume of the crystal was relatively small and yet the data still extend to high resolution. Furthermore, urate oxidase has one of the largest primitive unit-cell volumes (space group I222, unit-cell parameters a = 80, b = 96, c = 106 Å) and molecular weights (135 kDa for the homotetramer) so far successfully studied with neutrons.

  3. Genomic sequencing of uric acid metabolizing and clearing genes in relationship to xanthine oxidase inhibitor dose.

    Science.gov (United States)

    Carroll, Matthew B; Smith, Derek M; Shaak, Thomas L

    2017-03-01

    It remains unclear why the dose of xanthine oxidase inhibitors (XOI) allopurinol or febuxostat varies among patients though they reach similar serum uric acid (SUA) goal. We pursued genomic sequencing of XOI metabolism and clearance genes to identify single-nucleotide polymorphisms (SNPs) relate to differences in XOI dose. Subjects with a diagnosis of Gout based on the 1977 American College of Rheumatology Classification Criteria for the disorder, who were on stable doses of a XOI, and who were at their goal SUA level, were enrolled. The primary outcome was relationship between SNPs in any of these genes to XOI dose. The secondary outcome was relationship between SNPs and change in pre- and post-treatment SUA. We enrolled 100 subjects. The average patient age was 68.6 ± 10.6 years old. Over 80% were men and 77% were Caucasian. One SNP was associated with a higher XOI dose: rs75995567 (p = 0.031). Two SNPs were associated with 300 mg daily of allopurinol: rs11678615 (p = 0.022) and rs3731722 on Aldehyde Oxidase (AO) (His1297Arg) (p = 0.001). Two SNPs were associated with a lower dose of allopurinol: rs1884725 (p = 0.033) and rs34650714 (p = 0.006). For the secondary outcome, rs13415401 was the only SNP related to a smaller mean SUA change. Ten SNPs were identified with a larger change in SUA. Though multiple SNPs were identified in the primary and secondary outcomes of this study, rs3731722 is known to alter catalytic function for some aldehyde oxidase substrates.

  4. Heterologous expression of the Crassostrea gigas (Pacific oyster) alternative oxidase in the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Robertson, Aaron; Schaltz, Kyle; Neimanis, Karina; Staples, James F; McDonald, Allison E

    2016-10-01

    Alternative oxidase (AOX) is a terminal oxidase within the inner mitochondrial membrane (IMM) present in many organisms where it functions in the electron transport system (ETS). AOX directly accepts electrons from ubiquinol and is therefore capable of bypassing ETS Complexes III and IV. The human genome does not contain a gene coding for AOX, so AOX expression has been suggested as a gene therapy for a range of human mitochondrial diseases caused by genetic mutations that render Complex III and/or IV dysfunctional. An effective means of screening mutations amenable to AOX treatment remains to be devised. We have generated such a tool by heterologously expressing AOX from the Pacific oyster (Crassostrea gigas) in the yeast Saccharomyces cerevisiae under the control of a galactose promoter. Our results show that this animal AOX is monomeric and is correctly targeted to yeast mitochondria. Moreover, when expressed in yeast, Pacific oyster AOX is a functional quinol oxidase, conferring cyanide-resistant growth and myxothiazol-resistant oxygen consumption to yeast cells and isolated mitochondria. This system represents a high-throughput screening tool for determining which Complex III and IV genetic mutations in yeast will be amenable to AOX gene therapy. As many human genes are orthologous to those found in yeast, our invention represents an efficient and cost-effective way to evaluate viable research avenues. In addition, this system provides the opportunity to learn more about the localization, structure, and regulation of AOXs from animals that are not easily reared or manipulated in the lab.

  5. Proton translocation stoichiometry of cytochrome oxidase: use of a fast-responding oxygen electrode.

    Science.gov (United States)

    Reynafarje, B; Alexandre, A; Davies, P; Lehninger, A L

    1982-01-01

    The mechanistic stoichiometry of vectorial H+ ejection coupled to electron transport from added ferrocytochrome c to oxygen by the cytochrome oxidase (EC 1.9.3.1) of rat liver mitoplasts was determined from measurements of the initial rates of electron flow and H+ ejection in the presence of K+ (with valinomycin). Three different methods of measuring electron flow were used: (a) dual-wavelength spectrophotometry of ferrocytochrome c oxidation, (b) uptake of scalar H+ for the reduction of O2 in the presence of a protonophore, and (c) a fast-responding membraneless oxygen electrode. The reliability of the rate measurements was first established against the known stoichiometry of the scalar reaction of cytochrome oxidase (2ferrocytochrome c + 2H+ + 1/2O2 leads to 2ferricytochrome c + H2O) in the presence of excess protonophore. With all three methods the directly observed vectorial H+/O ejection ratios in the presence of K+ + valinomycin significantly exceeded 3.0. However, because the rate of backflow of the ejected H+ into the mitoplasts is very high and increases with the increasing delta pH generated across the membrane, there is a very rapid decline in the observed H+/O ratio from the beginning of the reaction. Kinetic analysis of ferrocytochrome c oxidation by the mitoplasts, carried out with a fast-responding membraneless oxygen electrode, showed the reaction to be first order in O2 and allowed accurate extrapolation of the rates of O2 uptake and H+ ejection to zero time. At this point, at which there is zero delta pH across the membrane, the H+/O ejection ratio of the cytochrome oxidase reaction, obtained from the rates at zero time, is close to 4.0. PMID:6296824

  6. Identification and characterization of an antennae-specific aldehyde oxidase from the navel orangeworm.

    Directory of Open Access Journals (Sweden)

    Young-Moo Choo

    Full Text Available Antennae-specific odorant-degrading enzymes (ODEs are postulated to inactivate odorant molecules after they convey their signal. Different classes of insect ODEs are specific to esters, alcohols, and aldehydes--the major functional groups of female-produced, hydrophobic sex pheromones from moth species. Esterases that rapidly inactive acetate and other esters have been well-studied, but less is known about aldehyde oxidases (AOXs. Here we report cloning of an aldehyde oxidase, AtraAOX2, from the antennae of the navel orangeworm (NOW, Amyelois transitella, and the first activity characterization of a recombinant insect AOX. AtraAOX2 gene spans 3,813 bp and encodes a protein with 1,270 amino acid residues. AtraAOX2 cDNA was expressed in baculovirus-infected insect Sf21 cells as a ≈280 kDa homodimer with 140 kDa subunits. Recombinant AtraAOX2 degraded Z11Z13-16Ald and plant volatile aldehydes as substrates. However, as expected for aldehyde oxidases, recombinant AtraAOX2 did not show specificity for Z11Z13-16Ald, the main constituent of the sex pheromone, but showed high activity for plant volatile aldehydes. Our data suggest AtraAOX2 might be involved in degradation of a diversity of aldehydes including sex pheromones, plant-derived semiochemicals, and chemical cues for oviposition sites. Additionally, AtraAOX2 could protect the insect's olfactory system from xenobiotics, including pesticides that might reach the sensillar lymph surrounding the olfactory receptor neurons.

  7. Protection by deferoxamine from endothelial injury: A possible link with inhibition of intracellular xanthine oxidase

    International Nuclear Information System (INIS)

    Rinaldo, J.E.; Gorry, M.

    1990-01-01

    Hydroxyl radical scavengers and xanthine oxidase inhibitors protect cultured bovine pulmonary endothelial cells (BPAEC) from lytic injury by the endotoxin lipopolysaccharide (LPS). We hypothesized that exposure of BPAEC to cytotoxic concentrations of LPS activated intracellular xanthine oxidase, and that intracellular iron-dependent hydroxyl radical formation (a Fenton reaction) ensued, resulting in cell lysis. To test this, the protective effects of deferoxamine against H2O2 and LPS-induced cytotoxicity to BPAEC was assessed by 51Cr release. Preincubation with 0.4 mM deferoxamine conferred 67 +/- 15% (mean +/- SE) protection from LPS-induced cytotoxicity but 48 h of preincubation were required to induce significant protection. Significant protection form a classical Fenton reaction model, injury by 50 microM H2O2, could be induced by a 1-h preincubation with a 0.4 mM deferoxamine. The dissociated time course suggested that deferoxamine might work by different mechanisms in these models. The effects of LPS and deferoxamine on BPAEC-associated xanthine oxidase (XO) and xanthine dehydrogenase (XD) activity were assessed using a spectrofluorophotometric measurement of the conversion of pterin to isoxanthopterin. BPAEC had 106 +/- 7 microU/mg XD+XO activity; XO activity constituted 48 +/- 1% of total XO+XD activity. LPS at a cytotoxic concentration did not alter XO, XD, or percent XO. Deferoxamine had striking proportional inhibitory effects on XO and XD in intact cells. XO+XD activity fell to 6 +/- 1% of control levels during a 48-h exposure of BPAEC to deferoxamine. Deferoxamine did not inhibit XO+XD ex vivo

  8. Gastric mucosal injury in the rat. Role of iron and xanthine oxidase

    International Nuclear Information System (INIS)

    Smith, S.M.; Grisham, M.B.; Manci, E.A.; Granger, D.N.; Kvietys, P.R.

    1987-01-01

    Recent studies have implicated oxygen free radicals in ischemia-reperfusion injury to the gastric mucosa. The aims of the present study were to test the hypothesis that the enzyme xanthine oxidase is the source of the oxygen radicals in the ischemic stomach and determine the importance of the iron-catalyzed Haber-Weiss reaction in generating the cytotoxic oxygen radicals. Gastric mucosal clearance of 51 Cr-labeled red blood cells was measured during a 30-min control period, a 30-min ischemic period (hemorrhage to 25 mmHg arterial pressure), and a 60-80-min reperfusion period (reinfusion of shed blood). In untreated (control) rats, a dramatic rise (100-fold) in the leakage of 51 Cr-labeled red blood cells into the gastric lumen was observed only during the reperfusion period. After the reperfusion period, gastric mucosal damage was further assessed using gross lesion area and histology. Rats were placed on a sodium tungstate diet (to inactivate xanthine oxidase), or treated with either deferoxamine (an iron chelating agent) or superoxide dismutase (a superoxide scavenger). All three interventions substantially reduced 51 Cr-labeled red blood cell clearance and gross lesion area relative to untreated rats. However, tissue injury assessed histologically was similar in both treated and untreated animals. The results of this study support the hypothesis that oxygen free radicals mediate the hemorrhagic shock-induced extravasation of red blood cells. The data also indicate that xanthine oxidase is the source of the oxy-radicals and that the iron-catalyzed Haber-Weiss reaction is largely responsible for hydroxyl radical generation in this model

  9. Expression of novel rice gibberellin 2-oxidase gene is under homeostatic regulation by biologically active gibberellins.

    Science.gov (United States)

    Sakai, Miho; Sakamoto, Tomoaki; Saito, Tamio; Matsuoka, Makoto; Tanaka, Hiroshi; Kobayashi, Masatomo

    2003-04-01

    We have cloned two genes for gibberellin (GA) 2-oxidase from rice ( Oryza sativa L.). Expression of OsGA2ox2 was not observed. The other gene, OsGA2ox3, was expressed in every tissue examined and was enhanced by the application of biologically active GA. Recombinant OsGA2ox3 protein catalyzed the metabolism of GA(1) to GA(8) and GA(20) to GA(29)-catabolite. These results indicate that OsGA2ox3 is involved in the homeostatic regulation of the endogenous level of biologically active GA in rice.

  10. Process considerations for use of galactose oxidase as an industrial biocatalyst

    DEFF Research Database (Denmark)

    Pedersen, Asbjørn Toftgaard; Rehn, Gustav; Woodley, John M.

    In nature galactose oxidase (GOase, EC.1.1.3.9) catalyses the oxidation of the C6 hydroxyl group of D-galactose to the corresponding aldehyde, while reducing molecular oxygen to hydrogen peroxide. In recent years a great effort has been made to broaden the substrate scope, enabling GOase to oxidize...... C6-OH of glucose and fructose, as well as secondary alcohols to ketones. The widened substrate scope of GOase opens up many important industrial applications, such as synthesis of industrially relevant compounds containing aldehydes and ketones (e.g. the oxidation of 5-hydroxymethylfurfural...

  11. Efficacy, safety, and patient preference of monoamine oxidase B inhibitors in the treatment of Parkinson's disease.

    Science.gov (United States)

    Robottom, Bradley J

    2011-01-20

    Parkinson's disease (PD) is the second most common neurodegenerative disease and the most treatable. Treatment of PD is symptomatic and generally focuses on the replacement or augmentation of levodopa. A number of options are available for treatment, both in monotherapy of early PD and to treat complications of advanced PD. This review focuses on rasagiline and selegiline, two medications that belong to a class of antiparkinsonian drugs called monoamine oxidase B (MAO-B) inhibitors. Topics covered in the review include mechanism of action, efficacy in early and advanced PD, effects on disability, the controversy regarding disease modification, safety, and patient preference for MAO-B inhibitors.

  12. Use of Fructosyl Peptide Oxidase for HbA1c Assay

    Science.gov (United States)

    Yonehara, Satoshi; Inamura, Norio; Fukuda, Miho; Sugiyama, Koji

    2015-01-01

    ARKRAY, Inc developed the world’s first automatic glycohemoglobin analyzer based on HPLC (1981). After that, ARKRAY developed enzymatic HbA1c assay “CinQ HbA1c” with the spread and diversification of HbA1c measurement (2007). CinQ HbA1c is the kit of Clinical Chemistry Analyzer, which uses fructosyl peptide oxidase (FPOX) for a measurement reaction. This report mainly indicates the developmental background, measurement principle, and future of the enzymatic method HbA1c reagent. PMID:25633966

  13. Brain monoamine oxidase B and A in human parkinsonian dopamine deficiency disorders.

    Science.gov (United States)

    Tong, Junchao; Rathitharan, Gausiha; Meyer, Jeffrey H; Furukawa, Yoshiaki; Ang, Lee-Cyn; Boileau, Isabelle; Guttman, Mark; Hornykiewicz, Oleh; Kish, Stephen J

    2017-09-01

    See Jellinger (doi:10.1093/awx190) for a scientific commentary on this article. The enzyme monoamine oxidases (B and A subtypes, encoded by MAOB and MAOA, respectively) are drug targets in the treatment of Parkinson's disease. Inhibitors of MAOB are used clinically in Parkinson's disease for symptomatic purposes whereas the potential disease-modifying effect of monoamine oxidase inhibitors is debated. As astroglial cells express high levels of MAOB, the enzyme has been proposed as a brain imaging marker of astrogliosis, a cellular process possibly involved in Parkinson's disease pathogenesis as elevation of MAOB in astrocytes might be harmful. Since brain monoamine oxidase status in Parkinson's disease is uncertain, our objective was to measure, by quantitative immunoblotting in autopsied brain homogenates, protein levels of both monoamine oxidases in three different degenerative parkinsonian disorders: Parkinson's disease (n = 11), multiple system atrophy (n = 11), and progressive supranuclear palsy (n = 16) and in matched controls (n = 16). We hypothesized that if MAOB is 'substantially' localized to astroglial cells, MAOB levels should be generally associated with standard astroglial protein measures (e.g. glial fibrillary acidic protein). MAOB levels were increased in degenerating putamen (+83%) and substantia nigra (+10%, non-significant) in multiple system atrophy; in caudate (+26%), putamen (+27%), frontal cortex (+31%) and substantia nigra (+23%) of progressive supranuclear palsy; and in frontal cortex (+33%), but not in substantia nigra of Parkinson's disease, a region we previously reported no increase in astrocyte protein markers. Although the magnitude of MAOB increase was less than those of standard astrocytic markers, significant positive correlations were observed amongst the astrocyte proteins and MAOB. Despite suggestions that MAOA (versus MAOB) is primarily responsible for metabolism of dopamine in dopamine neurons, there was no loss of the

  14. Rac1-NADPH oxidase signaling promotes CD36 activation under glucotoxic conditions in pancreatic beta cells.

    Science.gov (United States)

    Elumalai, Suma; Karunakaran, Udayakumar; Lee, In Kyu; Moon, Jun Sung; Won, Kyu Chang

    2017-04-01

    We recently reported that cluster determinant 36 (CD36), a fatty acid transporter, plays a pivotal role in glucotoxicity-induced β-cell dysfunction. However, little is known about how glucotoxicity influences CD36 expression. Emerging evidence suggests that the small GTPase Rac1 is involved in the pathogenesis of beta cell dysfunction in type 2 diabetes (T2D). The primary objective of the current study was to determine the role of Rac1 in CD36 activation and its impact on β-cell dysfunction in diabetes mellitus. To address this question, we subjected INS-1 cells and human beta cells (1.1B4) to high glucose conditions (30mM) in the presence or absence of Rac1 inhibition either by NSC23766 (Rac1 GTPase inhibitor) or small interfering RNA. High glucose exposure in INS-1 and human beta cells (1.1b4) resulted in the activation of Rac1 and induced cell apoptosis. Rac1 activation mediates NADPH oxidase (NOX) activation leading to elevated ROS production in both cells. Activation of the Rac1-NOX complex by high glucose levels enhanced CD36 expression in INS-1 and human 1.1b4 beta cell membrane fractions. The inhibition of Rac1 by NSC23766 inhibited NADPH oxidase activity and ROS generation induced by high glucose concentrations in INS-1 & human 1.1b4 beta cells. Inhibition of Rac1-NOX complex activation by NSC23766 significantly reduced CD36 expression in INS-1 and human 1.1b4 beta cell membrane fractions. In addition, Rac1 inhibition by NSC23766 significantly reduced high glucose-induced mitochondrial dysfunction. Furthermore, NADPH oxidase inhibition by VAS2870 also attenuated high glucose-induced ROS generation and cell apoptosis. These results suggest that Rac1-NADPH oxidase dependent CD36 expression contributes to high glucose-induced beta cell dysfunction and cell death. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  15. Electrophoresis of platelet monoamine oxidase in schizophrenia and manic-depressive illness

    International Nuclear Information System (INIS)

    Belmaker, R.H.; Ebstein, R.; Rimon, R.; Wyatt, R.J.; Murphy, D.L.

    1976-01-01

    Monoamine oxidase is an important enzyme in the catabolism of biogenic amines and can be measured in human platelets. Platelet MAO has been reported to be reduced in schizophrenic and manic-depressive patients, though other reports are contradictory. The present study evaluated the possibility that qualitative genetic enzyme abnormalities of MAO could be responsible for the different enzyme activities of platelet MAO in different populations. However, polyacrylamide gel electrophoresis of platelet MAO from 10 manic-depressive, 12 schizophrenic, and 11 normal individuals did not reveal any genetic mutant forms. (author)

  16. COI (cytochrome oxidase-I) sequence based studies of Carangid fishes from Kakinada coast, India.

    Science.gov (United States)

    Persis, M; Chandra Sekhar Reddy, A; Rao, L M; Khedkar, G D; Ravinder, K; Nasruddin, K

    2009-09-01

    Mitochondrial DNA, cytochrome oxidase-1 gene sequences were analyzed for species identification and phylogenetic relationship among the very high food value and commercially important Indian carangid fish species. Sequence analysis of COI gene very clearly indicated that all the 28 fish species fell into five distinct groups, which are genetically distant from each other and exhibited identical phylogenetic reservation. All the COI gene sequences from 28 fishes provide sufficient phylogenetic information and evolutionary relationship to distinguish the carangid species unambiguously. This study proves the utility of mtDNA COI gene sequence based approach in identifying fish species at a faster pace.

  17. Differences in activity of cytochrome C oxidase in brain between sleep and wakefulness.

    Science.gov (United States)

    Nikonova, Elena V; Vijayasarathy, Camasamudram; Zhang, Lin; Cater, Jacqueline R; Galante, Raymond J; Ward, Stephen E; Avadhani, Narayan G; Pack, Allan I

    2005-01-01

    Increased mRNA level of subunit 1 cytochrome c oxidase (COXI) during wakefulness and after short-term sleep deprivation has been described in brain. We hypothesized that this might contribute to increased activity of cytochrome oxidase (COX) enzyme during wakefulness, as part of the mechanisms to provide sufficient amounts of adenosine triphosphate to meet increased neuronal energy demands. COX activity was measured in isolated mitochondria from different brain regions in groups of rats with 3 hours of spontaneous sleep, 3 hours of spontaneous wake, and 3 hours of sleep deprivation. The group with 3 hours of spontaneous wake was added to delineate the circadian component of changes in the enzyme activity. Northern blot analysis was performed to examine the mRNA levels of 2 subunits of the enzyme COXI and COXIV, encoded by mitochondrial and nuclear DNA, respectively. Laboratory of Biochemistry, Department of Animal Biology, and Center for Sleep and Respiratory Neurobiology, University of Pennsylvania. 2-month-old male Fischer rats (N = 21) implanted for polygraphic recording. For COX activity, there was a main effect by analysis of variance of experimental group (P sleep-deprived groups as compared to the sleep group. A main effect of brain region was also significant (P sleep. There is an increase in COX activity after both 3 hours of spontaneous wake and 3 hours of sleep deprivation as compared with 3 hours of spontaneous sleep in diverse brain regions, which could be, in part, explained by the increased levels of bigenomic transcripts of the enzyme. This likely contributes to increased adenosine triphosphate production during wakefulness. ADP, adenosine diphosphate; ATP, adenosine triphosphate; COXI, cytochrome c oxidase subunit 1 mRNA; COX, cytochrome c oxidase (protein); CREB, cyclic AMP response element binding protein; DNA, deoxyribonucleic acid; EDTA, ethylenediaminetetraacetic acid; EEG, electroencephalography; EMG, electromyography; GABP, GA binding

  18. Galactose oxidase immobilized on silica in an analytical determination of galactose-containing carbohydrates.

    Science.gov (United States)

    Kondakova, Lyudmila; Yanishpolskii, Victor; Tertykh, Valentin; Buglova, Tat'yana

    2007-01-01

    Galactose oxidase from Fusarium graminearum IMV-1060 adsorbed on, and covalently bound to, silica carriers has been used for analytical determinations of D-galactose and galactose-containing sugars. Using a flowing oxygen electrode of the Clark-type, sensor system for enzymatic analysis of water solutions of galactose-containing carbohydrates was made. Measurements were taken both in the pulse and continuous modes of a substrate flowing through a column with an immobilized biocatalyst. The linear measurement ranges for galactose-containing carbohydrates concentrations were determined.

  19. Preliminary pharmacological studies on Eugenia uniflora leaves: xanthine oxidase inhibitory activity.

    Science.gov (United States)

    Schmeda-Hirschmann, G; Theoduloz, C; Franco, L; Ferro, E; de Arias, A R

    1987-11-01

    Eugenia uniflora is widely used in Paraguayan folk medicine. A hydroalcoholic extract of the leaves showed some central nervous system activity in hippocratic screening when given intraperitoneally, but little to no acute or subacute toxicity in doses up to 4200 mg/kg orally in BALB c mice. The LD50 of the extract was 220 mg/kg i.p. in mice. A decoction or infusion of the leaves is recommended for treating gout by native herbalists. The known flavonoids quercitrin, quercetin, myricitrin and myricetin were found to be responsible for the xanthine oxidase inhibitory action of the plant extract.

  20. Renal denervation attenuates NADPH oxidase-mediated oxidative stress and hypertension in rats with hydronephrosis

    DEFF Research Database (Denmark)

    Peleli, Maria; Al-Mashhadi, Ammar; Yang, Ting

    2016-01-01

    Hydronephrosis is associated with development of salt-sensitive hypertension. Studies suggest that increased sympathetic nerve activity (SNA) and oxidative stress play important roles in renovascular hypertension. This study aimed to investigate the link between renal SNA and NADPH oxidase (NOX......) regulation in the development of hypertension in rats with hydronephrosis. Hydronephrosis was induced by partial unilateral ureteral obstruction (PUUO) in young rats. Sham surgery or renal denervation was performed at the same time. Blood pressure was measured during normal, high and low salt diets. Renal...

  1. Activation of endothelial cells after exposure to ambient ultrafine particles: The role of NADPH oxidase

    International Nuclear Information System (INIS)

    Mo Yiqun; Wan Rong; Chien Sufan; Tollerud, David J.; Zhang Qunwei

    2009-01-01

    Several studies have shown that ultrafine particles (UFPs) may pass from the lungs to the circulation because of their very small diameter, and induce lung oxidative stress with a resultant increase in lung epithelial permeability. The direct effects of UFPs on vascular endothelium remain unknown. We hypothesized that exposure to UFPs leads to endothelial cell O 2 ·- generation via NADPH oxidase and results in activation of endothelial cells. Our results showed that UFPs, at a non-toxic dose, induced reactive oxygen species (ROS) generation in mouse pulmonary microvascular endothelial cells (MPMVEC) that was inhibited by pre-treatment with the ROS scavengers or inhibitors, but not with the mitochondrial inhibitor, rotenone. UFP-induced ROS generation in MPMVEC was abolished by p67 phox siRNA transfection and UFPs did not cause ROS generation in MPMVEC isolated from gp91 phox knock-out mice. UFP-induced ROS generation in endothelial cells was also determined in vivo by using a perfused lung model with imaging. Moreover, Western blot and immunofluorescence staining results showed that MPMVEC treated with UFPs resulted in the translocation of cytosolic proteins of NADPH oxidase, p47 phox , p67 phox and rac 1, to the plasma membrane. These results demonstrate that NADPH oxidase in the pulmonary endothelium is involved in ROS generation following exposure to UFPs. To investigate the activation of endothelial cells by UFP-induced oxidative stress, we determined the activation of the mitogen-activated protein kinases (MAPKs) in MPMVEC. Our results showed that exposure of MPMVEC to UFPs caused increased phosphorylation of p38 and ERK1/2 MAPKs that was blocked by pre-treatment with DPI or p67 phox siRNA. Exposure of MPMVEC obtained from gp91 phox knock-out mice to UFPs did not cause increased phosphorylation of p38 and ERK1/2 MAPKs. These findings confirm that UFPs can cause endothelial cells to generate ROS directly via activation of NADPH oxidase. UFP-induced ROS lead to

  2. Neutrophils to the ROScue: Mechanisms of NADPH Oxidase Activation and Bacterial Resistance

    Directory of Open Access Journals (Sweden)

    Giang T. Nguyen

    2017-08-01

    Full Text Available Reactive oxygen species (ROS generated by NADPH oxidase play an important role in antimicrobial host defense and inflammation. Their deficiency in humans results in recurrent and severe bacterial infections, while their unregulated release leads to pathology from excessive inflammation. The release of high concentrations of ROS aids in clearance of invading bacteria. Localization of ROS release to phagosomes containing pathogens limits tissue damage. Host immune cells, like neutrophils, also known as PMNs, will release large amounts of ROS at the site of infection following the activation of surface receptors. The binding of ligands to G-protein-coupled receptors (GPCRs, toll-like receptors, and cytokine receptors can prime PMNs for a more robust response if additional signals are encountered. Meanwhile, activation of Fc and integrin directly induces high levels of ROS production. Additionally, GPCRs that bind to the bacterial-peptide analog fMLP, a neutrophil chemoattractant, can both prime cells and trigger low levels of ROS production. Engagement of these receptors initiates intracellular signaling pathways, resulting in activation of downstream effector proteins, assembly of the NADPH oxidase complex, and ultimately, the production of ROS by this complex. Within PMNs, ROS released by the NADPH oxidase complex can activate granular proteases and induce the formation of neutrophil extracellular traps (NETs. Additionally, ROS can cross the membranes of bacterial pathogens and damage their nucleic acids, proteins, and cell membranes. Consequently, in order to establish infections, bacterial pathogens employ various strategies to prevent restriction by PMN-derived ROS or downstream consequences of ROS production. Some pathogens are able to directly prevent the oxidative burst of phagocytes using secreted effector proteins or toxins that interfere with translocation of the NADPH oxidase complex or signaling pathways needed for its activation

  3. Lysyl Oxidase Plays a Critical Role in Endothelial Cell Stimulation to Drive Tumor Angiogenesis

    DEFF Research Database (Denmark)

    Baker, Ann-Marie; Bird, Demelza; Welti, Jonathan C

    2013-01-01

    Identification of key molecules that drive angiogenesis is critical for the development of new modalities for the prevention of solid tumor progression. Using multiple models of colorectal cancer, we show that activity of the extracellular matrix-modifying enzyme lysyl oxidase (LOX) is essential...... for stimulating endothelial cells in vitro and angiogenesis in vivo. We show that LOX activates Akt through platelet-derived growth factor receptor ß (PDGFRß) stimulation, resulting in increased VEGF expression. LOX-driven angiogenesis can be abrogated through targeting LOX directly or using inhibitors of PDGFRß...

  4. Studies on the mechanism of action of 6-mercaptopurine. Interaction with copper and xanthine oxidase.

    OpenAIRE

    Kela, U; Vijayvargiya, R

    1981-01-01

    Interaction between 6-mercaptopurine, Cu2+ and the enzyme xanthine oxidase (EC 1.2.3.2.) was examined. Whereas Cu2+ was found to inhibit the enzyme, 6-mercaptopurine could protect as well as reverse the enzyme inhibition produced by the metal ion. The formation of a complex between 6-mercaptopurine and Cu2+ seems to be responsible for the observed effect. Job's [(1928) Ann. Chem. 9, 113] method has shown the composition of the complex to be 1:1. The apparent stability constant (log K value), ...

  5. Retrospective, multicentric study of 180 children with cytochrome C oxidase deficiency

    Czech Academy of Sciences Publication Activity Database

    Böhm, M.; Pronicka, E.; Karczmarewicz, E.; Pronicki, M.; Piekutowska-Abramczuk, D.; Sykut-Cegielska, J.; Mierzewska, H.; Hansíková, H.; Veselá, K.; Tesařová, M.; Houšťková, H.; Houštěk, Josef; Zeman, J.

    2006-01-01

    Roč. 59, č. 1 (2006), s. 21-26 ISSN 0031-3998 R&D Projects: GA ČR(CZ) GA303/03/0749; GA MŠk(CZ) 1M0520; GA MZd(CZ) NR8065 Grant - others:Framework Programme(XE) LSHM-CT-2004-503116; GA-(XE) GLG1-CT-2002-90358 Institutional research plan: CEZ:AV0Z50110509 Keywords : mitochondria * disease * cytochrome c oxidase Subject RIV: FG - Pediatrics Impact factor: 2.619, year: 2006

  6. Pseudo-bi-enzyme glucose sensor: ZnS hollow spheres and glucose oxidase concerted catalysis glucose.

    Science.gov (United States)

    Shuai, Ying; Liu, Changhua; Wang, Jia; Cui, Xiaoyan; Nie, Ling

    2013-06-07

    This work creatively uses peroxidase-like ZnS hollow spheres (ZnS HSs) to cooperate with glucose oxidase (GOx) for glucose determinations. This approach is that the ZnS HSs electrocatalytically oxidate the enzymatically generated H2O2 to O2, and then the O2 circularly participates in the previous glucose oxidation by glucose oxidase. Au nanoparticles (AuNPs) and carbon nanotubes (CNTs) are used as electron transfer and enzyme immobilization matrices, respectively. The biosensor of glucose oxidase-carbon nanotubes-Au nanoparticles-ZnS hollow spheres-gold electrode (GOx-CNT-AuNPs-ZnS HSs-GE) exhibits a rapid response, a low detection limit (10 μM), a wide linear range (20 μM to 7 mM) as well as good anti-interference, long-term longevity and reproducibility.

  7. Secreted fungal sulfhydryl oxidases: sequence analysis and characterisation of a representative flavin-dependent enzyme from Aspergillus oryzae

    Directory of Open Access Journals (Sweden)

    Faccio Greta

    2010-08-01

    Full Text Available Abstract Background Sulfhydryl oxidases are flavin-dependent enzymes that catalyse the formation of de novo disulfide bonds from free thiol groups, with the reduction of molecular oxygen to hydrogen peroxide. Sulfhydryl oxidases have been investigated in the food industry to remove the burnt flavour of ultraheat-treated milk and are currently studied as potential crosslinking enzymes, aiming at strengthening wheat dough and improving the overall bread quality. Results In the present study, potential sulfhydryl oxidases were identified in the publicly available fungal genome sequences and their sequence characteristics were studied. A representative sulfhydryl oxidase from Aspergillus oryzae, AoSOX1, was expressed in the fungus Trichoderma reesei. AoSOX1 was produced in relatively good yields and was purified and biochemically characterised. The enzyme catalysed the oxidation of thiol-containing compounds like glutathione, D/L-cysteine, beta-mercaptoethanol and DTT. The enzyme had a melting temperature of 57°C, a pH optimum of 7.5 and its enzymatic activity was completely inhibited in the presence of 1 mM ZnSO4. Conclusions Eighteen potentially secreted sulfhydryl oxidases were detected in the publicly available fungal genomes analysed and a novel proline-tryptophan dipeptide in the characteristic motif CXXC, where X is any amino acid, was found. A representative protein, AoSOX1 from A. oryzae, was produced in T. reesei in an active form and had the characteristics of sulfhydryl oxidases. Further testing of the activity on thiol groups within larger peptides and on protein level will be needed to assess the application potential of this enzyme.

  8. Boosting the oxidase mimicking activity of nanoceria by fluoride capping: rivaling protein enzymes and ultrasensitive F- detection

    Science.gov (United States)

    Liu, Biwu; Huang, Zhicheng; Liu, Juewen

    2016-07-01

    Nanomaterial-based enzyme mimics (nanozymes) are currently a new forefront of chemical research. However, the application of nanozymes is limited by their low catalytic activity and low turnover numbers. Cerium dioxide nanoparticles (nanoceria) are among the few with oxidase activity. Herein, we report an interesting finding addressing their limitations. The oxidase activity of nanoceria is improved by over 100-fold by fluoride capping, making it more close to real oxidases. The turnover number reached 700 in 15 min, drastically improved from ~15 turnovers for the naked particles. The mechanism is attributed to surface charge modulation and facilitated electron transfer by F- capping based on ζ-potential and free radical measurements. Ultrasensitive sensing of fluoride was achieved with a detection limit of 0.64 μM F- in water and in toothpastes, while no other tested anions can achieve the activity enhancement.Nanomaterial-based enzyme mimics (nanozymes) are currently a new forefront of chemical research. However, the application of nanozymes is limited by their low catalytic activity and low turnover numbers. Cerium dioxide nanoparticles (nanoceria) are among the few with oxidase activity. Herein, we report an interesting finding addressing their limitations. The oxidase activity of nanoceria is improved by over 100-fold by fluoride capping, making it more close to real oxidases. The turnover number reached 700 in 15 min, drastically improved from ~15 turnovers for the naked particles. The mechanism is attributed to surface charge modulation and facilitated electron transfer by F- capping based on ζ-potential and free radical measurements. Ultrasensitive sensing of fluoride was achieved with a detection limit of 0.64 μM F- in water and in toothpastes, while no other tested anions can achieve the activity enhancement. Electronic supplementary information (ESI) available: Methods, TMB oxidation kinetics and control experiments. See DOI: 10.1039/c6nr02730j

  9. Secreted fungal sulfhydryl oxidases: sequence analysis and characterisation of a representative flavin-dependent enzyme from Aspergillus oryzae.

    Science.gov (United States)

    Faccio, Greta; Kruus, Kristiina; Buchert, Johanna; Saloheimo, Markku

    2010-08-20

    Sulfhydryl oxidases are flavin-dependent enzymes that catalyse the formation of de novo disulfide bonds from free thiol groups, with the reduction of molecular oxygen to hydrogen peroxide. Sulfhydryl oxidases have been investigated in the food industry to remove the burnt flavour of ultraheat-treated milk and are currently studied as potential crosslinking enzymes, aiming at strengthening wheat dough and improving the overall bread quality. In the present study, potential sulfhydryl oxidases were identified in the publicly available fungal genome sequences and their sequence characteristics were studied. A representative sulfhydryl oxidase from Aspergillus oryzae, AoSOX1, was expressed in the fungus Trichoderma reesei. AoSOX1 was produced in relatively good yields and was purified and biochemically characterised. The enzyme catalysed the oxidation of thiol-containing compounds like glutathione, D/L-cysteine, beta-mercaptoethanol and DTT. The enzyme had a melting temperature of 57°C, a pH optimum of 7.5 and its enzymatic activity was completely inhibited in the presence of 1 mM ZnSO4. Eighteen potentially secreted sulfhydryl oxidases were detected in the publicly available fungal genomes analysed and a novel proline-tryptophan dipeptide in the characteristic motif CXXC, where X is any amino acid, was found. A representative protein, AoSOX1 from A. oryzae, was produced in T. reesei in an active form and had the characteristics of sulfhydryl oxidases. Further testing of the activity on thiol groups within larger peptides and on protein level will be needed to assess the application potential of this enzyme.

  10. Some enzymatic properties of cholesterol oxidase produced by Brevibacterium sp Algumas propriedades enzimáticas da colesterol oxidase produzida por Brevibacterium sp.

    Directory of Open Access Journals (Sweden)

    Terezinha J.G. Salva

    1999-12-01

    Full Text Available In this study we determined some properties of the cholesterol oxidase from a Brevibacterium strain isolated from buffalo's milk and identified the cholesterol degradation products by the bacterial cell. A small fraction of the enzyme synthesized by cells cultured in liquid medium for 7days was released into the medium whereas a larger fraction remained bound to the cell membrane. The extraction of this fraction was efficiently accomplished in 1 mM phosphate buffer, pH 7.0, containing 0.7% Triton X-100. The enzyme stability under freezing and at 45oC was improved by addition of 20% glycerol. The optimum temperature and pH for the enzyme activity were 53°C and 7.5, respectively. The only steroidal product from cholesterol oxidation by the microbial cell and by the crude extract of the membrane-bound enzyme was 4-colesten-3-one. Chromatographic analysis showed that minor no steroidal compounds as well as 4-colesten-3-one found in the reaction media arose during fermentation process and were extracted together with the enzyme in the buffer solution. Cholesterol oxidation by the membrane-bound enzyme was a first order reaction type.Neste trabalho foram definidas algumas propriedades da enzima colesterol oxidase produzida por uma linhagem de Brevibacterium sp. isolada de leite de búfala e foram identificados os compostos resultantes da degradação do colesterol pela bactéria. Uma pequena fração da enzima sintetizada pelas células cultivadas em meio líquido por 7 dias foi liberada no meio de cultura e uma fração maior permaneceu ligada à membrana celular. A extração desta fração foi eficientemente efetuada em tampão fosfato 1mM, pH 7,0, contendo 0,7% de triton X-100. A estabilidade da enzima congelada e a 45oC foi aumentada pela adição de 20% de glicerol. A temperatura ótima para a atividade enzimática esteve ao redor de 53(0C e o pH ótimo esteve ao redor de 7,5. O único produto da degradação do colesterol, causada pela a

  11. Identification, expression, and taxonomic distribution of alternative oxidases in non-angiosperm plants.

    Science.gov (United States)

    Neimanis, Karina; Staples, James F; Hüner, Norman P A; McDonald, Allison E

    2013-09-10

    Alternative oxidase (AOX) is a terminal ubiquinol oxidase present in the respiratory chain of all angiosperms investigated to date, but AOX distribution in other members of the Viridiplantae is less clear. We assessed the taxonomic distribution of AOX using bioinformatics. Multiple sequence alignments compared AOX proteins and examined amino acid residues involved in AOX catalytic function and post-translational regulation. Novel AOX sequences were found in both Chlorophytes and Streptophytes and we conclude that AOX is widespread in the Viridiplantae. AOX multigene families are common in non-angiosperm plants and the appearance of AOX1 and AOX2 subtypes pre-dates the divergence of the Coniferophyta and Magnoliophyta. Residues involved in AOX catalytic function are highly conserved between Chlorophytes and Streptophytes, while AOX post-translational regulation likely differs in these two lineages. We demonstrate experimentally that an AOX gene is present in the moss Physcomitrella patens and that the gene is transcribed. Our findings suggest that AOX will likely exert an influence on plant respiration and carbon metabolism in non-angiosperms such as green algae, bryophytes, liverworts, lycopods, ferns, gnetophytes, and gymnosperms and that further research in these systems is required. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. Glucose oxidase probe as a surface-enhanced Raman scattering sensor for glucose.

    Science.gov (United States)

    Qi, Guohua; Wang, Yi; Zhang, Biying; Sun, Dan; Fu, Cuicui; Xu, Weiqing; Xu, Shuping

    2016-10-01

    Glucose oxidase (GOx) possessing a Raman-active chromophore (flavin adenine dinucleotide) is used as a signal reporter for constructing a highly specific "turn off" surface-enhanced Raman scattering (SERS) sensor for glucose. This sensing chip is made by the electrostatic assembly of GOx over silver nanoparticle (Ag NP)-functionalized SERS substrate through a positively charged polyelectrolyte linker under the pH of 6.86. To trace glucose in blood serum, owing to the reduced pH value caused by the production of gluconic acid in the GOx-catalyzed oxidation reaction, the bonding force between GOx and polyelectrolyte weakens, making GOx drop off from the sensing chip. As a result, the SERS intensity of GOx on the chip decreases along with the concentration of glucose. This glucose SERS sensor exhibits excellent selectivity based on the specific GOx/glucose catalysis reaction and high sensitivity to 1.0 μM. The linear sensing range is 2.0-14.0 mM, which also meets the requirement on the working range of the human blood glucose detection. Using GOx as a probe shows superiority over other organic probes because GOx almost has no toxicity to the biological system. This sensing mechanism can be applied for intracellular in vivo SERS monitoring of glucose in the future. Graphical abstract Glucose oxidase is used as a Raman signal reporter for constructing a highly specific glucose surface-enhanced Raman scattering (SERS) sensor.

  13. Pyruvate oxidase influences the sugar utilization pattern and capsule production in Streptococcus pneumoniae.

    Directory of Open Access Journals (Sweden)

    Sandra M Carvalho

    Full Text Available Pyruvate oxidase is a key function in the metabolism and lifestyle of many lactic acid bacteria and its activity depends on the presence of environmental oxygen. In Streptococcus pneumoniae the protein has been suggested to play a major role in metabolism and has been implicated in virulence, oxidative stress survival and death in stationary phase. Under semi-aerobic conditions, transcriptomic and metabolite profiling analysis of a spxB mutant grown on glucose showed minor changes compared to the wild type, apart from the significant induction of two operons involved in carbohydrate uptake and processing. This induction leads to a change in the sugar utilization capabilities of the bacterium, as indicated by the analysis of the growth profiles of the D39 parent and spxB mutant on alternative carbohydrates. Metabolic analysis and growth experiments showed that inactivation of SpxB has no effect on the glucose fermentation pattern, except under aerobic conditions. More importantly, we show that mutation of spxB results in the production of increased amounts of capsule, the major virulence factor of S. pneumoniae. Part of this increase can be attributed to induction of capsule operon (cps transcription. Therefore, we propose that S. pneumoniae utilizes pyruvate oxidase as an indirect sensor of the oxygenation of the environment, resulting in the adaption of its nutritional capability and the amount of capsule to survive in the host.

  14. Direct electron transfer from glucose oxidase immobilized on a nano-porous glassy carbon electrode

    International Nuclear Information System (INIS)

    Haghighi, Behzad; Tabrizi, Mahmoud Amouzadeh

    2011-01-01

    Highlights: → A direct electron transfer reaction of glucose oxidase was observed on the surface of a nano-porous glassy carbon electrode. → A pair of well-defined and reversible redox peaks was observed at the formal potential of approximately -0.439 V. → The apparent electron transfer rate constant was measured to be 5.27 s -1 . → A mechanism for the observed direct electron transfer reaction was proposed, which consists of a two-electron and a two-proton transfer. - Abstract: A pair of well-defined and reversible redox peaks was observed for the direct electron transfer (DET) reaction of an immobilized glucose oxidase (GOx) on the surface of a nano-porous glassy carbon electrode at the formal potential (E o ') of -0.439 V versus Ag/AgCl/saturated KCl. The electron transfer rate constant (k s ) was calculated to be 5.27 s -1 . The dependence of E o ' on pH indicated that the direct electron transfer of the GOx was a two-electron transfer process, coupled with two-proton transfer. The results clearly demonstrate that the nano-porous glassy carbon electrode is a cost-effective and ready-to-use scaffold for the fabrication of a glucose biosensor.

  15. Direct electron transfer from glucose oxidase immobilized on a nano-porous glassy carbon electrode

    Energy Technology Data Exchange (ETDEWEB)

    Haghighi, Behzad, E-mail: haghighi@iasbs.ac.ir [Department of Chemistry, Institute for Advanced Studies in Basic Sciences, P.O. Box 45195-1159, Gava Zang, Zanjan (Iran, Islamic Republic of); Tabrizi, Mahmoud Amouzadeh [Department of Chemistry, Institute for Advanced Studies in Basic Sciences, P.O. Box 45195-1159, Gava Zang, Zanjan (Iran, Islamic Republic of)

    2011-11-30

    Highlights: > A direct electron transfer reaction of glucose oxidase was observed on the surface of a nano-porous glassy carbon electrode. > A pair of well-defined and reversible redox peaks was observed at the formal potential of approximately -0.439 V. > The apparent electron transfer rate constant was measured to be 5.27 s{sup -1}. > A mechanism for the observed direct electron transfer reaction was proposed, which consists of a two-electron and a two-proton transfer. - Abstract: A pair of well-defined and reversible redox peaks was observed for the direct electron transfer (DET) reaction of an immobilized glucose oxidase (GOx) on the surface of a nano-porous glassy carbon electrode at the formal potential (E{sup o}') of -0.439 V versus Ag/AgCl/saturated KCl. The electron transfer rate constant (k{sub s}) was calculated to be 5.27 s{sup -1}. The dependence of E{sup o}' on pH indicated that the direct electron transfer of the GOx was a two-electron transfer process, coupled with two-proton transfer. The results clearly demonstrate that the nano-porous glassy carbon electrode is a cost-effective and ready-to-use scaffold for the fabrication of a glucose biosensor.

  16. Localization of ascorbic acid, ascorbic acid oxidase, and glutathione in roots of Cucurbita maxima L.

    Science.gov (United States)

    Liso, Rosalia; De Tullio, Mario C; Ciraci, Samantha; Balestrini, Raffaella; La Rocca, Nicoletta; Bruno, Leonardo; Chiappetta, Adriana; Bitonti, Maria Beatrice; Bonfante, Paola; Arrigoni, Oreste

    2004-12-01

    To understand the function of ascorbic acid (ASC) in root development, the distribution of ASC, ASC oxidase, and glutathione (GSH) were investigated in cells and tissues of the root apex of Cucubita maxima. ASC was regularly distributed in the cytosol of almost all root cells, with the exception of quiescent centre (QC) cells. ASC also occurred at the surface of the nuclear membrane and correspondingly in the nucleoli. No ASC could be observed in vacuoles. ASC oxidase was detected by immunolocalization mainly in cell walls and vacuoles. This enzyme was particularly abundant in the QC and in differentiating vascular tissues and was absent in lateral root primordia. Administration of the ASC precursor L-galactono-gamma-lactone markedly increased ASC content in all root cells, including the QC. Root treatment with the ASC oxidized product, dehydroascorbic acid (DHA), also increased ASC content, but caused ASC accumulation only in peripheral tissues, where DHA was apparently reduced at the expense of GSH. The different pattern of distribution of ASC in different tissues and cell compartments reflects its possible role in cell metabolism and root morphogenesis.

  17. Screening of Bothrops snake venoms for L-amino acid oxidase activity

    Energy Technology Data Exchange (ETDEWEB)

    Pessati, M.L.; Fontana, J.D.; Guimaraes, M.F. [Federal Univ. of Parana, Curitiba (Brazil)

    1995-12-31

    Toxins, enzymes, and biologically active peptides are the main components of snake venoms from the genus Bothrops. Following the venom inoculation, the local effects are hemorrhage, edema, and myonecrosis. Nineteen different species of Brazilian Bothrops were screened for protein content and L-amino acid oxidase activity. B. cotiara, formerly found in the South of Brazil, is now threatened with extinction. Its venom contains a highly hemorrhagic fraction and, as expected from the deep yellow color of the corresponding lyophilized powder, a high L-amino acid oxidase (LAO) activity was also characterized. Flavin adenine dinucleotide (FAD) is its associate coenzyme. B. cotiara venom LAO catalyzed the oxidative deamination of several L-amino acids, and the best substrates were methionine, leucine, tryptophan, and phenylalanine, hence, its potential application for the use in biosensors for aspartame determination and for the removal of amino acids from plasma. High levels for LAO were also found in other species than B. cotiara. In addition, the technique of isoelectric focusing (IEF) was employed as a powerful tool to study the iso- or multi-enzyme distribution for LAO activity in the B. cotiara snake venom.

  18. Congruence between PM H+-ATPase and NADPH oxidase during root growth: a necessary probability.

    Science.gov (United States)

    Majumdar, Arkajo; Kar, Rup Kumar

    2018-07-01

    Plasma membrane (PM) H + -ATPase and NADPH oxidase (NOX) are two key enzymes responsible for cell wall relaxation during elongation growth through apoplastic acidification and production of ˙OH radical via O 2 ˙ - , respectively. Our experiments revealed a putative feed-forward loop between these enzymes in growing roots of Vigna radiata (L.) Wilczek seedlings. Thus, NOX activity was found to be dependent on proton gradient generated across PM by H + -ATPase as evident from pharmacological experiments using carbonyl cyanide m-chlorophenylhydrazone (CCCP; protonophore) and sodium ortho-vanadate (PM H + -ATPase inhibitor). Conversely, H + -ATPase activity retarded in response to different ROS scavengers [CuCl 2 , N, N' -dimethylthiourea (DMTU) and catalase] and NOX inhibitors [ZnCl 2 and diphenyleneiodonium (DPI)], while H 2 O 2 promoted PM H + -ATPase activity at lower concentrations. Repressing effects of Ca +2 antagonists (La +3 and EGTA) on the activity of both the enzymes indicate its possible mediation. Since, unlike animal NOX, the plant versions do not possess proton channel activity, harmonized functioning of PM H + -ATPase and NOX appears to be justified. Plasma membrane NADPH oxidase and H + -ATPase are functionally synchronized and they work cooperatively to maintain the membrane electrical balance while mediating plant cell growth through wall relaxation.

  19. Divergence and adaptive evolution of the gibberellin oxidase genes in plants.

    Science.gov (United States)

    Huang, Yuan; Wang, Xi; Ge, Song; Rao, Guang-Yuan

    2015-09-29

    The important phytohormone gibberellins (GAs) play key roles in various developmental processes. GA oxidases (GAoxs) are critical enzymes in GA synthesis pathway, but their classification, evolutionary history and the forces driving the evolution of plant GAox genes remain poorly understood. This study provides the first large-scale evolutionary analysis of GAox genes in plants by using an extensive whole-genome dataset of 41 species, representing green algae, bryophytes, pteridophyte, and seed plants. We defined eight subfamilies under the GAox family, namely C19-GA2ox, C20-GA2ox, GA20ox,GA3ox, GAox-A, GAox-B, GAox-C and GAox-D. Of these, subfamilies GAox-A, GAox-B, GAox-C and GAox-D are described for the first time. On the basis of phylogenetic analyses and characteristic motifs of GAox genes, we demonstrated a rapid expansion and functional divergence of the GAox genes during the diversification of land plants. We also detected the subfamily-specific motifs and potential sites of some GAox genes, which might have evolved under positive selection. GAox genes originated very early-before the divergence of bryophytes and the vascular plants and the diversification of GAox genes is associated with the functional divergence and could be driven by positive selection. Our study not only provides information on the classification of GAox genes, but also facilitates the further functional characterization and analysis of GA oxidases.

  20. Performance of optical biosensor using alcohol oxidase enzyme for formaldehyde detection

    Science.gov (United States)

    Sari, A. P.; Rachim, A.; Nurlely, Fauzia, V.

    2017-07-01

    The recent issue in the world is the long exposure of formaldehyde which is can increase the risk of human health, therefore, that is very important to develop a device and method that can be optimized to detect the formaldehyde elements accurately, have a long lifetime and can be fabricated and produced in large quantities. A new and simple prepared optical biosensor for detection of formaldehyde in aqueous solutions using alcohol oxidase (AOX) enzyme was successfully fabricated. The poly-n-butyl acrylic-co-N-acryloxysuccinimide (nBA-NAS) membranes containing chromoionophore ETH5294 were used for immobilization of alcohol oxidase enzyme (AOX). Biosensor response was based on the colour change of chromoionophore as a result of enzymatic oxidation of formaldehyde and correlated with the detection concentration of formaldehyde. The performance of biosensor parameters were measured through the optical absorption value using UV-Vis spectrophotometer including the repeatability, reproducibility, selectivity and lifetime. The results showed that the prepared biosensor has good repeatability (RSD = 1.9 %) and good reproducibility (RSD = 2.1 %). The biosensor was selective formaldehyde with no disturbance by methanol, ethanol, and acetaldehyde, and also stable before 49 days and decrease by 41.77 % after 49 days.