WorldWideScience

Sample records for membrane-bound guaiacol peroxidases

  1. Some biochemical properties of guaiacol peroxidases as modified ...

    African Journals Online (AJOL)

    Some biochemical properties of guaiacol peroxidases as modified by salt stress in leaves of salt-tolerant and salt-sensitive safflower ( Carthamus tinctorius L.cv.) ... The pH profile of GP activity in leaves extract of two cultivars in control and salt stressed plants showed different pattern of pH dependency with three maxima ...

  2. Guaiacol Peroxidase Zymography for the Undergraduate Laboratory

    Science.gov (United States)

    Wilkesman, Jeff; Castro, Diana; Contreras, Lellys M.; Kurz, Liliana

    2014-01-01

    This laboratory exercise presents a novel way to introduce undergraduate students to the specific detection of enzymatic activity by electrophoresis. First, students prepare a crude peroxidase extract and then analyze the homogenate via electrophoresis. Zymography, that is, a SDS-PAGE method to detect enzyme activity, is used to specifically…

  3. Dependence of Guaiacol Peroxidase Activity on pH in Officinal Plant Leaves

    Directory of Open Access Journals (Sweden)

    Zhivetyev, M. A.

    2013-02-01

    Full Text Available For the first time, the guaiacol peroxidase activity have been studied at different pH in lamina of Achillea asiatica Serg., Veronica chamaedrys L., Taraxacum officinale Wigg., Alchemilla subcrenata Buser.

  4. The role of ascorbate peroxidase, guaiacol peroxidase, and polysaccharides in cassava (Manihot esculenta Crantz) roots under postharvest physiological deterioration.

    Science.gov (United States)

    Uarrota, Virgílio Gavicho; Moresco, Rodolfo; Schmidt, Eder Carlos; Bouzon, Zenilda Laurita; Nunes, Eduardo da Costa; Neubert, Enilto de Oliveira; Peruch, Luiz Augusto Martins; Rocha, Miguel; Maraschin, Marcelo

    2016-04-15

    This study aimed to investigate the role of ascorbate peroxidase (APX), guaiacol peroxidase (GPX), polysaccharides, and protein contents associated with the early events of postharvest physiological deterioration (PPD) in cassava roots. Increases in APX and GPX activity, as well as total protein contents occurred from 3 to 5 days of storage and were correlated with the delay of PPD. Cassava samples stained with Periodic Acid-Schiff (PAS) highlighted the presence of starch and cellulose. Degradation of starch granules during PPD was also detected. Slight metachromatic reaction with toluidine blue is indicative of increasing of acidic polysaccharides and may play an important role in PPD delay. Principal component analysis (PCA) classified samples according to their levels of enzymatic activity based on the decision tree model which showed GPX and total protein amounts to be correlated with PPD. The Oriental (ORI) cultivar was more susceptible to PPD. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Betacyanin accumulation and guaiacol peroxidase activity in Beta vulgaris L. leaves following copper stress

    Directory of Open Access Journals (Sweden)

    Janet M. León Morales

    2012-07-01

    Full Text Available The effect of copper stress on betacyanin accumulation and guaiacol peroxidase (GPOD activity in leaves of different age was evaluated in red beet (Beta vulgaris L. var. Crosby Egyptian plants. In hydroponic culture, plants were treated with 0.3 μM (control, 50 μM, 100 μM, and 250 μM of CuSO4 for 6 days. Copper was taken up and accumulated in old roots but was not translocated to leaves. However in young leaves, the increase of lipid peroxidation and reduction of growth were evident from day 3 of copper exposure; whereas in old leaves, the lipid peroxidation and growth were the same from either copper-treated or control plants. In response to copper exposure, the betacyanin accumulation was evident in young leaves by day 3, and continued to increase until day 6. Betacyanin only were accumulated in old leaves until day 6, but the contents were from 4 to 5 times lower than those observed in young leaves at the same copper concentrations. GPOD activity increased 3.3- and 1.4-fold in young and old leaves from day 3 of copper treatment respectively, but only in the young leaves was sustained at the same level until day 6. Old roots shown betacyanin in the control plants, but the betacyanin level and growth were reduced with the copper exposure. In contrast, young roots emerged by copper effect also accumulated copper and showed the highest betacyanin content of all plant parts assayed. These results indicate that betacyanin accumulation and GPOD activity are defense responses to copper stress in actively growing organs.

  6. Biotransformation and Cytotoxic Activity of Guaiacol Dimer

    Directory of Open Access Journals (Sweden)

    Galuh Widiyarti

    2014-07-01

    Full Text Available Guaiacol, a phenolic compound is known as an anticancer. Dimerization of guaiacol has been done by biotransformation using peroxidase enzyme as biocatalyst. This enzyme was isolated from Indonesian plant, kailan (Brassica oleraceae var. alboglabra. Analysis of dimerization product was carried out by TLC, IR, LC-MS, and NMR. Whilst analysis of in-vitro cytotoxic activity was carried out by MTT method against breast cancer T47D and MCF7 cells. The result showed that the dimerization reaction gave O-para dehydroguaiacol. The in-vitro cytotoxic activity analysis showed that O-para dehydroguaiacol compound has potency as anti-breast cancer.

  7. Structure Biology of Membrane Bound Enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Fu, Dax [Johns Hopkins Univ., Baltimore, MD (United States). School of Medicine. Dept. of Physiology

    2016-11-30

    The overall goal of the proposed research is to understand the membrane-associated active processes catalyzed by an alkane $\\square$-hydroxylase (AlkB) from eubacterium Pseudomonase oleovorans. AlkB performs oxygenation of unactivated hydrocarbons found in crude oils. The enzymatic reaction involves energy-demanding steps in the membrane with the uses of structurally unknown metal active sites featuring a diiron [FeFe] center. At present, a critical barrier to understanding the membrane-associated reaction mechanism is the lack of structural information. The structural biology efforts have been challenged by technical difficulties commonly encountered in crystallization and structural determination of membrane proteins. The specific aims of the current budget cycle are to crystalize AlkB and initiate X-ray analysis to set the stage for structural determination. The long-term goals of our structural biology efforts are to provide an atomic description of AlkB structure, and to uncover the mechanisms of selective modification of hydrocarbons. The structural information will help elucidating how the unactivated C-H bonds of saturated hydrocarbons are oxidized to initiate biodegradation and biotransformation processes. The knowledge gained will be fundamental to biotechnological applications to biofuel transformation of non-edible oil feedstock. Renewable biodiesel is a promising energy carry that can be used to reduce fossil fuel dependency. The proposed research capitalizes on prior BES-supported efforts on over-expression and purification of AlkB to explore the inner workings of a bioenergy-relevant membrane-bound enzyme.

  8. Guaiacol production from ferulic acid, vanillin and vanillic acid by Alicyclobacillus acidoterrestris.

    Science.gov (United States)

    Witthuhn, R Corli; van der Merwe, Enette; Venter, Pierre; Cameron, Michelle

    2012-06-15

    Alicyclobacilli are thermophilic, acidophilic bacteria (TAB) that spoil fruit juice products by producing guaiacol. It is currently believed that guaiacol is formed by Alicyclobacillus in fruit juices as a product of ferulic acid metabolism. The aim of this study was to identify the precursors that can be metabolised by Alicyclobacillus acidoterrestris to produce guaiacol and to evaluate the pathway of guaiacol production. A. acidoterrestris FB2 was incubated at 45°C for 7days in Bacillus acidoterrestris (BAT) broth supplemented with ferulic acid, vanillin or vanillic acid, respectively. The samples were analysed every day to determine the cell concentration, the supplement concentration using high performance liquid chromatography with UV-diode array detection (HPLC-DAD) and the guaiacol concentration, using both the peroxidase enzyme colourimetric assay (PECA) and HPLC-DAD. The cell concentration of A. acidoterrestris FB2 during the 7days in all samples were above the critical cell concentration of 10(5)cfu/mL reportedly required for guaiacol production. The guaiacol produced by A. acidoterrestris FB2 increased with an increase in vanillin or vanillic acid concentration and a metabolic pathway of A. acidoterrestris FB2 directly from vanillin to guaiacol was established. The high concentration of vanillic acid (1000mg/L) resulted in an initial inhibitory effect on the cells, but the cell concentration increased after day 2. Guaiacol production did not occur in the absence of either a precursor or A. acidoterrestris FB2 and guaiacol was not produced by A. acidoterrestris FB2 in the samples supplemented with ferulic acid. The presence of Alicyclobacillus spp. that has the ability to produce guaiacol, as well as the substrates vanillin or vanillic acid is prerequisite for production of guaiacol. Copyright © 2012 Elsevier B.V. All rights reserved.

  9. Effect of Low Doses of Guaiacol and Ethanol on Enzymatic Activity of Fungal Cultures

    Science.gov (United States)

    Malarczyk, Elżbieta; Jarosz-Wilkołazka, Anna; Kochmanska-Rdest, Janina

    2003-01-01

    The influence of low doses of guaiacol and ethanol, the natural effectors of lignin and phenolics transformations, on laccase and peroxidase activities produced by two strains of Basidiomycetes, Pleurotus sajor-caju and Trametes versicolor, was evaluated. Fungal mycelia were grown for 2 weeks on liquid media containing serial dilutions of guaiacol or ethanol ranging from 100−1 to 100−20 mol/L. Laccase and peroxidase activities in the medium were measured at the end of 2 weeks. The effect of low doses of guaiacol and ethanol on enzyme activities was manifested in an oscillating manner. Similar response patterns were observed when pure enzymes were exposed to the same serial dilutions of guaiacol and ethanol. T. versicolor cultures enriched with 40 mmol guaiacol (simulating natural environmental conditions) also displayed oscillating enzyme activity patterns in response to serial dilutions of guaiacol, but the maximum enzyme activity values were increased compared to those observed in cultures not receiving 40 mmol guaiacol. The differences between maxima and minima varied among the experimental groups and depended on the species of fungus, type of effector, and kind of enzyme. The results suggest the possibility of subtle regulation of enzymatic activity on the molecular level. PMID:19330120

  10. Heterologous expression and purification of membrane-bound pyrophosphatases

    DEFF Research Database (Denmark)

    Kellosalo, J.; Kajander, T.; Palmgren, Michael Broberg

    2011-01-01

    Membrane-bound pyrophosphatases (M-PPases) are enzymes that couple the hydrolysis of inorganic pyrophosphate to pumping of protons or sodium ions. In plants and bacteria they are important for relieving stress caused by low energy levels during anoxia, drought, nutrient deficiency, cold and low l...

  11. Tunable Tensor Voting Improves Grouping of Membrane-Bound Macromolecules

    Energy Technology Data Exchange (ETDEWEB)

    Loss, Leandro A.; Bebis, George; Parvin, Bahram

    2009-04-15

    Membrane-bound macromolecules are responsible for structural support and mediation of cell-cell adhesion in tissues. Quantitative analysis of these macromolecules provides morphological indices for damage or loss of tissue, for example as a result of exogenous stimuli. From an optical point of view, a membrane signal may have nonuniform intensity around the cell boundary, be punctate or diffused, and may even be perceptual at certain locations along the boundary. In this paper, a method for the detection and grouping of punctate, diffuse curvilinear signals is proposed. Our work builds upon the tensor voting and the iterative voting frameworks to propose an efficient method to detect and refine perceptually interesting curvilinear structures in images. The novelty of our method lies on the idea of iteratively tuning the tensor voting fields, which allows the concentration of the votes only over areas of interest. We validate the utility of our system with synthetic and annotated real data. The effectiveness of the tunable tensor voting is demonstrated on complex phenotypic signals that are representative of membrane-bound macromolecular structures.

  12. Alteration of plasma membrane-bound redox systems of iron deficient pea roots by chitosan.

    Science.gov (United States)

    Meisrimler, Claudia-Nicole; Planchon, Sebastien; Renaut, Jenny; Sergeant, Kjell; Lüthje, Sabine

    2011-08-12

    Iron is essential for all living organisms and plays a crucial role in pathogenicity. This study presents the first proteome analysis of plasma membranes isolated from pea roots. Protein profiles of four different samples (+Fe, +Fe/Chitosan, -Fe, and -Fe/Chitosan) were compared by native IEF-PAGE combined with in-gel activity stains and DIGE. Using DIGE, 89 proteins of interest were detected in plasma membrane fractions. Data revealed a differential abundance of several spots in all samples investigated. In comparison to the control and -FeCh the abundance of six protein spots increased whereas 56 spots decreased in +FeCh. Altered protein spots were analyzed by MALDI-TOF-TOF mass spectrometry. Besides stress-related proteins, transport proteins and redox enzymes were identified. Activity stains after native PAGE and spectrophotometric measurements demonstrated induction of a ferric-chelate reductase (-Fe) and a putative respiratory burst oxidase homolog (-FeCh). However, the activity of the ferric-chelate reductase decreased in -Fe plants after elicitor treatment. The activity of plasma membrane-bound class III peroxidases increased after elicitor treatment and decreased under iron-deficiency, whereas activity of quinone reductases decreased mostly after elicitor treatment. Possible functions of proteins identified and reasons for a weakened pathogen response of iron-deficient plants were discussed. Copyright © 2011 Elsevier B.V. All rights reserved.

  13. Ficus sycomorus latex: A thermostable peroxidase

    African Journals Online (AJOL)

    ajl yemi

    2011-11-30

    Nov 30, 2011 ... glucanases, lipase, peroxidase and chitinase (Domsalla and Melzig, 2008; Mura ... for the production of polymers and for the biotransformation of .... POLI and lane 3 = Sephacryl S-200 POLI. Table 2. Relative activity of F. sycomorus latex POLI toward substrates. Substrate. % Relative activity. Guaiacol. 100.

  14. NMR spectroscopic studies of membrane-bound biological systems

    International Nuclear Information System (INIS)

    Hohlweg, W.

    2013-01-01

    In the course of this thesis, biological NMR spectroscopy was employed in studying membrane-bound peptides and proteins, for which structural information is still comparatively hard to obtain. Initial work focused on various model peptides bound to membrane-mimicking micelles, studying the protonation state of arginine in a membrane environment. Strong evidence for a cation-π complex was found in TM7, a peptide which forms the seventh transmembrane helix of subunit a of the vacuolar-type H+-ATPase (V-ATPase). V-ATPase is a physiologically highly relevant proton pump, which is present in intracellular membranes of all eukaryotic organisms, as well as the plasma membrane of several specialized cells. Loss of functional V-ATPase is associated with human diseases such as osteopetrosis, distal renal tubular acidosis or the spreading of cancer. V-ATPase is considered a potential drug target in the treatment of osteoporosis and cancer, or in the development of novel contraceptives. Results from NMR solution structure determination, NMR titration experiments, paramagnetic relaxation enhancement experiments and tryptophan fluorescence spectroscopy confirm the existence of a buried cation-? complex formed between arginine residue R735, which is essential for proton transport, and neighbouring tryptophan and tyrosine residues. In vivo experiments in the yeast Saccharomyces cerevisiae using selective growth tests and fluorescence microscopy showed that formation of the cation-π complex is essential for V-ATPase function. Deletion of both aromatic residues, as well as only the one tryptophan residue leads to growth defects and inability to maintain vacuolar pH homeostasis. These findings shine new light on the still elusive mechanism of proton transport in V-ATPase, and show that arginine R735 may be directly involved in proton transfer across the membrane. (author) [de

  15. Membrane-bound ATPase contributes to hop resistance of Lactobacillus brevis

    NARCIS (Netherlands)

    Sakamoto, K; van Veen, HW; Saito, H; Kobayashi, H; Konings, WN

    2002-01-01

    The activity of the membrane-bound H+-ATPase of the beer spoilage bacterium Lactobacillus brevis ABBC45 increased upon adaptation to bacteriostatic hop compounds. The ATPase activity was optimal around pH 5.6 and increased up to fourfold when L. brevis was exposed to 666 muM hop compounds. The

  16. Study on the changes in the levels of membrane-bound ATPases ...

    African Journals Online (AJOL)

    An attempt has been made to determine the deleterious effects of λ cyhalothrin- induced in fresh water tilapia (Oreochromis mossambicus) with respect to changes in the activities of membrane-bound ATPases (Na+/K+, Mg+ and Ca2+ ATPase) and mineral status ...

  17. Dissociation and purification of the endogenous membrane-bound Vo complex from Pichia pastoris.

    Science.gov (United States)

    Li, Sumei; Hong, Tao; Wang, Kun; Lu, Yinghong; Zhou, Min

    2017-10-01

    Most proteins occur and function in complexes rather than as isolated entities in membranes. In most cases macromolecules with multiple subunits are purified from endogenous sources. In this study, an endogenous membrane-protein complex was obtained from Pichia pastoris, which can be grown at high densities to significantly improve the membrane protein yield. We successfully isolated the membrane-bound Vo complex of V-ATPase from P. pastoris using a fusion FLAG tag attached to the C-terminus of subunit a to generate the vph-tag strain, which was used for dissociation and purification. After FLAG affinity and size exclusion chromatography purification, the production quantity and purity of the membrane-bound Vo complex was 20 μg l -1 and >98%, respectively. The subunits of the endogenous membrane-bound Vo complex observed in P. pastoris were similar to those obtained from S. cerevisiae, as demonstrated by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Therefore, successful dissociation and purification of the membrane-bound Vo complex at a high purity and sufficient quantity was achieved via a rapid and simple procedure that can be used to obtain the endogenous membrane-protein complexes from P. pastoris. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Steric Pressure among Membrane-Bound Polymers Opposes Lipid Phase Separation.

    Science.gov (United States)

    Imam, Zachary I; Kenyon, Laura E; Carrillo, Adelita; Espinoza, Isai; Nagib, Fatema; Stachowiak, Jeanne C

    2016-04-19

    Lipid rafts are thought to be key organizers of membrane-protein complexes in cells. Many proteins that interact with rafts have bulky polymeric components such as intrinsically disordered protein domains and polysaccharide chains. Therefore, understanding the interaction between membrane domains and membrane-bound polymers provides insights into the roles rafts play in cells. Multiple studies have demonstrated that high concentrations of membrane-bound polymeric domains create significant lateral steric pressure at membrane surfaces. Furthermore, our recent work has shown that lateral steric pressure at membrane surfaces opposes the assembly of membrane domains. Building on these findings, here we report that membrane-bound polymers are potent suppressors of membrane phase separation, which can destabilize lipid domains with substantially greater efficiency than globular domains such as membrane-bound proteins. Specifically, we created giant vesicles with a ternary lipid composition, which separated into coexisting liquid ordered and disordered phases. Lipids with saturated tails and poly(ethylene glycol) (PEG) chains conjugated to their head groups were included at increasing molar concentrations. When these lipids were sparse on the membrane surface they partitioned to the liquid ordered phase. However, as they became more concentrated, the fraction of GUVs that were phase-separated decreased dramatically, ultimately yielding a population of homogeneous membrane vesicles. Experiments and physical modeling using compositions of increasing PEG molecular weight and lipid miscibility phase transition temperature demonstrate that longer polymers are the most efficient suppressors of membrane phase separation when the energetic barrier to lipid mixing is low. In contrast, as the miscibility transition temperature increases, longer polymers are more readily driven out of domains by the increased steric pressure. Therefore, the concentration of shorter polymers required

  19. The impact of physiological crowding on the diffusivity of membrane bound proteins.

    Science.gov (United States)

    Houser, Justin R; Busch, David J; Bell, David R; Li, Brian; Ren, Pengyu; Stachowiak, Jeanne C

    2016-02-21

    Diffusion of transmembrane and peripheral membrane-bound proteins within the crowded cellular membrane environment is essential to diverse biological processes including cellular signaling, endocytosis, and motility. Nonetheless we presently lack a detailed understanding of the influence of physiological levels of crowding on membrane protein diffusion. Utilizing quantitative in vitro measurements, here we demonstrate that the diffusivities of membrane bound proteins follow a single linearly decreasing trend with increasing membrane coverage by proteins. This trend holds for homogenous protein populations across a range of protein sizes and for heterogeneous mixtures of proteins of different sizes, such that protein diffusivity is controlled by the total coverage of the surrounding membrane. These results demonstrate that steric exclusion within the crowded membrane environment can fundamentally limit the diffusive rate of proteins, regardless of their size. In cells this "speed limit" could be modulated by changes in local membrane coverage, providing a mechanism for tuning the rate of molecular interaction and assembly.

  20. Synthetic activity enhancement of membrane-bound lipase from Rhizopus chinensis by pretreatment with isooctane.

    Science.gov (United States)

    Wang, Dong; Xu, Yan; Teng, Yun

    2007-05-01

    The cell-bound lipase from Rhizopus chinensis CCTCC M201021 with high catalysis ability for ester synthesis was located as a membrane-bound lipase by the treatments of Yatalase firstly. In order to improve its synthetic activity in non-aqueous phase, the pretreatments of this enzyme with various organic solvents were investigated. The pretreatment with isooctane improved evidently the lipase synthetic activity, resulting in about 139% in relative synthetic activity and 115% in activity recovery. The morphological changes of mycelia caused by organic solvent pretreatments could influence the exposure of the membrane-bound enzyme from mycelia and the exhibition of the lipase activity. The pretreatment conditions with isooctane and acetone were further investigated, and the optimum effect was obtained by the isooctane pretreatment at 4 degrees C for 1 h, resulting in 156% in relative synthetic activity and 126% in activity recovery. When the pretreated lipases were employed as catalysts for the esterification production of ethyl hexanoate in heptane, higher initial reaction rate and higher final molar conversion were obtained using the lipase pretreated with isooctane, compared with the untreated lyophilized one. This result suggested that the pretreatment of the membrane-bound lipase with isooctane could be an effective method to substitute the lyophilization for preparing biocatalysts used in non-aqueous phase reactions.

  1. Deoxygenation Affects Composition of Membrane-Bound Proteins in Human Erythrocytes

    Directory of Open Access Journals (Sweden)

    Oksana G. Luneva

    2016-06-01

    Full Text Available Background/Aims: ATP release from erythrocyte plays a key role in hypoxia-induced elevation of blood flow in systematic circulation. We have previously shown that hemolysis contributes to erythrocyte ATP release triggered by several stimuli, including hypoxia, but the molecular mechanisms of hypoxia-increased membrane fragility remain unknown. Methods: In this study, we compared the action of hypoxia on hemolysis, ATP release and the composition of membrane-bound proteins in human erythrocytes. Results: Twenty minutes incubation of human erythrocytes in the oxygen-free environment increased the content of extracellular hemoglobin by ∼1.5 fold. Paired measurements of hemoglobin and ATP content in the same samples, showed a positive correlation between hemolysis and ATP release. Comparative analysis of SDS-PAGE electrophoresis of erythrocyte ghosts obtained under control and deoxygenated conditions revealed a ∼2-fold elevation of the content of membrane-bound protein with Mr of ∼60 kDa. Conclusion: Deoxygenation of human erythrocytes affects composition of membrane-bound proteins. Additional experiments should be performed to identify the molecular origin of 60 kDa protein and its role in the attenuation of erythrocyte integrity and ATP release in hypoxic conditions.

  2. Compartmentalized system with membrane-bound glycerol kinase. Activity and product distribution versus asymmetrical substrate supply.

    Science.gov (United States)

    Girard, A; Merchie, B; Maïsterrena, B

    1991-03-15

    An artificial-membrane-bound glycerokinase chosen as a membrane-bound two-substrate-enzyme model has been used to separate two unequal compartments of a specially designed diffusion cell. An interesting feature is the asymmetry of compartments and the existence of a diffusion layer adjacent to only one face of the enzymic membrane. In such a situation the apparent enzyme activity and the product distribution in the system have been studied versus all the possibilities of combination of ATP and glycerol supply. Our approach has lead us to differentiate two different roles played by a diffusion layer adjacent to a permeable enzymic membrane. Depending on the spatial origin of the enzymic substrates (i.e. from which compartment they derive), the diffusion layer can play either the role of a passive additional resistance to that of the membrane or the role of a third compartment in which the reaction product can partially accumulate before splitting on both parts of the membrane. Our results mainly demonstrate that a membrane-bound enzyme activity and the resulting product distribution occurring in a compartmentalized system may be regulated by the cumulative effect due to the asymmetry in volumes of the compartments, the presence of a diffusion layer and the different possibilities of substrate supply. With the topography studied, which is close to that reported for many 'in vivo' situations, the product may be diffused lead to vectorial metabolism processes.

  3. Enzyme catalytic resonance scattering spectral detection of trace hydrogen peroxide using guaiacol as substrate

    Directory of Open Access Journals (Sweden)

    Shiwen Huang

    2011-08-01

    Full Text Available Hydrogen peroxide oxidized guaiacol to form tetramer particles that exhibited a strong resonance scattering (RS peak at 530 nm in the presence of horseradish peroxidase (HRP in citric acid-Na2HPO4 buffer solution of pH 4.4. The RS peak increased when the concentration of hydrogen peroxide increased. The increased RS intensity (ΔI530 nm was linear to the hydrogen peroxide concentration in the range of 0.55-27.6 μM, with a linear regression equation of ΔI530 nm = 17.1C + 1.6, a relative coefficient of 0.9996 and a detection limit of 0.03 μM H2O2. This proposed method was applied to detect hydrogen peroxide in rain water, with sensitivity, selectivity, rapidity, and recovery of 98.0-104 %.

  4. Precursors and metabolic pathway for guaiacol production by Alicyclobacillus acidoterrestris.

    Science.gov (United States)

    Cai, Rui; Yuan, Yahong; Wang, Zhouli; Guo, Chunfeng; Liu, Bin; Liu, Laping; Wang, Yutang; Yue, Tianli

    2015-12-02

    Alicyclobacillus acidoterrestris has recently received much attention due to its implication in the spoilage of pasteurized fruit juices, which was manifested by the production of guaiacol. Vanillic acid and vanillin have been accepted as the biochemical precursors of guaiacol in fruit juices. The purpose of this study was to try to find other precursors and elucidate details about the conversion of vanillic acid and vanillin to guaiacol by A. acidoterrestris. Four potential substrates including ferulic acid, catechol, phenylalanine and tyrosine were analyzed, but they could not be metabolized to guaiacol by all the thirty A. acidoterrestris strains tested. Resting cell studies and enzyme assays demonstrated that vanillin was reduced to vanillyl alcohol by NADPH-dependent vanillin reductase and oxidized to vanillic acid by NAD(P)(+)-dependent vanillin dehydrogenases in A. acidoterrestris DSM 3923. Vanillic acid underwent a nonoxidative decarboxylation to guaiacol. The reversible vanillic acid decarboxylase involved was oxygen insensitive and pyridine nucleotide-independent. Copyright © 2015. Published by Elsevier B.V.

  5. Hydrogen Production by a Hyperthermophilic Membrane-Bound Hydrogenase in Soluble Nanolipoprotein Particles

    Energy Technology Data Exchange (ETDEWEB)

    Baker, S E; Hopkins, R C; Blanchette, C; Walsworth, V; Sumbad, R; Fischer, N; Kuhn, E; Coleman, M; Chromy, B; Letant, S; Hoeprich, P; Adams, M W; Henderson, P T

    2008-10-22

    Hydrogenases constitute a promising class of enzymes for ex vivo hydrogen production. Implementation of such applications is currently hindered by oxygen sensitivity and, in the case of membrane-bound hydrogenases (MBH), poor water solubility. Nanolipoprotein particles (NLPs), formed from apolipoproteins and phospholipids, offer a novel means to incorporate MBH into in a well-defined water-soluble matrix that maintains the enzymatic activity and is amenable to incorporation into more complex architectures. We report the synthesis, hydrogen-evolving activity and physical characterization of the first MBH-NLP assembly. This may ultimately lead to the development of biomimetic hydrogen production devices.

  6. Optimisation of the Factor VIII yield in mammalian cell cultures by reducing the membrane bound fraction

    DEFF Research Database (Denmark)

    Kolind, Mille Petersen; Nørby, Peder Lisby; Berchtold, Martin Werner

    2011-01-01

    In vivo, clotting Factor VIII (FVIII) circulates in plasma bound to von Willebrand factor (vWF), and the vWF:FVIII complex prevents binding of FVIII to phosphatidylserine (PS). Activation of FVIII by thrombin releases FVIII from vWF, and subsequently FVIII binds to PS exposed on activated platelets...... or ortho-phospho-L-serine (OPLS) demonstrated that annexin V and OPLS were able to reduce the membrane bound fraction of rFVIII by 70% and 30%, respectively. Finally, adding OPLS to CHO cells stably expressing FVIII increased the yield by 50%. Using this new knowledge, the recovery of rFVIII could...

  7. Transferred nuclear Overhauser effect analyses of membrane-bound enkephalin analogues by sup 1 H nuclear magnetic resonance: Correlation between activities and membrane-bound conformations

    Energy Technology Data Exchange (ETDEWEB)

    Milon, Alain; Miyazawa, Tatsuo; Higashijima, Tsutomu (Univ. of Tokyo (Japan))

    1990-01-09

    Leu-enkephalin, (D-Ala{sup 2})Leu-enkephalin, and (D-Ala{sup 2})Leu-enkephalinamide (agonists) and (L-Ala{sup 2})Leu-enkephalin (inactive analogue) bind to lipid bilayer consisting of phosphatidylcholine and phosphatidylserine. The conformations that these compounds assume, once bound to perdeuterated phospholipid bilayer, have been shown to be unique, as shown by the transferred nuclear Overhauser effect (TRNOE) of {sup 1}H NMR spectroscopy. In addition, their location in the bilayer was analyzed by TRNOE in the presence of spin-labeled phospholipids. These analyses showed a clear relationship between the activity and the peptide-membrane interaction. The three active peptides, when bound to membranes, adopt the same conformation, characterized by a type II{prime} {beta}-turn around Gly{sup 3}-Phe and a {gamma}-turn around Gly{sup 2} (or D-Ala{sup 2}). The inactive analogue, (L-Ala{sup 2})Leu-enkephalin, displayed a completely different TRNOE pattern corresponding to a different conformation in the membrane-bound state. The tyrosine residue of the active compounds is not inserted into the interior of membrane, but it is inserted into the bilayer for the L-Ala{sup 2} analogue. According to these results, (L-Ala{sup 2})Leu-enkephalin may be explained to be inactive because the mode of binding to the membranes is different from that of active compounds.

  8. Membrane-bound globin X protects the cell from reactive oxygen species.

    Science.gov (United States)

    Koch, Jonas; Burmester, Thorsten

    2016-01-08

    Globin X (GbX) is a member of the globin family that emerged early in the evolution of Metazoa. In vertebrates, GbX is restricted to lampreys, fish, amphibians and some reptiles, and is expressed in neurons. Unlike any other metazoan globin, GbX is N-terminally acylated and anchored in the cell membrane via myristoyl and palmitoyl groups, suggesting a unique function. Here, we compared the capacity of GbX to protect a mouse neuronal cell line from hypoxia and reactive oxygen species (ROS) with that of myoglobin. To evaluate the contribution of membrane-binding, we generated a mutated version of GbX without acyl groups. All three globins enhanced cell viability under hypoxia, with myoglobin having the most pronounced effect. GbX but not myoglobin protected the cells from hydrogen peroxide (H2O2)-induced stress. Membrane-bound GbX was significantly more efficient than its mutated, soluble form. Furthermore, myoglobin and mutated GbX increased production of ROS upon H2O2-treatment, while membrane-bound GbX did not. The results indicate that myoglobin enhances O2 supply while GbX protects the cell membrane from ROS-stress. The ancient origin of GbX suggests that ROS-protection reflects the function of the early globins before they acquired a respiratory role. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Biotransformation of ferulic acid to 4-vinyl guaiacol by Lactobacillus ...

    African Journals Online (AJOL)

    Continuously growing demand for natural flavors has led to a tremendous increase in biotransformation process employing microorganisms of different genera using ferulic acid (FA) as the precursor. In this study, potential of Lactobacillus farciminis (ATCC 29644) for biotransformation of FA to 4-vinyl guaiacol (4VG) was ...

  10. Allosteric activation of membrane-bound glutamate receptors using coordination chemistry within living cells

    Science.gov (United States)

    Kiyonaka, Shigeki; Kubota, Ryou; Michibata, Yukiko; Sakakura, Masayoshi; Takahashi, Hideo; Numata, Tomohiro; Inoue, Ryuji; Yuzaki, Michisuke; Hamachi, Itaru

    2016-10-01

    The controlled activation of proteins in living cells is an important goal in protein-design research, but to introduce an artificial activation switch into membrane proteins through rational design is a significant challenge because of the structural and functional complexity of such proteins. Here we report the allosteric activation of two types of membrane-bound neurotransmitter receptors, the ion-channel type and the G-protein-coupled glutamate receptors, using coordination chemistry in living cells. The high programmability of coordination chemistry enabled two His mutations, which act as an artificial allosteric site, to be semirationally incorporated in the vicinity of the ligand-binding pockets. Binding of Pd(2,2‧-bipyridine) at the allosteric site enabled the active conformations of the glutamate receptors to be stabilized. Using this approach, we were able to activate selectively a mutant glutamate receptor in live neurons, which initiated a subsequent signal-transduction pathway.

  11. Effect of narcotics on membrane-bound mitochondrial processes in fish

    DEFF Research Database (Denmark)

    Vergauwen, Lucia; Nørgaard Schmidt, Stine; Michiels, Ellen

    in vivo (zebrafish embryo) and in vitro tests. We applied a passive dosing method to expose zebrafish embryos up to 5 days post fertilization to linear dilution series of a set of non-polar narcotics (phenanthrene and three chlorobenzene structure analogues). In addition to increasing mortality, we...... membrane-bound process and is therefore a potential target. We found that in zebrafish embryos ETC activity was increased at low exposure concentrations, suggesting a compensatory response, while it decreased when exposure concentrations reached levels causing reduced motility, heart rate and eventually...... mortality. The effect of narcotic compounds on ETC activity was confirmed in vitro: we observed inhibition of the ETC after adding the compounds directly to a homogenate of control embryos. To further investigate effects on the energy production system, and to characterize the observed compensatory response...

  12. Membrane-bound alcohol dehydrogenase is essential for glyceric acid production in Acetobacter tropicalis.

    Science.gov (United States)

    Habe, Hiroshi; Sato, Shun; Fukuoka, Tokuma; Kitamoto, Dai; Yakushi, Toshiharu; Matsushita, Kazunobu; Sakaki, Keiji

    2011-01-01

    Acetobacter tropicalis NBRC16470 can produce highly enantiomerically pure D-glyceric acid (D-GA; >99 % enantiomeric excess) from glycerol. To investigate whether membrane-bound alcohol dehydrogenase (mADH) is involved in GA production in A. tropicalis, we amplified part of the gene encoding mADH subunit I (adhA) using polymerase chain reaction and constructed an adhA-disrupted mutant of A. tropicalis (ΔadhA). Because ΔadhA did not produce GA, we confirmed that mADH is essential for the conversion of glycerol to GA. We also cloned and sequenced the entire region corresponding to adhA and adhB, which encodes mADH subunit II. The sequences showed high identities (84-86 %) with the equivalent mADH subunits from other Acetobacter spp.

  13. Free and membrane-bound ribosomes and polysomes in hippocampal neurons during a learning experiment.

    Science.gov (United States)

    Wenzel, J; David, H; Pohle, W; Marx, I; Matthies, H

    1975-01-24

    The ribosomes of the CA1 and CA3 pyramidal cells of hipocampus were investigated by morphometric methods after the acquisition of a shock-motivated brightness discrimination in rats. A significant increase in the total number of ribosomes was observed in CA1 cells of trained animals and in CA3 cells of both active controls and trained rats. A significant increase in membrane-bound ribosomes was obtained in CA1 and CA3 cells after training only. The results confirm the suggestion of an increased protein synthesis in hippocampal neurons during and after the acquisition of a brightness discrimination, as we have concluded from out previous investigations on the incorporation of labeled amino acids under identical experimental conditions. The results lead to the assumption that the protein synthesis in some neuronal cells may probably differ not only quantitatively, but also qualitatively in trained and untrained animals.

  14. Biochemical and spectroscopic characterization of the membrane-bound nitrate reductase from Marinobacter hydrocarbonoclasticus 617.

    Science.gov (United States)

    Correia, Cristina; Besson, Stéphane; Brondino, Carlos D; González, Pablo J; Fauque, Guy; Lampreia, Jorge; Moura, Isabel; Moura, José J G

    2008-11-01

    Membrane-bound nitrate reductase from Marinobacter hydrocarbonoclasticus 617 can be solubilized in either of two ways that will ultimately determine the presence or absence of the small (Iota) subunit. The enzyme complex (NarGHI) is composed of three subunits with molecular masses of 130, 65, and 20 kDa. This enzyme contains approximately 14 Fe, 0.8 Mo, and 1.3 molybdopterin guanine dinucleotides per enzyme molecule. Curiously, one heme b and 0.4 heme c per enzyme molecule have been detected. These hemes were potentiometrically characterized by optical spectroscopy at pH 7.6 and two noninteracting species were identified with respective midpoint potentials at Em=+197 mV (heme c) and -4.5 mV (heme b). Variable-temperature (4-120 K) X-band electron paramagnetic resonance (EPR) studies performed on both as-isolated and dithionite-reduced nitrate reductase showed, respectively, an EPR signal characteristic of a [3Fe-4S]+ cluster and overlapping signals associated with at least three types of [4Fe-4S]+ centers. EPR of the as-isolated enzyme shows two distinct pH-dependent Mo(V) signals with hyperfine coupling to a solvent-exchangeable proton. These signals, called "low-pH" and "high-pH," changed to a pH-independent Mo(V) signal upon nitrate or nitrite addition. Nitrate addition to dithionite-reduced samples at pH 6 and 7.6 yields some of the EPR signals described above and a new rhombic signal that has no hyperfine structure. The relationship between the distinct EPR-active Mo(V) species and their plausible structures is discussed on the basis of the structural information available to date for closely related membrane-bound nitrate reductases.

  15. Characterization of a membrane-bound C-glucosyltransferase responsible for carminic acid biosynthesis in Dactylopius coccus Costa

    DEFF Research Database (Denmark)

    Kannangara, Rubini; Siukstaite, Lina; Borch-Jensen, Jonas

    2017-01-01

    Carminic acid, a glucosylated anthraquinone found in scale insects like Dactylopius coccus, has since ancient times been used as a red colorant in various applications. Here we show that a membrane-bound C-glucosyltransferase, isolated from D. coccus and designated DcUGT2, catalyzes the glucosyla......Carminic acid, a glucosylated anthraquinone found in scale insects like Dactylopius coccus, has since ancient times been used as a red colorant in various applications. Here we show that a membrane-bound C-glucosyltransferase, isolated from D. coccus and designated DcUGT2, catalyzes...

  16. Membrane-bound organelles versus membrane-less compartments and their control of anabolic pathways in Drosophila

    NARCIS (Netherlands)

    Aguilera-Gomez, Angelica; Rabouille, Catherine

    2017-01-01

    Classically, we think of cell compartmentalization as being achieved by membrane-bound organelles. It has nevertheless emerged that membrane-less assemblies also largely contribute to this compartmentalization. Here, we compare the characteristics of both types of compartmentalization in term of

  17. Targeting Membrane-Bound Viral RNA Synthesis Reveals Potent Inhibition of Diverse Coronaviruses Including the Middle East Respiratory Syndrome Virus

    NARCIS (Netherlands)

    Lundin, A.; Dijkman, R.; Bergstrom, T.; Kann, N.; Adamiak, B.; Hannoun, C.; Kindler, E.; Jonsdottir, H.R.; Muth, D.; Kint, J.; Forlenza, M.

    2014-01-01

    Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound

  18. Optimized Production of Lignin Peroxidase, Manganese Peroxidase ...

    African Journals Online (AJOL)

    The effect of temperature, pH, carbon, nitrogen, Cu2+, 2,5-xylidine, ferulic acid, varatryl alcohol and Mn2+ in submerged culture fermentations were investigated for maximum enzymes production. After 7 days of incubation, 72-100% oxidation of RBBR, ABTS and guaiacol was observed. With optimized culture conditions, ...

  19. Analysis of the Peroxidase Activity of Rice (Oryza Sativa) Recombinant Hemoglobin 1: Implications for the In Vivo Function of Hexacoordinate Non-Symbiotic Hemoglobins in Plants

    Science.gov (United States)

    In plants, it has been proposed that hexacoordinate (class 1) non-symbiotic Hbs (nsHb-1) function in vivo as peroxidases. However, little is known about the peroxidase activity of nsHb-1. We evaluated the peroxidase activity of rice recombinant Hb1 (a nsHb-1) by using the guaiacol/H2O2 system at pH ...

  20. The morphogenetic MreBCD proteins of Escherichia coli form an essential membrane-bound complex

    DEFF Research Database (Denmark)

    Kruse, Torben; Bork-Jensen, J.; Gerdes, Kenn

    2005-01-01

    . subtilis and C. crescentus, the mreB gene is essential. However, in E. coli, mreB was inferred not to be essential. Using a tight, conditional gene depletion system, we systematically investigated whether the E. coli mreBCD-encoded components were essential. We found that cells depleted of mreBCD became......D. In contrast, MreB and MreD did not interact in this assay. Thus, we conclude that the E. coli MreBCD form an essential membrane-bound complex. Curiously, MreB did not form cables in cell depleted for MreC, MreD or RodA, indicating a mutual interdependency between MreB filament morphology and cell shape. Based......MreB proteins of Escherichia coli, Bacillus subtilis and Caulobacter crescentus form actin-like cables lying beneath the cell surface. The cables are required to guide longitudinal cell wall synthesis and their absence leads to merodiploid spherical and inflated cells prone to cell lysis. In B...

  1. Ligand-modulated conformational switching in a fully synthetic membrane-bound receptor

    Science.gov (United States)

    Lister, Francis G. A.; Le Bailly, Bryden A. F.; Webb, Simon J.; Clayden, Jonathan

    2017-05-01

    Signal transduction through G-protein-coupled receptors (GPCRs) involves binding to signalling molecules at the cell surface, which leads to global changes in molecular conformation that are communicated through the membrane. Artificial mechanisms for communication involving ligand binding and global conformational switching have been demonstrated so far only in the solution phase. Here, we report a membrane-bound synthetic receptor that responds to binding of a ligand by undergoing a conformational change that is propagated over several nanometres, deep into the phospholipid bilayer. Our design uses a helical foldamer core, with structural features borrowed from a class of membrane-active fungal antibiotics, ligated to a water-compatible, metal-centred binding site and a conformationally responsive fluorophore. Using the fluorophore as a remote reporter of conformational change, we find that binding of specific carboxylate ligands to a Cu(II) cofactor at the binding site perturbs the foldamer's global conformation, mimicking the conformational response of a GPCR to ligand binding.

  2. Electrostatic control by lipids upon the membrane-bound (Na+ + K+)-ATPase.

    Science.gov (United States)

    Ahrens, M L

    1981-04-06

    In this paper, the membrane-bound (Na+ + K+)-ATPase from bovine brain is shown to be controlled by electrostatic alterations of the charged lipids surrounding the enzyme. The properties under investigation are the enzymatic activity, activation energy and the response of the enzymatic system to temperature. Arrhenius plots of the ATPase activity are biphasic with a break at temperature Ti. The temperature Ti, the activation energies at temperatures above and below Ti, and the enzymatic activity at any constant temperature have been shown to depend upon the concentrations of alkali and alkaline-earth metal ions in the solution. These electrolyte dependencies are ascribed to changes of electrostatic conditions at the lipids surrounding the ATPase. If the higher electrostatic screening ability of divalent ions is taken into account, the results in the presence of mono- and divalent ions become virtually the same. As a result of this work, it is concluded that electrostatic alterations are transmitted to the ATPase from the lipids of the membrane in which the enzyme is embedded. Inhibition and activation of the enzyme by mono-and divalent metal ions may thus be explained without any auxiliary hypothesis, particularly without postulating specific binding sites for the different ionic species at the protein. In addition, the specific lipid requirement of the ATPase may be understood better in the light of this interpretation.

  3. Steric pressure between membrane-bound proteins opposes lipid phase separation.

    Science.gov (United States)

    Scheve, Christine S; Gonzales, Paul A; Momin, Noor; Stachowiak, Jeanne C

    2013-01-30

    Cellular membranes are densely crowded with a diverse population of integral and membrane-associated proteins. In this complex environment, lipid rafts, which are phase-separated membrane domains enriched in cholesterol and saturated lipids, are thought to organize the membrane surface. Specifically, rafts may help to concentrate proteins and lipids locally, enabling cellular processes such as assembly of caveolae, budding of enveloped viruses, and sorting of lipids and proteins in the Golgi. However, the ability of rafts to concentrate protein species has not been quantified experimentally. Here we show that when membrane-bound proteins become densely crowded within liquid-ordered membrane regions, steric pressure arising from collisions between proteins can destabilize lipid phase separations, resulting in a homogeneous distribution of proteins and lipids over the membrane surface. Using a reconstituted system of lipid vesicles and recombinant proteins, we demonstrate that protein-protein steric pressure creates an energetic barrier to the stability of phase-separated membrane domains that increases in significance as the molecular weight of the proteins increases. Comparison with a simple analytical model reveals that domains are destabilized when the steric pressure exceeds the approximate enthalpy of membrane mixing. These results suggest that a subtle balance of free energies governs the stability of phase-separated cellular membranes, providing a new perspective on the role of lipid rafts as concentrators of membrane proteins.

  4. A Redundant Role of Human Thyroid Peroxidase Propeptide for Cellular, Enzymatic, and Immunological Activity

    OpenAIRE

    Godlewska, Marlena; Góra, Monika; Buckle, Ashley M.; Porebski, Benjamin T.; Kemp, E. Helen; Sutton, Brian J.; Czarnocka, Barbara; Banga, J. Paul

    2014-01-01

    Background: Thyroid peroxidase (TPO) is a dimeric membrane-bound enzyme of thyroid follicular cells, responsible for thyroid hormone biosynthesis. TPO is also a common target antigen in autoimmune thyroid disease (AITD). With two active sites, TPO is an unusual enzyme, and thus there is much interest in understanding its structure and role in AITD. Homology modeling has shown TPO to be composed of different structural modules, as well as a propeptide sequence. During the course of studies to ...

  5. Optimized Production of Lignin Peroxidase, Manganese Peroxidase

    African Journals Online (AJOL)

    Mgina

    OPTIMIZED PRODUCTION OF LIGNIN PEROXIDASE, MANGANESE. PEROXIDASE AND LACCASE IN SUBMERGED CULTURES OF. TRAMETES TROGII USING VARIOUS GROWTH MEDIA. COMPOSITIONS. F Patrick*, G Mtui, AM Mshandete and A Kivaisi. Department of Molecular Biology and Biotechnology, College ...

  6. Molecular characterization of soluble and membrane-bound trehalases of the whitefly, Bemisia tabaci.

    Science.gov (United States)

    Wang, Jia; He, Wen-Bo; Su, Yun-Lin; Bing, Xiao-Li; Liu, Shu-Sheng

    2014-04-01

    Trehalases (Tres) have been demonstrated to be the key enzymes that are involved in various trehalose-associated physiological processes in insects. However, little attention has been devoted to the Tres in the whitefly, Bemisia tabaci. In this study, a soluble Tre (BtTre-1) and a membrane-bound Tre (BtTre-2) were cloned in the invasive cryptic species Middle East-Asia Minor 1 (MEAM1) of the whitefly B. tabaci complex. Alignment of deduced amino acids sequences of both BtTres revealed that they share common consensus regions and residues with Tres of other insect species. Levels of BtTres expression in various stages and tissues of the whitefly suggested that BtTre-2 may play a key role in trehalose catabolism during development of the whitefly, especially for oocyte development, while BtTre-1 may prevent trehalose in salivary gland from leaking and entering into plants along with saliva. Potential roles of trehalose catabolism in response to direct and/or plant-mediated indirect effects of Tomato Yellow Leaf Curl China Virus (TYLCCNV) were also detected. Whiteflies feeding on virus-infected tobacco plants showed higher BtTres expressions and accordingly higher BtTres activity but lower trehalose content than those feeding on uninfected plants. The enhanced trehalose catabolism may be beneficial to oocyte development in ovary and attenuate plant defensive responses induced by trehalose in saliva. Viruliferous and nonviruliferous whiteflies feeding on cotton, a nonhost plant for TYLCCNV, differed significantly only in trehalose content. The higher trehalose content in viruliferous whiteflies may be conducive to resisting the stress inflicted by TYLCCNV. © 2014 Wiley Periodicals, Inc.

  7. Membrane-bound state of the colicin E1 channel domain as an extended two-dimensional helical array.

    Science.gov (United States)

    Zakharov, S D; Lindeberg, M; Griko, Y; Salamon, Z; Tollin, G; Prendergast, F G; Cramer, W A

    1998-04-14

    Atomic level structures have been determined for the soluble forms of several colicins and toxins, but the structural changes that occur after membrane binding have not been well characterized. Changes occurring in the transition from the soluble to membrane-bound state of the C-terminal 190-residue channel polypeptide of colicin E1 (P190) bound to anionic membranes are described. In the membrane-bound state, the alpha-helical content increases from 60-64% to 80-90%, with a concomitant increase in the average length of the helical segments from 12 to 16 or 17 residues, close to the length required to span the membrane bilayer in the open channel state. The average distance between helical segments is increased and interhelix interactions are weakened, as shown by a major loss of tertiary structure interactions, decreased efficiency of fluorescence resonance energy transfer from an energy donor on helix V of P190 to an acceptor on helix IX, and decreased resonance energy transfer at higher temperatures, not observed in soluble P190, implying freedom of motion of helical segments. Weaker interactions are also shown by a calorimetric thermal transition of low cooperativity, and the extended nature of the helical array is shown by a 3- to 4-fold increase in the average area subtended per molecule to 4,200 A2 on the membrane surface. The latter, with analysis of the heat capacity changes, implies the absence of a developed hydrophobic core in the membrane-bound P190. The membrane interfacial layer thus serves to promote formation of a highly helical extended two-dimensional flexible net. The properties of the membrane-bound state of the colicin channel domain (i.e., hydrophobic anchor, lengthened and loosely coupled alpha-helices, and close association with the membrane interfacial layer) are plausible structural features for the state that is a prerequisite for voltage gating, formation of transmembrane helices, and channel opening.

  8. Mechanism of biological denitrification inhibition: procyanidins induce an allosteric transition of the membrane-bound nitrate reductase through membrane alteration.

    Science.gov (United States)

    Bardon, Clément; Poly, Franck; Piola, Florence; Pancton, Muriel; Comte, Gilles; Meiffren, Guillaume; Haichar, Feth el Zahar

    2016-05-01

    Recently, it has been shown that procyanidins from Fallopia spp. inhibit bacterial denitrification, a phenomenon called biological denitrification inhibition (BDI). However, the mechanisms involved in such a process remain unknown. Here, we investigate the mechanisms of BDI involving procyanidins, using the model strain Pseudomonas brassicacearum NFM 421. The aerobic and anaerobic (denitrification) respiration, cell permeability and cell viability of P. brassicacearum were determined as a function of procyanidin concentration. The effect of procyanidins on the bacterial membrane was observed using transmission electronic microscopy. Bacterial growth, denitrification, NO3- and NO2-reductase activity, and the expression of subunits of NO3- (encoded by the gene narG) and NO2-reductase (encoded by the gene nirS) under NO3 or NO2 were measured with and without procyanidins. Procyanidins inhibited the denitrification process without affecting aerobic respiration at low concentrations. Procyanidins also disturbed cell membranes without affecting cell viability. They specifically inhibited NO3- but not NO2-reductase.Pseudomonas brassicacearum responded to procyanidins by over-expression of the membrane-bound NO3-reductase subunit (encoded by the gene narG). Our results suggest that procyanidins can specifically inhibit membrane-bound NO3-reductase inducing enzymatic conformational changes through membrane disturbance and that P. brassicacearum responds by over-expressing membrane-bound NO3-reductase. Our results lead the way to a better understanding of BDI. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  9. Influence of kaempferol, a flavonoid compound, on membrane-bound ATPases in streptozotocin-induced diabetic rats.

    Science.gov (United States)

    Al-Numair, Khalid S; Veeramani, Chinnadurai; Alsaif, Mohammed A; Chandramohan, Govindasamy

    2015-01-01

    Kaempferol is a flavonoid found in many edible plants (e.g. tea, cabbage, beans, tomato, strawberries, and grapes) and in plants or botanical products commonly used in traditional medicine. Numerous preclinical studies have shown that kaempferol have a wide range of pharmacological activities, including antioxidant, anti-inflammatory, anticancer, cardioprotective, neuroprotective, and antidiabetic activities. The present study investigates the effect of kaempferol on membrane-bound ATPases in erythrocytes and in liver, kidney, and heart of streptozotocin (STZ)-induced diabetic rats. Diabetes was induced into adult male albino rats of the Wistar strain, by intraperitoneal administration of STZ (40 mg/kg body weight (BW)). Kaempferol (100 mg/kg BW) or glibenclamide (600 µg/kg BW) was administered orally once daily for 45 d to normal and STZ-induced diabetic rats. The effects of kaempferol on membrane-bound ATPases (total ATPase, Na(+)/K(+)-ATPase, Ca(2+)-ATPase, and Mg(2+)-ATPase) activity in erythrocytes and in liver, kidney, and heart were determined. In our study, diabetic rats had significantly (p kaempferol (100 mg/kg BW) or glibenclamide (600 µg/kg BW) for a period of 45 d resulted in significant (p kaempferol has the potential to restore deranged activity of membrane-bound ATPases in STZ-induced diabetic rats. Further detailed investigation is necessary to discover kaempferol's action mechanism.

  10. The catalytic function of cytochrome P450 is entwined with its membrane-bound nature [version 1; referees: 4 approved

    Directory of Open Access Journals (Sweden)

    Carlo Barnaba

    2017-05-01

    Full Text Available Cytochrome P450, a family of monooxygenase enzymes, is organized as a catalytic metabolon, which requires enzymatic partners as well as environmental factors that tune its complex dynamic. P450 and its reducing counterparts—cytochrome P450-reductase and cytochrome b5—are membrane-bound proteins located in the cytosolic side of the endoplasmic reticulum. They are believed to dynamically associate to form functional complexes. Increasing experimental evidence signifies the role(s played by both protein-protein and protein-lipid interactions in P450 catalytic function and efficiency. However, the biophysical challenges posed by their membrane-bound nature have severely limited high-resolution understanding of the molecular interfaces of these interactions. In this article, we provide an overview of the current knowledge on cytochrome P450, highlighting the environmental factors that are entwined with its metabolic function. Recent advances in structural biophysics are also discussed, setting up the bases for a new paradigm in the study of this important class of membrane-bound enzymes.

  11. Evaluation of membrane-bound and soluble forms of HLA-G in Systemic Sclerosis.

    Science.gov (United States)

    Contini, Paola; Negrini, Simone; Murdaca, Giuseppe; Borro, Matteo; Puppo, Francesco

    2018-04-16

    Systemic sclerosis (SSc) is a complex disease characterized by immune dysregulation, extensive vascular damage and wide-spread fibrosis. Human leukocyte antigen-G (HLA-G) is a non-classic class I major histocompatibility complex (MHC) molecule characterized by complex immuno-modulating properties. HLA-G is expressed on the membrane of different cell lineages in both physiological and pathological conditions. HLA-G is also detectable in soluble form (sHLA-G) deriving from the shedding of surface isoforms (sHLA-G1) or the secretion of soluble isoforms (HLA-G5). Several immunosuppressive functions have been attributed to both membrane-bound and soluble HLA-G molecules. The plasma levels of sHLA-G were higher in SSc patients (444.27 ± 304.84 U/ml) as compared to controls (16.74 ± 20.58 U/ml) (p G (r: 0.65; p G1 (r: 0.60; p = 0.003) and HLA-G5 (r: 0.47; p = 0.02). The percentage of HLA-G-positive monocytes (0.98 ± 1.72), CD4+ (0.37 ± 0.68), CD8+ (2.05 ± 3.74) and CD4+CD8+ double positive cells (14.53 ± 16.88) was higher in SSc patients than in controls (0.11 ± 0.08, 0.01 ± 0.01, 0.01 ± 0.01 and 0.39 ± 0.40, respectively) (p G molecules and the membrane expression of HLA-G by PBMC is clearly elevated suggesting an involvement of HLA-G molecules in the immune dysregulation of SSc. This article is protected by copyright. All rights reserved. © 2018 British Society for Immunology.

  12. Structural and dynamical insights into the membrane-bound α-synuclein.

    Directory of Open Access Journals (Sweden)

    Neha Jain

    Full Text Available Membrane-induced disorder-to-helix transition of α-synuclein, a presynaptic protein, has been implicated in a number of important neuronal functions as well as in the etiology of Parkinson's disease. In order to obtain structural insights of membrane-bound α-synuclein at the residue-specific resolution, we took advantage of the fact that the protein is devoid of tryptophan and incorporated single tryptophan at various residue positions along the sequence. These tryptophans were used as site-specific markers to characterize the structural and dynamical aspects of α-synuclein on the negatively charged small unilamellar lipid vesicles. An array of site-specific fluorescence readouts, such as the spectral-shift, quenching efficiency and anisotropy, allowed us to discern various features of the conformational rearrangements occurring at different locations of α-synuclein on the lipid membrane. In order to define the spatial localization of various regions of the protein near the membrane surface, we utilized a unique and sensitive indicator, namely, red-edge excitation shift (REES, which originates when a fluorophore is located in a highly ordered micro-environment. The extent of REES observed at different residue positions allowed us to directly identify the residues that are localized at the membrane-water interface comprising a thin (∼ 15 Å layer of motionally restrained water molecules and enabled us to construct a dynamic hydration map of the protein. The combination of site-specific fluorescence readouts allowed us to unravel the intriguing molecular details of α-synuclein on the lipid membrane in a direct model-free fashion. Additionally, the combination of methodologies described here are capable of distinguishing subtle but important structural alterations of α-synuclein bound to different negatively charged lipids with varied head-group chemistry. We believe that the structural modulations of α-synuclein on the membrane could

  13. Generation and characterization of tabalumab, a human monoclonal antibody that neutralizes both soluble and membrane-bound B-cell activating factor

    Science.gov (United States)

    Manetta, Joseph; Bina, Holly; Ryan, Paul; Fox, Niles; Witcher, Derrick R; Kikly, Kristine

    2014-01-01

    B-cell activating factor (BAFF) is a B-cell survival factor with a key role in B-cell homeostasis and tolerance. Dysregulated BAFF expression may contribute to autoimmune diseases or B-cell malignancies via effects on abnormal B-lymphocyte activation, proliferation, survival, and immunoglobulin secretion. Monoclonal antibodies were generated against human BAFF, characterized for species specificity and affinity, and screened for the ability to neutralize both membrane-bound and soluble BAFF. In addition, studies were undertaken to determine the relative potency of membrane-bound and soluble BAFF. Tabalumab has a high affinity for human, cynomolgus monkey, and rabbit BAFF. No binding to mouse BAFF was detected. Tabalumab was able to neutralize soluble human, cynomolgus monkey, or rabbit BAFF with equal potency. Our data demonstrate that membrane-bound BAFF can be a more potent stimulus for B-cells than soluble BAFF, and tabalumab also neutralized membrane-bound BAFF. Tabalumab prevented BAFF from binding to BAFF receptors and demonstrated pharmacodynamic effects in human BAFF transgenic mice. Tabalumab is a high-affinity human antibody with neutralizing activity against membrane-bound and soluble BAFF. Given our findings that membrane-bound BAFF can have greater in vitro potency than soluble BAFF, neutralization of both forms of BAFF is likely to be important for optimal therapeutic effect. PMID:25258549

  14. Generation and characterization of tabalumab, a human monoclonal antibody that neutralizes both soluble and membrane-bound B-cell activating factor.

    Science.gov (United States)

    Manetta, Joseph; Bina, Holly; Ryan, Paul; Fox, Niles; Witcher, Derrick R; Kikly, Kristine

    2014-01-01

    B-cell activating factor (BAFF) is a B-cell survival factor with a key role in B-cell homeostasis and tolerance. Dysregulated BAFF expression may contribute to autoimmune diseases or B-cell malignancies via effects on abnormal B-lymphocyte activation, proliferation, survival, and immunoglobulin secretion. Monoclonal antibodies were generated against human BAFF, characterized for species specificity and affinity, and screened for the ability to neutralize both membrane-bound and soluble BAFF. In addition, studies were undertaken to determine the relative potency of membrane-bound and soluble BAFF. Tabalumab has a high affinity for human, cynomolgus monkey, and rabbit BAFF. No binding to mouse BAFF was detected. Tabalumab was able to neutralize soluble human, cynomolgus monkey, or rabbit BAFF with equal potency. Our data demonstrate that membrane-bound BAFF can be a more potent stimulus for B-cells than soluble BAFF, and tabalumab also neutralized membrane-bound BAFF. Tabalumab prevented BAFF from binding to BAFF receptors and demonstrated pharmacodynamic effects in human BAFF transgenic mice. Tabalumab is a high-affinity human antibody with neutralizing activity against membrane-bound and soluble BAFF. Given our findings that membrane-bound BAFF can have greater in vitro potency than soluble BAFF, neutralization of both forms of BAFF is likely to be important for optimal therapeutic effect.

  15. Characterization of a membrane-bound C-glucosyltransferase responsible for carminic acid biosynthesis in Dactylopius coccus Costa

    OpenAIRE

    Kannangara, Rubini; Siukstaite, Lina; Borch-Jensen, Jonas; Madsen, Bjørn; Kongstad, Kenneth T.; Staerk, Dan; Bennedsen, Mads; Okkels, Finn T.; Rasmussen, Silas A.; Larsen, Thomas O.; Frandsen, Rasmus J. N.; Møller, Birger Lindberg

    2017-01-01

    Carminic acid, a glucosylated anthraquinone found in scale insects like Dactylopius coccus, has since ancient times been used as a red colorant in various applications. Here we show that a membrane-bound C-glucosyltransferase, isolated from D. coccus and designated DcUGT2, catalyzes the glucosylation of flavokermesic acid and kermesic acid into their respective C-glucosides dcII and carminic acid. DcUGT2 is predicted to be a type I integral endoplasmic reticulum (ER) membrane protein, contain...

  16. A genetic screen for anchorage-independent proliferation in mammalian cells identifies a membrane-bound neuregulin.

    Directory of Open Access Journals (Sweden)

    Davide Danovi

    2010-07-01

    Full Text Available Anchorage-independent proliferation is a hallmark of oncogenic transformation and is thought to be conducive to proliferation of cancer cells away from their site of origin. We have previously reported that primary Schwann cells expressing the SV40 Large T antigen (LT are not fully transformed in that they maintain a strict requirement for attachment, requiring a further genetic change, such as oncogenic Ras, to gain anchorage-independence. Using the LT-expressing cells, we performed a genetic screen for anchorage-independent proliferation and identified Sensory and Motor Neuron Derived Factor (SMDF, a transmembrane class III isoform of Neuregulin 1. In contrast to oncogenic Ras, SMDF induced enhanced proliferation in normal primary Schwann cells but did not trigger cellular senescence. In cooperation with LT, SMDF drove anchorage-independent proliferation, loss of contact inhibition and tumourigenicity. This transforming ability was shared with membrane-bound class III but not secreted class I isoforms of Neuregulin, indicating a distinct mechanism of action. Importantly, we show that despite being membrane-bound signalling molecules, class III neuregulins transform via a cell intrinsic mechanism, as a result of constitutive, elevated levels of ErbB signalling at high cell density and in anchorage-free conditions. This novel transforming mechanism may provide new targets for cancer therapy.

  17. Extracción y medida de peroxidasa en pulpa de arazá (Eugenia stipitata MC Vaugh) Extraction and activity of peroxidase in arazá (Eugenia stipitata Mc Vaugh) pulp fruit

    OpenAIRE

    Carlos Eduardo Narváez-Cuenca

    2008-01-01

    A technique for both extraction and activity measurement of peroxidase extracted from arazá (Eugenia stipitata Mc Vaugh) is described. Peroxidase from arazá pulp fruit was extracted using a combination of protein precipitation with acetone and extraction with 50 mM sodium buffer phosphate (pH 6.0). Optimum activity using guaiacol as H-donor was obtained at pH from 5.0 to 6.5, temperature from 60 to 75 °C, H2O2 between 10 to 15 mM and guaiacol from 80 to 160 mM. Thermal inactivation showed a f...

  18. Protective effect of Lagenaria siceraria (Mol) against membrane-bound enzyme alterations in isoproterenol-induced cardiac damage in rats.

    Science.gov (United States)

    Vijayakumar, M; Selvi, V; Krishnakumari, S

    2012-01-01

    This study was aimed at evaluating the preventive role of the ethanolic extract of Lagenaria siceraria (Mol) fruit on membrane-bound enzymes, such as sodium potassium-dependent adenosine triphosphatase (Na(+)/K(+) ATPase), calcium-dependent adenosine triphosphatase (Ca(2+) ATPase) and magnesium-dependent adenosine triphosphatase (Mg(2+) ATPase) on isoproterenol (ISO)-induced myocardial infarction (MI) in rats. Male albino Wistar rats were pretreated with the ethanolic extract of L. siceraria (Mol) fruit (125, 250 and 500 mg kg(-1) body weight) for a period of 30 days. After the treatment period, ISO (85mg kg(-1) body weight) was subcutaneously injected into rats at 24-h intervals for 2 days. ISO-induced rats showed a significant (p Mol) fruit for a period of 30 days exhibited a significant (p Mol) fruit has membrane-stabilising role in ISO-induced MI in rats.

  19. Targeting of a chimeric human histone fusion mRNA to membrane-bound polysomes in HeLa cells

    International Nuclear Information System (INIS)

    Zambetti, G.; Stein, J.; Stein, G.

    1987-01-01

    The subcellular location of histone mRNA-containing polysomes may play a key role in the posttranscriptional events that mediate histone mRNA turnover following inhibition of DNA synthesis. Previously, it has been shown that histone mRNA is found primarily on free polysomes that are associated with the cytoskeleton. The authors report here the construction of an Escherichia coli pBR322 β-lactamase signal peptide-human H3 histone fusion gene. The fusion transcript is targeted to membrane-bound polysomes and remains stable following interruption of DNA replication. Relocating mRNA within the cell may provide a procedure for studying the posttranscriptional regulation of gene expression

  20. Release of membrane-bound vesicles and inhibition of tumor cell adhesion by the peptide Neopetrosiamide A.

    Directory of Open Access Journals (Sweden)

    Pamela Austin

    2010-05-01

    Full Text Available Neopetrosiamide A (NeoA is a 28-amino acid tricyclic peptide originally isolated from a marine sponge as a tumor cell invasion inhibitor whose mechanism of action is unknown.We show that NeoA reversibly inhibits tumor cell adhesion, disassembles focal adhesions in pre-attached cells, and decreases the level of beta1 integrin subunits on the cell surface. NeoA also induces the formation of dynamic, membrane-bound protrusions on the surface of treated cells and the release of membrane-bound vesicles into the culture medium. Proteomic analysis indicates that the vesicles contain EGF and transferrin receptors as well as a number of proteins involved in adhesion and migration including: beta1 integrin and numerous alpha integrin subunits; actin and actin-binding proteins such as cofilin, moesin and myosin 1C; and membrane modulating eps15 homology domain (EHD proteins. Surface labeling, trafficking inhibition, and real-time imaging experiments all suggest that beta1 integrin-containing vesicles are released directly from NeoA-induced cell surface protrusions rather than from vesicles generated intracellularly. The biological activity of NeoA is dependent on its disulfide bond pattern and NMR spectroscopy indicates that the peptide is globular with a continuous ridge of hydrophobic groups flanked by charged amino acid residues that could facilitate a simultaneous interaction with lipids and proteins in the membrane.NeoA is an anti-adhesive peptide that decreases cell surface integrin levels through a novel, yet to be elucidated, mechanism that involves the release of adhesion molecule-containing vesicles from the cell surface.

  1. Generation and characterization of tabalumab, a human monoclonal antibody that neutralizes both soluble and membrane-bound B-cell activating factor

    Directory of Open Access Journals (Sweden)

    Manetta J

    2014-08-01

    Full Text Available Joseph Manetta, Holly Bina, Paul Ryan, Niles Fox, Derrick R Witcher, Kristine Kikly Biotechnology Discovery Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN, USA Abstract: B-cell activating factor (BAFF is a B-cell survival factor with a key role in B-cell homeostasis and tolerance. Dysregulated BAFF expression may contribute to autoimmune diseases or B-cell malignancies via effects on abnormal B-lymphocyte activation, proliferation, survival, and immunoglobulin secretion. Monoclonal antibodies were generated against human BAFF, characterized for species specificity and affinity, and screened for the ability to neutralize both membrane-bound and soluble BAFF. In addition, studies were undertaken to determine the relative potency of membrane-bound and soluble BAFF. Tabalumab has a high affinity for human, cynomolgus monkey, and rabbit BAFF. No binding to mouse BAFF was detected. Tabalumab was able to neutralize soluble human, cynomolgus monkey, or rabbit BAFF with equal potency. Our data demonstrate that membrane-bound BAFF can be a more potent stimulus for B-cells than soluble BAFF, and tabalumab also neutralized membrane-bound BAFF. Tabalumab prevented BAFF from binding to BAFF receptors and demonstrated pharmacodynamic effects in human BAFF transgenic mice. Tabalumab is a high-affinity human antibody with neutralizing activity against membrane-bound and soluble BAFF. Given our findings that membrane-bound BAFF can have greater in vitro potency than soluble BAFF, neutralization of both forms of BAFF is likely to be important for optimal therapeutic effect. Keywords: autoimmunity, B-cell malignancies, B-cell survival factor, BAFF

  2. Activated Carbon, Carbon Nanofiber and Carbon Nanotube Supported Molybdenum Carbide Catalysts for the Hydrodeoxygenation of Guaiacol

    Directory of Open Access Journals (Sweden)

    Eduardo Santillan-Jimenez

    2015-03-01

    Full Text Available Molybdenum carbide was supported on three types of carbon support—activated carbon; multi-walled carbon nanotubes; and carbon nanofibers—using ammonium molybdate and molybdic acid as Mo precursors. The use of activated carbon as support afforded an X-ray amorphous Mo phase, whereas crystalline molybdenum carbide phases were obtained on carbon nanofibers and, in some cases, on carbon nanotubes. When the resulting catalysts were tested in the hydrodeoxygenation (HDO of guaiacol in dodecane, catechol and phenol were obtained as the main products, although in some instances significant amounts of cyclohexane were produced. The observation of catechol in all reaction mixtures suggests that guaiacol was converted into phenol via sequential demethylation and HDO, although the simultaneous occurrence of a direct demethoxylation pathway cannot be discounted. Catalysts based on carbon nanofibers generally afforded the highest yields of phenol; notably, the only crystalline phase detected in these samples was Mo2C or Mo2C-ζ, suggesting that crystalline Mo2C is particularly selective to phenol. At 350 °C, carbon nanofiber supported Mo2C afforded near quantitative guaiacol conversion, the selectivity to phenol approaching 50%. When guaiacol HDO was performed in the presence of acetic acid and furfural, guaiacol conversion decreased, although the selectivity to both catechol and phenol was increased.

  3. NMR Studies of Peroxidases.

    Science.gov (United States)

    Veitch, Nigel Charles

    Available from UMI in association with The British Library. Requires signed TDF. Peroxidases are a haem-containing group of enzymes with a wide diversity of function within biological systems. While a common characteristic is the ability to catalyse the conversion of hydrogen peroxide to water, it is the accompanying processes of hormone synthesis and degradation which have generated such a high level of interest. However, information at the molecular level is limited to a single well-resolved crystal structure, that of yeast cytochrome c peroxidase. This thesis presents a strategy for the investigation of peroxidase structure and function based on proton nuclear magnetic resonance spectroscopy, a technique which has the ability to address aspects of both protein structure and protein dynamics in solution. The application of one- and two-dimensional NMR techniques has been developed in the context of plant peroxidases, notably the isoenzyme HRP-C derived from the horseradish root. Characterisation of the proton NMR spectra of HRP -C in resting and ligated states provided new information enabling the structure of the binding site for aromatic donor molecules, such as indole-3-propionic, ferulic and benzhydroxamic acids, to be resolved. In order to overcome difficulties encountered with a protein of the complexity of peroxidase, additional information was obtained from chemical shift parameters and the use of peroxidase variants produced by site-directed mutagenesis. A comparative study using NMR spectroscopy was undertaken for wild-type recombinant HRP-C expressed in Escherichia coli, and two protein variants with substitutions made to residues located on the distal side of the haem pocket, Phe41 to Val and Arg38 to Lys. NMR analyses of a plant peroxidase from barley grains and the fungal peroxidase from Coprinus cinereus were also successful using methods conceived with HRP-C. Examination of three specifically constructed recombinant protein variants of C. cinereus

  4. Identification and characterization of a naturally occurring, efficiently cleaved, membrane-bound, clade A HIV-1 Env, suitable for immunogen design, with properties comparable to membrane-bound BG505.

    Science.gov (United States)

    Das, Supratik; Boliar, Saikat; Samal, Sweety; Ahmed, Shubbir; Shrivastava, Tripti; Shukla, Brihaspati N; Goswami, Sandeep; Bansal, Manish; Chakrabarti, Bimal K

    2017-10-01

    Efficient cleavage of HIV-1 Env gp160 into its constituent subunits correlates with selective binding to neutralizing antibodies and are the closest mimetic of native, functional Envs. This was first demonstrated with the clade B Env, JRFL. The correlation between efficient cleavage and selective binding to neutralizing antibodies is the guiding principle for immunogen design for HIV vaccine. We have recently reported that Envs 4-2.J41 (clade C) and JRCSF (clade B) are also efficiently cleaved and show similar properties. However, an efficiently cleaved, membrane-bound clade A Env suitable for genetic vaccination has not been directly demonstrated. Here we report that BG505 and a new clade A Env, QB726.70M.ENV.C4 (or A5) are efficiently cleaved on cell membrane. A5 shows desirable antigenic properties comparable with BG505 on cell surface. A5SOSIP in supernatant displays majority of bNAb binding epitopes. Thus, both BG505 and A5 Envs can be used in DNA prime-protein boost vaccination studies. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Molecular Dynamics Simulations to Provide Insights into Epitopes Coupled to the Soluble and Membrane-Bound MHC-II Complexes

    Science.gov (United States)

    Bello, Martiniano; Correa-Basurto, Jose

    2013-01-01

    Epitope recognition by major histocompatibility complex II (MHC-II) is essential for the activation of immunological responses to infectious diseases. Several studies have demonstrated that this molecular event takes place in the MHC-II peptide-binding groove constituted by the α and β light chains of the heterodimer. This MHC-II peptide-binding groove has several pockets (P1-P11) involved in peptide recognition and complex stabilization that have been probed through crystallographic experiments and in silico calculations. However, most of these theoretical calculations have been performed without taking into consideration the heavy chains, which could generate misleading information about conformational mobility both in water and in the membrane environment. Therefore, in absence of structural information about the difference in the conformational changes between the peptide-free and peptide-bound states (pMHC-II) when the system is soluble in an aqueous environment or non-covalently bound to a cell membrane, as the physiological environment for MHC-II is. In this study, we explored the mechanistic basis of these MHC-II components using molecular dynamics (MD) simulations in which MHC-II was previously co-crystallized with a small epitope (P7) or coupled by docking procedures to a large (P22) epitope. These MD simulations were performed at 310 K over 100 ns for the water-soluble (MHC-IIw, MHC-II-P7w, and MHC-II-P22w) and 150 ns for the membrane-bound species (MHC-IIm, MHC-II-P7m, and MHC-II-P22m). Our results reveal that despite the different epitope sizes and MD simulation environments, both peptides are stabilized primarily by residues lining P1, P4, and P6-7, and similar noncovalent intermolecular energies were observed for the soluble and membrane-bound complexes. However, there were remarkably differences in the conformational mobility and intramolecular energies upon complex formation, causing some differences with respect to how the two peptides are

  6. Differential expression of growth factor receptors and membrane-bound tumor markers for imaging in male and female breast cancer.

    Directory of Open Access Journals (Sweden)

    Jeroen F Vermeulen

    Full Text Available INTRODUCTION: Male breast cancer accounts for 0.5-1% of all breast cancers and is generally diagnosed at higher stage than female breast cancers and therefore might benefit from earlier detection and targeted therapy. Except for HER2 and EGFR, little is known about expression of growth factor receptors in male breast cancer. We therefore investigated expression profiles of growth factor receptors and membrane-bound tumor markers in male breast cancer and gynecomastia, in comparison with female breast cancer. METHODS: Tissue microarrays containing 133 male breast cancer and 32 gynecomastia cases were stained by immunohistochemistry for a panel of membrane-bound targets and compared with data on 266 female breast cancers. RESULTS: Growth factor receptors were variably expressed in 4.5% (MET up to 38.5% (IGF1-R of male breast cancers. Compared to female breast cancer, IGF1-R and carbonic anhydrase 12 (CAXII were more frequently and CD44v6, MET and FGFR2 less frequently expressed in male breast cancer. Expression of EGFR, HER2, CAIX, and GLUT1 was not significantly different between male and female breast cancer. Further, 48.1% of male breast cancers expressed at least one and 18.0% expressed multiple growth factor receptors. Since individual membrane receptors are expressed in only half of male breast cancers, a panel of membrane markers will be required for molecular imaging strategies to reach sensitivity. A potential panel of markers for molecular imaging, consisting of EGFR, IGF1-R, FGFR2, CD44v6, CAXII, GLUT1, and CD44v6 was positive in 77% of male breast cancers, comparable to female breast cancers. CONCLUSIONS: Expression patterns of growth factor receptors and hypoxia membrane proteins in male breast cancer are different from female breast cancer. For molecular imaging strategies, a putative panel consisting of markers for EGFR, IGF1-R, FGFR2, GLUT1, CAXII, CD44v6 was positive in 77% of cases and might be considered for development of

  7. Different functions of the insect soluble and membrane-bound trehalase genes in chitin biosynthesis revealed by RNA interference.

    Directory of Open Access Journals (Sweden)

    Jie Chen

    Full Text Available BACKGROUND: Trehalase, an enzyme that hydrolyzes trehalose to yield two glucose molecules, plays a pivotal role in various physiological processes. In recent years, trehalase proteins have been purified from several insect species and are divided into soluble (Tre-1 and membrane-bound (Tre-2 trehalases. However, no functions of the two trehalases in chitin biosynthesis in insects have yet been reported. PRINCIPAL FINDINGS: The membrane-bound trehalase of Spodoptera exigua (SeTre-2 was characterized in our laboratory previously. In this study, we cloned the soluble trehalase gene (SeTre-1 and investigated the tissue distribution and developmental expression pattern of the two trehalase genes. SeTre-1 was expressed highly in cuticle and Malpighian tubules, while SeTre-2 was expressed in tracheae and fat body. In the midgut, the two trehalase genes were expressed in different locations. Additionally, the expression profiles of both trehalase mRNAs and their enzyme activities suggest that they may play different roles in chitin biosynthesis. The RNA interference (RNAi of either SeTre-1 or SeTre-2 was gene-specific and effective, with efficiency rates up to 83% at 72 h post injection. After RNAi of SeTre-1 and SeTre-2, significant higher mortality rates were observed during the larva-pupa stage and pupa-adult stage, and the lethal phenotypes were classified and analyzed. Additionally, the change trends of concentration of trehalose and glucose appeared reciprocally in RNAi-mutants. Moreover, knockdown of SeTre-1 gene largely inhibited the expression of chitin synthase gene A (CHSA and reduced the chitin content in the cuticle to two-thirds relative to the control insects. The chitin synthase gene B (CHSB expression, however, was inhibited more by the injection of dsRNA for SeTre-2, and the chitin content in the midgut decreased by about 25%. CONCLUSIONS: SeTre-1 plays a major role in CHSA expression and chitin synthesis in the cuticle, and SeTre-2

  8. Depression of membrane-bound Na+-K+-ATPase activity induced by free radicals and by ischemia of kidney

    International Nuclear Information System (INIS)

    Kako, K.; Kato, M.; Matsuoka, T.; Mustapha, A.

    1988-01-01

    A partially purified, membrane-bound Na + -K + -ATPase fraction, prepared from the outer medulla of porcine kidney, was incubated in the presence of 0.1-100 mM H 2 O 2 for either 15 or 30 min at 37 degree C. The activity of ouabain-sensitive Na + -K + -ATPase was reduced proportionally to the concentration of H 2 O 2 and the duration of incubation. There were decreases in SH contents and turnover rates of the Na + -K + -ATPase preparation, while malondialdehyde (MDA) and conjugated dienes were generated from the membrane lipids in the course of the incubation. The concentrations of ethanolamine (E) plasmalogen and of arachidonic acid in the E glycerophospholipid molecules were reduced by the free radical reaction. Similarly, a reduction in Na + K + -ATPase activity and the formation of MDA and conjugated dienes, together with a decrease in E glycerophospholipids, were observed when the membrane fraction was exposed to ultraviolet irradiation (254 nm) for 30 min at 4 degree C. Microsomal fractions, prepared from the outer medulla of canine kidney after 1 h of unilateral ischemia and 1 h of reperfusion, showed a decreased Na + -K + -ATPase activity, a reduced amount of SH groups, and an increased MDA. These changes were normalized by the infusion of N-mercaptopropionylglycine. These results support the view (1) that free radical generation affects the enzyme protein as well as membrane lipids, and (2) that free radicals may be formed in the ischemic reperfused kidney

  9. Structure and dynamics of the membrane-bound form of the filamentous bacteriophage coat proteins by NMR spectroscopy

    International Nuclear Information System (INIS)

    Bogusky, M.J.

    1987-01-01

    The structure and dynamics of the Pf1 and fd bacteriophage coat proteins in detergent micelles are characterized in solution by nuclear magnetic resonance spectroscopy. The coat proteins are found to exist within the bacterial inner cell membrane during viral infection and assembly. The coat proteins serve as a model system to investigate integral membrane proteins as well as the viral infection and assembly processes. The coat protein is insoluble in aqueous or organic solvents and can only be effectively solubilized in the presence of detergents that form micelles or phospholipids that form vesicles. The effective molecular weight of the detergent-micelle complex is ca. 30K daltons. Sequential assignment strategies were ineffective due to short T/sub 2s/ and severe resonance degeneracy. The backbone resonance assignments were completed by the combination of several homo- and heteronuclear correlation techniques with biosynthetic 15 N labelling. 2D NOE experiments were used to locate and characterize the secondary structure of the membrane bound form of the proteins showing them to be largely helical with the hydrophobic core existing in a very stable helix

  10. Membrane-bound Dickkopf-1 in Foxp3+ regulatory T cells suppresses T-cell-mediated autoimmune colitis.

    Science.gov (United States)

    Chae, Wook-Jin; Park, Jong-Hyun; Henegariu, Octavian; Yilmaz, Saliha; Hao, Liming; Bothwell, Alfred L M

    2017-10-01

    Induction of tolerance is a key mechanism to maintain or to restore immunological homeostasis. Here we show that Foxp3 + regulatory T (Treg) cells use Dickkopf-1 (DKK-1) to regulate T-cell-mediated tolerance in the T-cell-mediated autoimmune colitis model. Treg cells from DKK-1 hypomorphic doubleridge mice failed to control CD4 + T-cell proliferation, resulting in CD4 T-cell-mediated autoimmune colitis. Thymus-derived Treg cells showed a robust expression of DKK-1 but not in naive or effector CD4 T cells. DKK-1 expression in Foxp3 + Treg cells was further increased upon T-cell receptor stimulation in vitro and in vivo. Interestingly, Foxp3 + Treg cells expressed DKK-1 in the cell membrane and the functional inhibition of DKK-1 using DKK-1 monoclonal antibody abrogated the suppressor function of Foxp3 + Treg cells. DKK-1 expression was dependent on de novo protein synthesis and regulated by the mitogen-activated protein kinase pathway but not by the canonical Wnt pathway. Taken together, our results highlight membrane-bound DKK-1 as a novel Treg-derived mediator to maintain immunological tolerance in T-cell-mediated autoimmune colitis. © 2017 The Authors. Immunology Published by John Wiley & Sons Ltd.

  11. Reduced levels of membrane-bound alkaline phosphatase are common to lepidopteran strains resistant to Cry toxins from Bacillus thuringiensis.

    Science.gov (United States)

    Jurat-Fuentes, Juan Luis; Karumbaiah, Lohitash; Jakka, Siva Rama Krishna; Ning, Changming; Liu, Chenxi; Wu, Kongming; Jackson, Jerreme; Gould, Fred; Blanco, Carlos; Portilla, Maribel; Perera, Omaththage; Adang, Michael

    2011-03-01

    Development of insect resistance is one of the main concerns with the use of transgenic crops expressing Cry toxins from the bacterium Bacillus thuringiensis. Identification of biomarkers would assist in the development of sensitive DNA-based methods to monitor evolution of resistance to Bt toxins in natural populations. We report on the proteomic and genomic detection of reduced levels of midgut membrane-bound alkaline phosphatase (mALP) as a common feature in strains of Cry-resistant Heliothis virescens, Helicoverpa armigera and Spodoptera frugiperda when compared to susceptible larvae. Reduced levels of H. virescens mALP protein (HvmALP) were detected by two dimensional differential in-gel electrophoresis (2D-DIGE) analysis in Cry-resistant compared to susceptible larvae, further supported by alkaline phosphatase activity assays and Western blotting. Through quantitative real-time polymerase chain reaction (qRT-PCR) we demonstrate that the reduction in HvmALP protein levels in resistant larvae are the result of reduced transcript amounts. Similar reductions in ALP activity and mALP transcript levels were also detected for a Cry1Ac-resistant strain of H. armigera and field-derived strains of S. frugiperda resistant to Cry1Fa. Considering the unique resistance and cross-resistance phenotypes of the insect strains used in this work, our data suggest that reduced mALP expression should be targeted for development of effective biomarkers for resistance to Cry toxins in lepidopteran pests.

  12. Membrane-bound catechol-O-methyl transferase in cortical neurons and glial cells is intracellularly oriented

    Directory of Open Access Journals (Sweden)

    Björn H Schott

    2010-10-01

    Full Text Available Catechol-O-methyl transferase (COMT is involved in the inactivation of dopamine in brain regions in which the dopamine transporter (DAT1 is sparsely expressed. The membrane-bound isoform of COMT (MB-COMT is the predominantly expressed form in the mammalian central nervous system (CNS. It has been a matter of debate whether in neural cells of the CNS the enzymatic domain of MB-COMT is oriented towards the cytoplasmic or the extracellular compartment. Here we used live immunocytochemistry on cultured neocortical neurons and glial cells to investigate the expression and membrane orientation of native COMT and of transfected MB-COMT fused to green fluorescent protein (GFP. After live staining, COMT immunoreactivity was reliably detected in both neurons and glial cells after permeabilization, but not on unpermeabilized cells. Similarly, autofluorescence of COMT-GFP fusion protein and antibody fluorescence showed overlap only in permeabilized neurons. Our data provide converging evidence for an intracellular membrane orientation of MB-COMT in neurons and glial cells, suggesting the presence of a DAT1-independent postsynaptic uptake mechanism for dopamine, prior to its degradation via COMT.

  13. Molecular cloning, mRNA expression and characterization of membrane-bound hemoglobin in oriental river prawn Macrobrachium nipponense.

    Science.gov (United States)

    Sun, Shengming; Xuan, Fujun; Fu, Hongtuo; Zhu, Jian; Ge, Xianping; Wu, Xugan

    2017-05-01

    Most hemoglobins are respiratory proteins and are ubiquitous in animals, bacteria, fungi, protists, and plants. In this study, we describe a membrane-bound hemoglobin in the oriental river prawn Macrobrachium nipponense (MnHb), which also expresses hemocyanin. MnHb cDNA was cloned using the rapid amplification of cDNA ends (RACE) approach, which afforded a 1201bp gene encoding a 193 amino acid polypeptide. Bioinformatic evaluation suggested MnHb is membrane anchored by N-myristoylation, and immunofluorescence confirmed its location in the membrane of chief cells in the gill. The effect of hypoxia on MnHb expression was investigated, and reverse transcription PCR (RT-PCR) and Western blotting showed that MnHb was expressed almost exclusively in the gill. Quantitative RT-PCR revealed a significant increase in expression after 6h of hypoxia, and levels peaked at 24h due to oxidative stress. Exposure of cultured prawns to the stress inducer H 2 O 2 significantly up-regulated the expression of MnHb in a dose-dependent manner. MnHb may have a role in protecting cell membrane lipids from damage by reactive oxygen species. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Characterization of a membrane-bound C-glucosyltransferase responsible for carminic acid biosynthesis in Dactylopius coccus Costa.

    Science.gov (United States)

    Kannangara, Rubini; Siukstaite, Lina; Borch-Jensen, Jonas; Madsen, Bjørn; Kongstad, Kenneth T; Staerk, Dan; Bennedsen, Mads; Okkels, Finn T; Rasmussen, Silas A; Larsen, Thomas O; Frandsen, Rasmus J N; Møller, Birger Lindberg

    2017-12-07

    Carminic acid, a glucosylated anthraquinone found in scale insects like Dactylopius coccus, has since ancient times been used as a red colorant in various applications. Here we show that a membrane-bound C-glucosyltransferase, isolated from D. coccus and designated DcUGT2, catalyzes the glucosylation of flavokermesic acid and kermesic acid into their respective C-glucosides dcII and carminic acid. DcUGT2 is predicted to be a type I integral endoplasmic reticulum (ER) membrane protein, containing a cleavable N-terminal signal peptide and a C-terminal transmembrane helix that anchors the protein to the ER, followed by a short cytoplasmic tail. DcUGT2 is found to be heavily glycosylated. Truncated DcUGT2 proteins synthesized in yeast indicate the presence of an internal ER-targeting signal. The cleavable N-terminal signal peptide is shown to be essential for the activity of DcUGT2, whereas the transmembrane helix/cytoplasmic domains, although important, are not crucial for its catalytic function.

  15. Aluminium and Acrylamide Disrupt Cerebellum Redox States, Cholinergic Function and Membrane-Bound ATPase in Adult Rats and Their Offspring.

    Science.gov (United States)

    Ghorbel, Imen; Amara, Ibtissem Ben; Ktari, Naourez; Elwej, Awatef; Boudawara, Ons; Boudawara, Tahia; Zeghal, Najiba

    2016-12-01

    Accumulation of aluminium and acrylamide in food is a major source of human exposure. Their adverse effects are well documented, but there is no information about the health problems arising from their combined exposure. The aim of the present study was to examine the possible neurotoxic effects after co-exposure of pregnant and lactating rats to aluminium and acrylamide in order to evaluate redox state, cholinergic function and membrane-bound ATPases in the cerebellum of adult rats and their progeny. Pregnant female rats have received aluminium (50 mg/kg body weight) via drinking water and acrylamide (20 mg/kg body weight) by gavage, either individually or in combination from the 14th day of pregnancy until day 14 after delivery. Exposure to these toxicants provoked an increase in malondialdehyde (MDA) and advanced oxidation protein product (AOPP) levels and a decrease in SOD, CAT, GPx, Na + K + -ATPase, Mg 2+ -ATPase and AChE activities in the cerebellum of mothers and their suckling pups. A reduction in GSH, NPSH and vitamin C levels was also observed. These changes were confirmed by histological results. Interestingly, co-exposure to these toxicants exhibited synergism based on physical and biochemical variables in the cerebellum of mothers and their progeny.

  16. Reduced levels of membrane-bound alkaline phosphatase are common to lepidopteran strains resistant to Cry toxins from Bacillus thuringiensis.

    Directory of Open Access Journals (Sweden)

    Juan Luis Jurat-Fuentes

    Full Text Available Development of insect resistance is one of the main concerns with the use of transgenic crops expressing Cry toxins from the bacterium Bacillus thuringiensis. Identification of biomarkers would assist in the development of sensitive DNA-based methods to monitor evolution of resistance to Bt toxins in natural populations. We report on the proteomic and genomic detection of reduced levels of midgut membrane-bound alkaline phosphatase (mALP as a common feature in strains of Cry-resistant Heliothis virescens, Helicoverpa armigera and Spodoptera frugiperda when compared to susceptible larvae. Reduced levels of H. virescens mALP protein (HvmALP were detected by two dimensional differential in-gel electrophoresis (2D-DIGE analysis in Cry-resistant compared to susceptible larvae, further supported by alkaline phosphatase activity assays and Western blotting. Through quantitative real-time polymerase chain reaction (qRT-PCR we demonstrate that the reduction in HvmALP protein levels in resistant larvae are the result of reduced transcript amounts. Similar reductions in ALP activity and mALP transcript levels were also detected for a Cry1Ac-resistant strain of H. armigera and field-derived strains of S. frugiperda resistant to Cry1Fa. Considering the unique resistance and cross-resistance phenotypes of the insect strains used in this work, our data suggest that reduced mALP expression should be targeted for development of effective biomarkers for resistance to Cry toxins in lepidopteran pests.

  17. Effect of Temperature on Electrical Conductivity of Guaiacol-Guanidine Hydrochloride-Formaldehyde Copolymer Resin

    Science.gov (United States)

    Kukade, S. D.; Bawankar, S. V.

    2018-02-01

    The purpose of the present paper is to report temperature dependence of electrical conductivity on Guaiacol-guanidine hydrochloride-formaldehyde copolymer resin. By using a microwave irradiation technique, various ratios of copolymer resin were synthesized from the reacting monomers, i.e., guaiacol, guanidine hydrochloride and formaldehyde. The characterization of the copolymer resins has been fulfilled by spectral methods viz. ultraviolet visible (UV visible), infrared and proton nuclear magnetic spectroscopy (1H-NMR). The solid state direct current electrical conductivity of synthesized copolymer resins has been measured as a function of temperature. The electrical conductivity values of all the copolymers have been found in the range of a semiconductor.

  18. Nitrate reduction associated with respiration in Sinorhizobium meliloti 2011 is performed by a membrane-bound molybdoenzyme.

    Science.gov (United States)

    Ferroni, Felix M; Rivas, María G; Rizzi, Alberto C; Lucca, María E; Perotti, Nora I; Brondino, Carlos D

    2011-10-01

    The purification and biochemical characterization of the respiratory membrane-bound nitrate reductase from Sinorhizobium meliloti 2011 (Sm NR) is reported together with the optimal conditions for cell growth and enzyme production. The best biomass yield was obtained under aerobic conditions in a fed-batch system using Luria-Bertani medium with glucose as carbon source. The highest level of Sm NR production was achieved using microaerobic conditions with the medium supplemented with both nitrate and nitrite. Sm NR is a mononuclear Mo-protein belonging to the DMSO reductase family isolated as a heterodimeric enzyme containing two subunits of 118 and 45 kDa. Protein characterization by mass spectrometry showed homology with respiratory nitrate reductases. UV-Vis spectra of as-isolated and dithionite reduced Sm NR showed characteristic absorption bands of iron-sulfur and heme centers. Kinetic studies indicate that Sm NR follows a Michaelis-Menten mechanism (K (m) = 97 ± 11 μM, V = 9.4 ± 0.5 μM min(-1), and k (cat) = 12.1 ± 0.6 s(-1)) and is inhibited by azide, chlorate, and cyanide with mixed inhibition patterns. Physiological and kinetic studies indicate that molybdenum is essential for NR activity and that replacement of this metal for tungsten inhibits the enzyme. Although no narGHI gene cluster has been annotated in the genome of rhizobia, the biochemical characterization indicates that Sm NR is a Mo-containing NR enzyme with molecular organization similar to NarGHI.

  19. Membrane-bound mucins and mucin terminal glycans expression in idiopathic or Helicobacter pylori, NSAID associated peptic ulcers.

    Science.gov (United States)

    Niv, Yaron; Boltin, Doron; Halpern, Marisa; Cohen, Miriam; Levi, Zohar; Vilkin, Alex; Morgenstern, Sara; Manugian, Vahig; St Lawrence, Erica; Gagneux, Pascal; Kaur, Sukhwinder; Sharma, Poonam; Batra, Surinder K; Ho, Samuel B

    2014-10-28

    To determine the expression of membrane-bound mucins and glycan side chain sialic acids in Helicobacter pylori (H. pylori)-associated, non-steroidal inflammatory drug (NSAID)-associated and idiopathic-gastric ulcers. We studied a cohort of randomly selected patients with H. pylori (group 1, n = 30), NSAID (group 2, n = 18), combined H. pylori and NSAID associated gastric ulcers (group 3, n = 24), and patients with idiopathic gastric ulcers (group 4, n = 20). Immunohistochemistry for MUC1, MUC4, MUC17, and staining for Erythrina cristagalli agglutinin and Sambucus nigra agglutinin (SNA) lectins was performed on sections from the ulcer margins. Staining intensity of MUC17 was higher in H. pylori ulcers (group 1) than in idiopathic ulcers (group 4), 11.05 ± 3.67 vs 6.93 ± 4.00 for foveola cells, and 10.29 ± 4.67 vs 8.00 ± 3.48 for gland cells, respectively (P < 0.0001). In contrast, MUC1 expression was higher in group 4 compared group 1, 9.89 ± 4.17 vs 2.93 ± 5.13 in foveola cells and 7.63 ± 4.60 vs 2.57± 4.50 for glands, respectively (P < 0.0001). SNA lectin staining was increased in group 4, in parallel to elevated MUC1 expression, indicating more abundant α2-6 sialylation in that group. Cytoplasmic MUC17 staining was significantly decreased in the cases with idiopathic ulcer. The opposite was observed for both MUC1 and SNA lectin. This observation may reflect important pathogenic mechanisms, since different mucins with altered sialylation patterns may differ in their protection efficiency against acid and pepsin.

  20. Identification and characterization of GmPDIL7, a soybean ER membrane-bound protein disulfide isomerase family protein.

    Science.gov (United States)

    Okuda, Aya; Matsusaki, Motonori; Masuda, Taro; Urade, Reiko

    2017-02-01

    Most proteins synthesized in the endoplasmic reticulum (ER) possess intramolecular and intermolecular disulfide bonds, which play an important role in the conformational stability and function of proteins. Hence, eukaryotic cells contain protein disulfide bond formation pathways such as the protein disulfide isomerase (PDI)-ER oxidoreductin 1 (Ero1) system in the ER lumen. In this study, we identified soybean PDIL7 (GmPDIL7), a novel soybean ER membrane-bound PDI family protein, and determined its enzymatic properties. GmPDIL7 has a putative N-terminal signal sequence, a thioredoxin domain with an active center motif (CGHC), and a putative C-terminal transmembrane region. Likewise, we demonstrated that GmPDIL7 is ubiquitously expressed in soybean tissues and is localized in the ER membrane. Furthermore, GmPDIL7 associated with other soybean PDI family proteins in vivo and GmPDIL7 mRNA was slightly upregulated under ER stress. The redox potential of recombinant GmPDIL7 expressed in Escherichia coli was -187 mV, indicating that GmPDIL7 could oxidize unfolded proteins. GmPDIL7 exhibited a dithiol oxidase activity level that was similar to other soybean PDI family proteins. However, the oxidative refolding activity of GmPDIL7 was lower than other soybean PDI family proteins. GmPDIL7 was well oxidized by GmERO1. Taken together, our results indicated that GmPDIL7 primarily plays a role as a supplier of disulfide bonds in nascent proteins for oxidative folding on the ER membrane. The nucleotide sequence data for the GmPDIL7 cDNA are available in the DNA Data Bank of Japan (DDBJ) databases under the accession numbers LC158001. Protein disulfide isomerase: EC 5.3.4.1. © 2016 Federation of European Biochemical Societies.

  1. Survival, mobility, and membrane-bound enzyme activities of freshwater planarian, Dugesia japonica, exposed to synthetic and natural surfactants.

    Science.gov (United States)

    Li, Mei-Hui

    2012-04-01

    Surfactants are a major class of emerging pollutants widely used in large quantities in everyday life and commonly found in surface waters worldwide. Freshwater planarian was selected to examine the effects of different surfactants by measuring mortality, mobility, and membrane-bound enzyme activities. Among the 10 surfactants tested, the acute toxicities of betaine and polyethylene glycol (PEG-200) to planarians were relatively low, with a median lethal concentration (LC50) greater than 10,000 mg/L. The toxicity to planarians of the other eight surfactants based on 48-h LC50 could be arranged in the descending order of cetylpyridinum chloride (CPC) > 4-tert-octylphenol (4-tert-OP) > ammonium lauryl sulfate > benzalkonium chloride > saponin > sodium lauroylsarcosinate > dioctyl sulfosuccinate > dodecyl trimethyl ammonium bromide (DTAB). Both CPC and 4-tert-OP were very toxic to planarians, with 48-h LC50 values <1 mg/L. The median effective concentrations (EC50s) of planarian mobility were in the 0.1 to 50 mg/L range and were in the same range as the 24-h LC50 of planarians exposed to different surfactants, except for DTAB. In addition, significant inhibition of cholinesterase activity activities was found in planarians exposed to 4-tert-OP at 2.5 and 5 mg/L and to saponin at 10 mg/L after 2-h treatments. This result suggests that planarian mobility responses can be used as an alternative indicator for acute toxicity of surfactants after a very short exposure period. Copyright © 2012 SETAC.

  2. The structure of Serratia marcescens Lip, a membrane-bound component of the type VI secretion system

    International Nuclear Information System (INIS)

    Rao, Vincenzo A.; Shepherd, Sharon M.; English, Grant; Coulthurst, Sarah J.; Hunter, William N.

    2011-01-01

    The high-resolution crystal structure of S. marcescens Lip reveals a new member of the transthyretin family of proteins. Lip, a core component of the type VI secretion apparatus, is localized to the outer membrane and is positioned to interact with other proteins forming this complex system. Lip is a membrane-bound lipoprotein and a core component of the type VI secretion system found in Gram-negative bacteria. The structure of a Lip construct (residues 29–176) from Serratia marcescens (SmLip) has been determined at 1.92 Å resolution. Experimental phases were derived using a single-wavelength anomalous dispersion approach on a sample cocrystallized with iodide. The membrane localization of the native protein was confirmed. The structure is that of the globular domain lacking only the lipoprotein signal peptide and the lipidated N-terminus of the mature protein. The protein fold is dominated by an eight-stranded β-sandwich and identifies SmLip as a new member of the transthyretin family of proteins. Transthyretin and the only other member of the family fold, 5-hydroxyisourate hydrolase, form homotetramers important for their function. The asymmetric unit of SmLip is a tetramer with 222 symmetry, but the assembly is distinct from that previously noted for the transthyretin protein family. However, structural comparisons and bacterial two-hybrid data suggest that the SmLip tetramer is not relevant to its role as a core component of the type VI secretion system, but rather reflects a propensity for SmLip to participate in protein–protein interactions. A relatively low level of sequence conservation amongst Lip homologues is noted and is restricted to parts of the structure that might be involved in interactions with physiological partners

  3. Aldopentoses as new substrates for the membrane-bound, pyrroloquinoline quinone-dependent glycerol (polyol) dehydrogenase of Gluconobacter sp.

    Science.gov (United States)

    Yakushi, Toshiharu; Terada, Yuka; Ozaki, Seishiro; Kataoka, Naoya; Akakabe, Yoshihiko; Adachi, Osao; Matsutani, Minenosuke; Matsushita, Kazunobu

    2018-04-01

    Membrane-bound, pyrroloquinoline quinone (PQQ)-dependent glycerol dehydrogenase (GLDH, or polyol dehydrogenase) of Gluconobacter sp. oxidizes various secondary alcohols to produce the corresponding ketones, such as oxidation of D-sorbitol to L-sorbose in vitamin C production. Substrate specificity of GLDH is considered limited to secondary alcohols in the D-erythro configuration at the next to the last carbon. Here, we suggest that L-ribose, D- and L-lyxoses, and L-tagatose are also substrates of GLDH, but these sugars do not meet the substrate specificity rule of GLDH. The oxygen consumption activity of wild-type Gluconobacter frateurii cell membranes depends on several kinds of sugars as compared with that of the membranes of a GLDH-negative variant. Biotransformation of those sugars with the membranes was examined to determine the reaction products. A time course measuring the pH in the reaction mixture and the increase or decrease in substrates and products on TLC suggested that oxidation products of L-lyxose and L-tagatose were ketones with unknown structures, but those of L-ribose and D-lyxose were acids. The oxidation product of L-ribose was purified and revealed to be L-ribonate by HRMS and NMR analysis. Biotransformation of L-ribose with the membranes and also with the whole cells produced L-ribonate in nearly stoichiometric amounts, indicating that the specific oxidation site in L-ribose is recognized by GLDH. Since purified GLDH produced L-ribonate without any intermediate-like compounds, we propose here a reaction model where the first carbon in the pyranose form of L-ribose is oxidized by GLDH to L-ribonolactone, which is further hydrolyzed spontaneously to produce L-ribonate.

  4. Depression of membrane-bound Na sup + -K sup + -ATPase activity induced by free radicals and by ischemia of kidney

    Energy Technology Data Exchange (ETDEWEB)

    Kako, K.; Kato, M.; Matsuoka, T.; Mustapha, A. (Univ. of Ottawa, Ontario (Canada))

    1988-02-01

    A partially purified, membrane-bound Na{sup +}-K{sup +}-ATPase fraction, prepared from the outer medulla of porcine kidney, was incubated in the presence of 0.1-100 mM H{sub 2}O{sub 2} for either 15 or 30 min at 37{degree}C. The activity of ouabain-sensitive Na{sup +}-K{sup +}-ATPase was reduced proportionally to the concentration of H{sub 2}O{sub 2} and the duration of incubation. There were decreases in SH contents and turnover rates of the Na{sup +}-K{sup +}-ATPase preparation, while malondialdehyde (MDA) and conjugated dienes were generated from the membrane lipids in the course of the incubation. The concentrations of ethanolamine (E) plasmalogen and of arachidonic acid in the E glycerophospholipid molecules were reduced by the free radical reaction. Similarly, a reduction in Na{sup +}K{sup +}-ATPase activity and the formation of MDA and conjugated dienes, together with a decrease in E glycerophospholipids, were observed when the membrane fraction was exposed to ultraviolet irradiation (254 nm) for 30 min at 4{degree}C. Microsomal fractions, prepared from the outer medulla of canine kidney after 1 h of unilateral ischemia and 1 h of reperfusion, showed a decreased Na{sup +}-K{sup +}-ATPase activity, a reduced amount of SH groups, and an increased MDA. These changes were normalized by the infusion of N-mercaptopropionylglycine. These results support the view (1) that free radical generation affects the enzyme protein as well as membrane lipids, and (2) that free radicals may be formed in the ischemic reperfused kidney.

  5. Membrane-bound Na,K-ATPase: target size and radiation inactivation size of some of its enzymatic reactions

    Energy Technology Data Exchange (ETDEWEB)

    Jensen, J.; Norby, J.G.

    1988-12-05

    Frozen samples of membrane-bound pig kidney Na,K-ATPase were subjected to target size analysis by radiation inactivation with 10-MeV electrons at -15 degrees C. The various properties investigated decreased monoexponentially with radiation dose, and the decay constants, gamma, were independent of the presence of other proteins and of sucrose concentrations above 0.25 M. The temperature factor was the same as described by others. Irradiation of four proteins of known molecular mass, m, showed that gamma for protein integrity was proportional to m with a proportionality factor about 20% higher than that conventionally used. By this standard curve, glucose-6-phosphate dehydrogenase activity used as internal standard gave a radiation inactivation size of 110 +/- 5 kDa, very close to m = 104-108 kDa for the dimer, as expected. For Na+/K+-transporting ATPase the following target sizes and radiation inactivation size values were very close to m = 112 kDa for the alpha-peptide: peptide integrity of alpha, 115 kDa; unmodified binding sites for ATP and vanadate, 108 kDa; K+-activated p-nitrophenylphosphatase activity, 106 kDa. There was thus no sign of dimerization of the alpha-peptide or involvement of the beta-peptide. In contrast, optimal Na+/K+-transporting ATPase activity had a radiation inactivation size = 189 +/- 7 kDa, and total nucleotide binding capacity corresponded to 72 +/- 3 kDa. These latter results will be extended and discussed in a forthcoming paper.

  6. Membrane-bound Na,K-ATPase: target size and radiation inactivation size of some of its enzymatic reactions

    International Nuclear Information System (INIS)

    Jensen, J.; Norby, J.G.

    1988-01-01

    Frozen samples of membrane-bound pig kidney Na,K-ATPase were subjected to target size analysis by radiation inactivation with 10-MeV electrons at -15 degrees C. The various properties investigated decreased monoexponentially with radiation dose, and the decay constants, gamma, were independent of the presence of other proteins and of sucrose concentrations above 0.25 M. The temperature factor was the same as described by others. Irradiation of four proteins of known molecular mass, m, showed that gamma for protein integrity was proportional to m with a proportionality factor about 20% higher than that conventionally used. By this standard curve, glucose-6-phosphate dehydrogenase activity used as internal standard gave a radiation inactivation size of 110 +/- 5 kDa, very close to m = 104-108 kDa for the dimer, as expected. For Na+/K+-transporting ATPase the following target sizes and radiation inactivation size values were very close to m = 112 kDa for the alpha-peptide: peptide integrity of alpha, 115 kDa; unmodified binding sites for ATP and vanadate, 108 kDa; K+-activated p-nitrophenylphosphatase activity, 106 kDa. There was thus no sign of dimerization of the alpha-peptide or involvement of the beta-peptide. In contrast, optimal Na+/K+-transporting ATPase activity had a radiation inactivation size = 189 +/- 7 kDa, and total nucleotide binding capacity corresponded to 72 +/- 3 kDa. These latter results will be extended and discussed in a forthcoming paper

  7. The structure of Serratia marcescens Lip, a membrane-bound component of the type VI secretion system

    Energy Technology Data Exchange (ETDEWEB)

    Rao, Vincenzo A.; Shepherd, Sharon M.; English, Grant; Coulthurst, Sarah J.; Hunter, William N., E-mail: w.n.hunter@dundee.ac.uk [College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland (United Kingdom)

    2011-12-01

    The high-resolution crystal structure of S. marcescens Lip reveals a new member of the transthyretin family of proteins. Lip, a core component of the type VI secretion apparatus, is localized to the outer membrane and is positioned to interact with other proteins forming this complex system. Lip is a membrane-bound lipoprotein and a core component of the type VI secretion system found in Gram-negative bacteria. The structure of a Lip construct (residues 29–176) from Serratia marcescens (SmLip) has been determined at 1.92 Å resolution. Experimental phases were derived using a single-wavelength anomalous dispersion approach on a sample cocrystallized with iodide. The membrane localization of the native protein was confirmed. The structure is that of the globular domain lacking only the lipoprotein signal peptide and the lipidated N-terminus of the mature protein. The protein fold is dominated by an eight-stranded β-sandwich and identifies SmLip as a new member of the transthyretin family of proteins. Transthyretin and the only other member of the family fold, 5-hydroxyisourate hydrolase, form homotetramers important for their function. The asymmetric unit of SmLip is a tetramer with 222 symmetry, but the assembly is distinct from that previously noted for the transthyretin protein family. However, structural comparisons and bacterial two-hybrid data suggest that the SmLip tetramer is not relevant to its role as a core component of the type VI secretion system, but rather reflects a propensity for SmLip to participate in protein–protein interactions. A relatively low level of sequence conservation amongst Lip homologues is noted and is restricted to parts of the structure that might be involved in interactions with physiological partners.

  8. Guaiacol hydrodeoxygenation mechanism on Pt(111): Insights from density functional theory and linear free energy relations

    Science.gov (United States)

    In this study density functional theory (DFT) was used to study the adsorption of guaiacol and its initial hydrodeoxygenation (HDO) reactions on Pt(111). Previously reported Brønsted–Evans–Polanyi (BEP) correlations for small open chain molecules are found to be inadequate in estimating the reaction...

  9. Carbon Nanofiber Supported Transition-Metal Carbide Catalysts for the Hydrodeoxygenation of Guaiacol

    NARCIS (Netherlands)

    Jongerius, A.; Gosselink, R.W.; Dijkstra, J.; Bitter, J.H.; Bruijnincx, P.C.A.; Weckhuysen, B.M.

    2013-01-01

    Hydrodeoxygenation (HDO) studies over carbon nanofiber-supported (CNF) W2C and Mo2C catalysts were performed on guaiacol, a prototypical substrate to evaluate the potential of a catalyst for valorization of depolymerized lignin streams. Typical reactions were executed at 55 bar hydrogen pressure

  10. Tumor Cell Clone Expressing the Membrane-bound Form of IL-12p35 Subunit Stimulates Antitumor Immune Responses Dominated by CD8(+) T Cells.

    Science.gov (United States)

    Lim, Hoyong; Do, Seon Ah; Park, Sang Min; Kim, Young Sang

    2013-04-01

    IL-12 is a secretory heterodimeric cytokine composed of p35 and p40 subunits. IL-12 p35 and p40 subunits are sometimes produced as monomers or homodimers. IL-12 is also produced as a membrane-bound form in some cases. In this study, we hypothesized that the membrane-bound form of IL-12 subunits may function as a costimulatory signal for selective activation of TAA-specific CTL through direct priming without involving antigen presenting cells and helper T cells. MethA fibrosarcoma cells were transfected with expression vectors of membrane-bound form of IL-12p35 (mbIL-12p35) or IL-12p40 subunit (mbIL-12p40) and were selected under G418-containing medium. The tumor cell clones were analyzed for the expression of mbIL-12p35 or p40 subunit and for their stimulatory effects on macrophages. The responsible T-cell subpopulation for antitumor activity of mbIL-12p35 expressing tumor clone was also analyzed in T cell subset-depleted mice. Expression of transfected membrane-bound form of IL-12 subunits was stable during more than 3 months of in vitro culture, and the chimeric molecules were not released into culture supernatants. Neither the mbIL-12p35-expressing tumor clones nor mbIL-12p40-expressing tumor clones activated macrophages to secrete TNF-α. Growth of mbIL-12p35-expressing tumor clones was more accelerated in the CD8(+) T cell-depleted mice than in CD4(+) T cell-depleted or normal mice. These results suggest that CD8(+) T cells could be responsible for the rejection of mbIL-12p35-expressing tumor clone, which may bypass activation of antigen presenting cells and CD4(+) helper T cells.

  11. Arabidopsis thaliana peroxidase N

    DEFF Research Database (Denmark)

    Mirza, Osman Asghar; Henriksen, A; Ostergaard, L

    2000-01-01

    The structure of the neutral peroxidase from Arabidopsis thaliana (ATP N) has been determined to a resolution of 1.9 A and a free R value of 20.5%. ATP N has the expected characteristic fold of the class III peroxidases, with a C(alpha) r.m.s.d. of 0.82 A when compared with horseradish peroxidase C...... (HRP C). HRP C is 54% identical to ATP N in sequence. When the structures of four class III plant peroxidases are superimposed, the regions with structural differences are non-randomly distributed; all are located in one half of the molecule. The architecture of the haem pocket of ATP N is very similar...... to that of HRP C, in agreement with the low small-molecule substrate specificity of all class III peroxidases. The structure of ATP N suggests that the pH dependence of the substrate turnover will differ from that of HRP C owing to differences in polarity of the residues in the substrate-access channel. Since...

  12. Adsorption of guaiacol on Fe (110) and Pd (111) from first principles

    Science.gov (United States)

    Hensley, Alyssa J. R.; Wang, Yong; McEwen, Jean-Sabin

    2016-06-01

    The catalytic properties of surfaces are highly dependent upon the effect said surfaces have on the geometric and electronic structure of adsorbed reactants, products, and intermediates. It is therefore crucial to have a surface-level understanding of the adsorption of the key species in a reaction in order to design active and selective catalysts. Here, we study the adsorption of guaiacol on Fe (110) and Pd (111) using dispersion-corrected density functional theory calculations as both of these metals are of interest as hydrodeoxygenation catalysts for the conversion of bio-oils to useable biofuels. Both vertical (via the oxygen functional groups) and horizontal (via the aromatic ring) adsorption configurations were examined and the resulting adsorption and molecular distortion energies showed that the vertical sites were only physisorbed while the horizontal sites were chemisorbed on both metal surfaces. A comparison of guaiacol's horizontal adsorption on Fe (110) and Pd (111) showed that guaiacol had a stronger adsorption on Pd (111) while the Fe (110) surface distorted the Csbnd O bonds to a greater degree. Electronic analyses on the horizontal systems showed that the greater adsorption strength for guaiacol on Pd (111) was likely due to the greater charge transfer between the aromatic ring and the surface Pd atoms. Additionally, the greater distortion of the Csbnd O bonds in adsorbed guaiacol on Fe (110) is likely due to the greater degree of interaction between the oxygen and surface Fe atoms. Overall, our results show that the Fe (110) surface has a greater degree of interaction with the functional groups and the Pd (111) surface has a greater degree of interaction with the aromatic ring.

  13. Spectroscopic and Kinetic Characterization of Peroxidase-Like π-Cation Radical Pinch-Porphyrin-Iron(III Reaction Intermediate Models of Peroxidase Enzymes

    Directory of Open Access Journals (Sweden)

    Samuel Hernández Anzaldo

    2016-06-01

    Full Text Available The spectroscopic and kinetic characterization of two intermediates from the H2O2 oxidation of three dimethyl ester [(proto, (meso, (deuteroporphyrinato (picdien]Fe(III complexes ([FePPPic], [FeMPPic] and [FeDPPic], respectively pinch-porphyrin peroxidase enzyme models, with s = 5/2 and 3/2 Fe(III quantum mixed spin (qms ground states is described herein. The kinetic study by UV/Vis at λmax = 465 nm showed two different types of kinetics during the oxidation process in the guaiacol test for peroxidases (1–3 + guaiacol + H2O2 → oxidation guaiacol products. The first intermediate was observed during the first 24 s of the reaction. When the reaction conditions were changed to higher concentration of pinch-porphyrins and hydrogen peroxide only one type of kinetics was observed. Next, the reaction was performed only between pinch-porphyrins-Fe(III and H2O2, resulting in only two types of kinetics that were developed during the first 0–4 s. After this time a self-oxidation process was observed. Our hypotheses state that the formation of the π-cation radicals, reaction intermediates of the pinch-porphyrin-Fe(III family with the ligand picdien [N,N’-bis-pyridin-2-ylmethyl-propane-1,3-diamine], occurred with unique kinetics that are different from the overall process and was involved in the oxidation pathway. UV-Vis, 1H-NMR and ESR spectra confirmed the formation of such intermediates. The results in this paper highlight the link between different spectroscopic techniques that positively depict the kinetic traits of artificial compounds with enzyme-like activity.

  14. An artificial neural network for membrane-bound catechol-O-methyltransferase biosynthesis with Pichia pastoris methanol-induced cultures.

    Science.gov (United States)

    Pedro, Augusto Q; Martins, Luís M; Dias, João M L; Bonifácio, Maria J; Queiroz, João A; Passarinha, Luís A

    2015-08-07

    Membrane proteins are important drug targets in many human diseases and gathering structural information regarding these proteins encourages the pharmaceutical industry to develop new molecules using structure-based drug design studies. Specifically, membrane-bound catechol-O-methyltransferase (MBCOMT) is an integral membrane protein that catalyzes the methylation of catechol substrates and has been linked to several diseases such as Parkinson's disease and Schizophrenia. Thereby, improvements in the clinical outcome of the therapy to these diseases may come from structure-based drug design where reaching MBCOMT samples in milligram quantities are crucial for acquiring structural information regarding this target protein. Therefore, the main aim of this work was to optimize the temperature, dimethylsulfoxide (DMSO) concentration and the methanol flow-rate for the biosynthesis of recombinant MBCOMT by Pichia pastoris bioreactor methanol-induced cultures using artificial neural networks (ANN). The optimization trials intended to evaluate MBCOMT expression by P. pastoris bioreactor cultures led to the development of a first standard strategy for MBCOMT bioreactor biosynthesis with a batch growth on glycerol until the dissolved oxygen spike, 3 h of glycerol feeding and 12 h of methanol induction. The ANN modeling of the aforementioned fermentation parameters predicted a maximum MBCOMT specific activity of 384.8 nmol/h/mg of protein at 30°C, 2.9 mL/L/H methanol constant flow-rate and with the addition of 6% (v/v) DMSO with almost 90% of healthy cells at the end of the induction phase. These results allowed an improvement of MBCOMT specific activity of 6.4-fold in comparison to that from the small-scale biosynthesis in baffled shake-flasks. The ANN model was able to describe the effects of temperature, DMSO concentration and methanol flow-rate on MBCOMT specific activity, as shown by the good fitness between predicted and observed values. This experimental procedure

  15. Novel Applications of Peroxidase

    Science.gov (United States)

    Rob, Abdul; Ball, Andrew S.; Tuncer, Munir; Wilson, Michael T.

    1997-02-01

    The article entitled "Novel Biocatalysts Will Work Even Better for Industry" published recently in this Journal (1) was informative and interesting. However it touched only briefly on the application of peroxidase as catalyst. Here, we would like to mention in more detail the novel applications of peroxidase in agricultural, paper pulp, water treatment, pharmaceutical, and medical situations. Firstly, the peroxidase isolated from Phanerochaete chyrosporium has been shown to detoxify herbicides such as atrazine to less toxic compounds and would certainly find potential application in agriculture (2). Secondly, the peroxidase produced by Streptomyces thermoviolaceus may find application in the paper pulp industry as a delignifying agent (3). Thirdly, it has been shown that extracellular peroxidase produced by Streptomyces avermitilis can remove the intense color from paper-mill effluent obtained after semichemical alkaline pulping of wheat straw (4), and thus this enzyme might find application as a catalyst in water treatment plants. Fourthly, the heme-containing horseradish peroxidase enzyme has been exploited in several diagnostic applications in pharmaceutics and medicine, such as the detection of human immunodeficiency virus and cystic fibrosis (5-10). Finally, recent work from our laboratory has suggested that thermophilic nonheme peroxidase produced by Thermomonospora fusca BD25 may find medical use in the diagnosis of myocardial infarction (11, 12). Literature Cited 1. Wiseman, A. J. Chem. Educ. 1996, 73, 55-58. 2. Mougin, C. Appl. Environ. Microbiol. 1994, 60, 705-708. 3. McCarthy A. J.; Peace, W.; Broda, P. Appl. Microbiol. Technol. 1985, 23, 238-244. 4. Hernandez, M; Rodriguez J; Soliveri, J; Copa, J. L; Perez, M. I; Arias, M. E. Appl. Environ. Microbiol. 1994, 60, 3909-3913. 5. Hopfer, S. M.; Aslanzadeh, J. Ann. Clin. Lab. Sci. 1995, 25, 475-480. 6. Suzuki, K; Iman, M. J. Virol. Methods 1995, 55, 347-356. 7. Nielsen, K. J. Immunoassay 1995, 16, 183-197. 8

  16. [Expression and characterization of Coprinus cinereus peroxidase].

    Science.gov (United States)

    Dong, Bingxue; Niu, Qiuhong; Zhang, Wei; Geng, Sanchun; Li, Peng; Yuan, Wanli; Gong, Yifeng; Liang, Kewei

    2015-03-04

    The aim of our study is to express Coprinus cinereus peroxidase (CIP) in Pichia Pastori efficiently. We synthesized CIP gene with P. pastori codon bias by our Gene Synthesis and site-specific mutagenesis platform, using DNAWorks 3.1 program to design and optimize primers. Then, we sequenced the PCR products, inserted the correct gene into expression vector pPICZαA and transformed the linearized pPICZαA-Cip DNA into P. pastori GS115. We integrated CIP gene into the genome of P. pastori, using the α-mating factor from Sacchoramyces cerevisiae as signal peptide to direct the secretion of the recombinant protein. To obtain transformants with high CIP activity, we checked transformants by nested PCR and stained 82 positive ones on YPD agar plate with 1000 mg/L Zeocin. Then, we got 6 transforments with high resistance to Zeocin and expressed them in small scale; the one exhibiting the highest activity was chosen as engineered strain and named CIP/Gs115. We purified CIP from culture medium after induction with ethanol, the maximum activity reached 487.5 U/mL on the 4th day. The purified CIP exhibited maximal activity at pH 5.0 and 25 degrees C with ABTS as substrate. The enzyme had 61.5% of the maximal activity at 45 degrees C and was stable below 40 degrees C. However, the stability was drastically reduced above 45 degrees C. The recombinant CIP remained stable between pH 4.5 and 6.5. We studied the substrate specificity on different substrates with the purified enzyme, and the optimal substrates were in the order of ABTS > 2, 6-Dimethoxyphenol > guaiacol > 2, 4-Dichlorophenol > phenol. The highly secretory expression of CIP and high special activity lay the good foundation for it' s industrial applications in waste water treatment, decolouration of dyestuffs.

  17. Horseradish peroxidase embedded in polyacrylamide nanoparticles enables optical detection of reactive oxygen species

    DEFF Research Database (Denmark)

    Poulsen, A.K.; Scharff-Poulsen, Anne Marie; Olsen, L.F.

    2007-01-01

    enough for substrates to enter and products to leave the polymer matrix containing the enzyme. The polymer matrix protects the embedded enzyme from proteolytic digestion, which is demonstrated by treating the particles with a mixture of the two proteases trypsin and proteinase K. The particles allow......We have synthesized and characterized new nanometer-sized polyacrylamide particles containing horseradish peroxidase and fluorescent dyes. Proteins and dyes are encapsulated by radical polymerization in inverse microemulsion. The activity of the encapsulated enzyme has been examined...... and it maintains its ability to catalyze the oxidation of guaiacol with hydrogen peroxide as the electron acceptor, although at a slightly lower rate compared to that of the free enzyme in solution. The embedded enzyme is also capable of catalyzing the peroxidase-oxidase reaction. However, the rate is decreased...

  18. Two specific membrane-bound aminopeptidase N isoforms from Aedes aegypti larvae serve as functional receptors for the Bacillus thuringiensis Cry4Ba toxin implicating counterpart specificity.

    Science.gov (United States)

    Aroonkesorn, Aratee; Pootanakit, Kusol; Katzenmeier, Gerd; Angsuthanasombat, Chanan

    2015-05-29

    The interaction between Bacillus thuringiensis Cry toxins and their receptors on midgut cells of susceptible insect larvae is the critical determinant in toxin specificity. Besides GPI-linked alkaline phosphatase in Aedes aegypti mosquito-larval midguts, membrane-bound aminopeptidase N (AaeAPN) is widely thought to serve as a Cry4Ba receptor. Here, two full-length AaeAPN isoforms, AaeAPN2778 and AaeAPN2783, predicted to be GPI-linked were cloned and successfully expressed in Spodoptera frugiperda (Sf9) cells as 112- and 107-kDa membrane-bound proteins, respectively. In the cytotoxicity assay, Sf9 cells expressing each of the two AaeAPN isoforms showed increased sensitivity to the Cry4Ba mosquito-active toxin. Double immunolocalization revealed specific binding of Cry4Ba to each individual AaeAPN expressed on the cell membrane surface. Sequence analysis and homology-based modeling placed these two AaeAPNs to the M1 aminopeptidase family as they showed similar four-domain structures, with the most conserved domain II being the catalytic component. Additionally, the most variable domain IV containing negatively charged surface patches observed only in dipteran APNs could be involved in insect specificity. Overall results demonstrated that these two membrane-bound APN isoforms were responsible for mediating Cry4Ba toxicity against AaeAPN-expressed Sf9 cells, suggesting their important role as functional receptors for the toxin counterpart in A. aegypti mosquito larvae. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Membrane-bound steel factor maintains a high local concentration for mouse primordial germ cell motility, and defines the region of their migration.

    Directory of Open Access Journals (Sweden)

    Ying Gu

    Full Text Available Steel factor, the protein product of the Steel locus in the mouse, is a multifunctional signal for the primordial germ cell population. We have shown previously that its expression accompanies the germ cells during migration to the gonads, forming a "travelling niche" that controls their survival, motility, and proliferation. Here we show that these functions are distributed between the alternatively spliced membrane-bound and soluble forms of Steel factor. The germ cells normally migrate as individuals from E7.5 to E11.5, when they aggregate together in the embryonic gonads. Movie analysis of Steel-dickie mutant embryos, which make only the soluble form, at E7.5, showed that the germ cells fail to migrate normally, and undergo "premature aggregation" in the base of the allantois. Survival and directionality of movement is not affected. Addition of excess soluble Steel factor to Steel-dickie embryos rescued germ cell motility, and addition of Steel factor to germ cells in vitro showed that a fourfold higher dose was required to increase motility, compared to survival. These data show that soluble Steel factor is sufficient for germ cell survival, and suggest that the membrane-bound form provides a higher local concentration of Steel factor that controls the balance between germ cell motility and aggregation. This hypothesis was tested by addition of excess soluble Steel factor to slice cultures of E11.5 embryos, when migration usually ceases, and the germ cells aggregate. This reversed the aggregation process, and caused increased motility of the germ cells. We conclude that the two forms of Steel factor control different aspects of germ cell behavior, and that membrane-bound Steel factor controls germ cell motility within a "motility niche" that moves through the embryo with the germ cells. Escape from this niche causes cessation of motility and death by apoptosis of the ectopic germ cells.

  20. Role of Lactobacillus plantarum MTCC1325 in membrane-bound transport ATPases system in Alzheimer’s disease-induced rat brain

    Directory of Open Access Journals (Sweden)

    Nimgampalle Mallikarjuna

    2016-12-01

    Results: Chronic injection of D-Galactose caused lipid peroxidation, oxidative stress, and mitochondrial dysfunction leading to the damage of neurons in the brain, finally bringing a significant decrease (-20% in the brain total membrane bound ATPases over the controls. Contrary to this, treatment of AD-induced rats with L. plantarum MTCC1325 reverted all the constituents of ATPase enzymes to near normal levels within 30 days. Conclusion: Lactobacillus plantarum MTCC1325 exerted a beneficial action on the entire ATPases system in AD-induced rat brain by delaying neurodegeneration.

  1. Direct evidence from in situ FTIR spectroscopy that o-quinonemethide is a key intermediate during the pyrolysis of guaiacol.

    Science.gov (United States)

    Cheng, Hao; Wu, Shubin; Huang, Jinbao; Zhang, Xiaohua

    2017-04-01

    Although o-quinonemethide (6-methylene-2,4-cyclohexadien-1-one) has been proposed as a key intermediate in char formation during the pyrolysis of guaiacol (2-methoxyphenol), direct evidence of this (e.g., spectroscopic data) has not yet been provided. Using in situ FTIR spectroscopy, the pyrolysis of guaiacol was investigated from 30 °C to 630 °C at 40 °C/min. The IR profiles showed direct evidence of o-quinonemethide production at about 350 °C, which vanished rapidly at around 420 °C in the vapor phase, indicating char formation. In addition, at 400 °C, salicyl aldehyde was observed, which decomposed slowly at about 500 °C. In combination with the known products of guaiacol pyrolysis, these results allowed the major reaction pathways of guaiacol pyrolysis to be discerned. Density functional theory calculations were performed, and the results were found to be in good agreement with the experimentally obtained IR profiles. These findings provide guidance on how to suppress secondary reactions of guaiacol during lignin pyrolysis. Graphical abstract On-line analysis of pyrolysis process of guaiacol using in situ FTIR.

  2. Peroxidase production and ligninolytic potentials of fresh water bacteria Raoultella ornithinolytica and Ensifer adhaerens

    Directory of Open Access Journals (Sweden)

    Ayodeji O. Falade

    2017-12-01

    Full Text Available Interest in novel ligninolytic bacteria has remained topical due to, in part, the maneuverability of the bacterial genome. Conversely, the fungal genome lacks the dexterity for similar maneuverability thus, posing challenges in the fungal enzyme yield optimization process. Some impact of this situation includes the inability to commercialize the bio-catalytic process of lignin degradation by fungi. Consequently, this study assessed some fresh water bacteria isolates for ligninolytic and peroxidase properties through the utilization and degradation of model lignin compounds (guaiacol and veratryl alcohol and the decolourization of selected ligninolytic indicator dyes; Azure B (AZB, Remazol Brilliant Blue R (RBBR and Congo Red (CR. Bacterial strains with appreciable ligninolytic and peroxidase production potentials were identified through 16S rDNA sequence analysis and the nucleotide sequences deposited in the GenBank. About 5 isolates were positive for the degradation of both guaiacol (GA and veratryl alcohol (VA thus, accounting for about 17% of the test isolates. Similarly, AZB, RBBR and CR were respectively decolorized by 3, 2 and 5 bacterial strains thus, accounting for 10%, 7% and 17% of the test isolates. Two of the test bacterial strains were able to decolourize AZB, RBBR and CR respectively and these bacterial strains were identified as Raoultella ornithinolytica OKOH-1 and Ensifer adhaerens NWODO-2 with respective accession numbers as KX640917 and KX640918. Upon quantitation of the peroxidase activities; 5250 ± 0.00 U/L was recorded against Raoultella ornithinolytica OKOH-1 and 5833 ± 0.00 U/L against Ensifer adhaerens NWODO-2. The ligninolytic and dye decolourization properties of Raoultella ornithinolytica OKOH-1 and Ensifer adhaerens NWODO-2 marks for novelty particularly, as dyes with arene substituents were decolourized. Consequently, the potentials for the industrial applicability of these test bacterial strains abound as

  3. An organelle-free assay for pea chloroplast Mg-chelatase: Resolution of the activity into soluble and membrane bound fractions

    Energy Technology Data Exchange (ETDEWEB)

    Walker, C.J.; Weinstein, J.D. (Clemson Univ, SC (United States))

    1991-05-01

    Mg-chelatase, which catalyzes the insertion of magnesium into protoporphyrin, lies at the branchpoint of heme and chlorophyll biosynthesis in chloroplasts. Since magnesium chelation is the first step unique to chlorophyll synthesis, one would expect this step to be highly regulated. However, to date little is known about the enzymology or regulation of Mg-chelatase due mostly to an inability to assay it's activity outside of the intact plastid. Here the authors report the first truly in vitro i.e. organelle-free, assay for Mg-chelatase. Mg-chelatase activity in intact pea chloroplasts which is 3 to 4 fold higher than in cucumber chloroplasts, survived chloroplast lysis and could be fractionated, by centrifugation, into supernatant and pellet components. Both of these fractions were required to reconstitute Mg-chelatase activity and both were inactivated by boiling; indicating that the enzyme is composed of soluble and membrane bound protein(s). The specific activity of the reconstituted system was typically 1 nmol Mg-Deuteroporphyrin/h/mg protein and activity was linear for at least 60 min under our assay conditions. ATP and magnesium were required for Mg-chelatase activity. The soluble component could be fractionated with ammonium sulfate. The product of the reaction was confirmed fluorometrically as the magnesium chelate of the porphyrin substrate. Crude separation of chloroplast membranes into thylakoids and envelopes, suggested that the membrane-bound component of Mg-chelatase is probably located in the envelope.

  4. Catalytic profile of Arabidopsis peroxidases, AtPrx-2, 25 and 71, contributing to stem lignification.

    Directory of Open Access Journals (Sweden)

    Jun Shigeto

    Full Text Available Lignins are aromatic heteropolymers that arise from oxidative coupling of lignin precursors, including lignin monomers (p-coumaryl, coniferyl, and sinapyl alcohols, oligomers, and polymers. Whereas plant peroxidases have been shown to catalyze oxidative coupling of monolignols, the oxidation activity of well-studied plant peroxidases, such as horseradish peroxidase C (HRP-C and AtPrx53, are quite low for sinapyl alcohol. This characteristic difference has led to controversy regarding the oxidation mechanism of sinapyl alcohol and lignin oligomers and polymers by plant peroxidases. The present study explored the oxidation activities of three plant peroxidases, AtPrx2, AtPrx25, and AtPrx71, which have been already shown to be involved in lignification in the Arabidopsis stem. Recombinant proteins of these peroxidases (rAtPrxs were produced in Escherichia coli as inclusion bodies and successfully refolded to yield their active forms. rAtPrx2, rAtPrx25, and rAtPrx71 were found to oxidize two syringyl compounds (2,6-dimethoxyphenol and syringaldazine, which were employed here as model monolignol compounds, with higher specific activities than HRP-C and rAtPrx53. Interestingly, rAtPrx2 and rAtPrx71 oxidized syringyl compounds more efficiently than guaiacol. Moreover, assays with ferrocytochrome c as a substrate showed that AtPrx2, AtPrx25, and AtPrx71 possessed the ability to oxidize large molecules. This characteristic may originate in a protein radical. These results suggest that the plant peroxidases responsible for lignin polymerization are able to directly oxidize all lignin precursors.

  5. Simultaneous analysis of guaiacol and vanillin in a vanilla extract by using high-performance liquid chromatography with electrochemical detection.

    Science.gov (United States)

    Takahashi, Makoto; Sakamaki, Shizuka; Fujita, Akira

    2013-01-01

    We developed and validated a new high-performance liquid chromatographic analysis for electrochemically detecting guaiacol and vanillin as important components in vanilla extract. Separation was achieved with Capcell Pak C-18 MG, the potential of the working electrode being set at +1000 mV. The respective calibration curves for guaiacol and vanillin were linear in the range of 1.60-460 µg/L and 5.90-1180 µg/L. The respective limits for the quantities of guaiacol and vanillin were 1.60 µg/L and 2.36 µg/L. The related standard deviations of the intra- and inter-day precision of the retention time and peak area were all less than 4%. The recovery of guaiacol and vanillin was both more than 97%, all of the validation data being within an acceptable range. This analysis method is well suited for the simultaneous and convenient analysis of guaiacol and vanillin in a vanilla extract to evaluate the quality of the vanilla extract.

  6. Influence of glyceryl guaiacolate ether on anesthetics in tilapia compared to benzocaine and eugenol

    Directory of Open Access Journals (Sweden)

    Geovana R. Cosenza

    2014-03-01

    Full Text Available Objective. The study aimed to investigate the effectiveness of glyceryl guaiacolate ether (GGE and compare the times of induction, recovery, hematological changes, total protein and glycaemia among anesthetics in Nile tilapia, Oreochromis niloticus. Materials and methods. A total of 60 tilapia distributed in 3 aquariums (N=20 were used, which formed the group benzocaine (100 mg/L, eugenol (50 mg/L and guaiacol glyceryl ether (9.000 mg/L. After the induction of anesthesia fish blood samples were collected to determine the complete hemogram and glycemia. Then the animals were placed in aquariums with running water for assessing the anesthesia recovery. Results. It was verified that GGE showed longer induction and recovery times as well a significant increase (p0.05. An increase in the number of monocytes in the group treated with benzocaine (p <0.05 was observed in the analysis of the hematological parameters with no difference between groups for other variables. Conclusions. Eugenol and benzocaine allow rapid induction and recovery in Nile tilapia, without evidence of stress during handling and GGE showed high induction and recovery times, being inadequate for anesthetic use in Nile tilapia.

  7. Influence of the chemical structure on odor qualities and odor thresholds of halogenated guaiacol-derived odorants

    Science.gov (United States)

    Juhlke, Florian; Lorber, Katja; Wagenstaller, Maria; Buettner, Andrea

    2017-12-01

    Chlorinated guaiacol derivatives are found in waste water of pulp mills using chlorine in the bleaching process of wood pulp. They can also be detected in fish tissue, possibly causing off-odors. To date, there is no systematic investigation on the odor properties of halogenated guaiacol derivatives. To close this gap, odor thresholds in air and odor qualities of 14 compounds were determined by gas chromatography-olfactometry. Overall, the investigated compounds elicited smells that are characteristic for guaiacol, namely smoky, sweet, vanilla-like, but also medicinal and plaster-like. Their odor thresholds in air were, however, very low, ranging from 0.00072 to 23 ng/Lair. The lowest thresholds were found for 5-chloro- and 5-bromoguaiacol, followed by 4,5-dichloro- and 6-chloroguaiacol. Moreover, some inter-individual differences in odor threshold values could be observed, with the highest variations having been recorded for the individual values of 5-iodo- and 4-bromoguaiacol.

  8. Occlusion of 22Na+ and 86Rb+ in membrane-bound and soluble protomeric alpha beta-units of Na,K-ATPase

    International Nuclear Information System (INIS)

    Vilsen, B.; Andersen, J.P.; Petersen, J.; Jorgensen, P.L.

    1987-01-01

    In this work, we examined occlusion of 22 Na+ and 86 Rb+ in membranous and detergent-solubilized Na,K-ATPase from outer renal medulla. Optimum conditions for occlusion of 22 Na+ were provided by formation of the phosphorylated complex from the beta,gamma-bidentate complex of chromium (III) with ATP (CrATP). Release of occluded cations occurred at equally slow rates in soluble and membrane-bound Na,K-ATPase. Values of 22 Na+ occlusion as high as 11 nmol/mg of protein were measured, corresponding to 1.8-2.7 mol of Na+/mol of phosphorylated Na,K-ATPase as determined by 32 P incorporation from [gamma- 32 P]CrATP. Maximum capacity for phosphorylation from [gamma- 32 P]CrATP was 6 nmol/mg of protein and equal to capacities for binding of [48V]vanadate and [ 3 H]ouabain. The stoichiometry for occlusion of Rb+ was close to 2 Rb+ ions/phosphorylation site. In an analytical ultracentrifuge, the soluble Na+- or Rb+-occluded complexes showed sedimentation velocities (S20,w = 6.8-7.4) consistent with monomeric alpha beta-units. The data show that soluble monomeric alpha beta-units of Na,K-ATPase can occlude Rb+ or Na+ with the same stoichiometry as the membrane-bound enzyme. The structural basis for occlusion of cations in Na,K-ATPase is suggested to be the formation of a cavity inside a monomeric alpha beta-unit constituting the minimum protein unit required for active Na,K-transport

  9. Incorporation of membrane-bound, mammalian-derived immunomodulatory proteins into influenza whole virus vaccines boosts immunogenicity and protection against lethal challenge

    Directory of Open Access Journals (Sweden)

    Roberts Paul C

    2009-04-01

    Full Text Available Abstract Background Influenza epidemics continue to cause morbidity and mortality within the human population despite widespread vaccination efforts. This, along with the ominous threat of an avian influenza pandemic (H5N1, demonstrates the need for a much improved, more sophisticated influenza vaccine. We have developed an in vitro model system for producing a membrane-bound Cytokine-bearing Influenza Vaccine (CYT-IVAC. Numerous cytokines are involved in directing both innate and adaptive immunity and it is our goal to utilize the properties of individual cytokines and other immunomodulatory proteins to create a more immunogenic vaccine. Results We have evaluated the immunogenicity of inactivated cytokine-bearing influenza vaccines using a mouse model of lethal influenza virus challenge. CYT-IVACs were produced by stably transfecting MDCK cell lines with mouse-derived cytokines (GM-CSF, IL-2 and IL-4 fused to the membrane-anchoring domain of the viral hemagglutinin. Influenza virus replication in these cell lines resulted in the uptake of the bioactive membrane-bound cytokines during virus budding and release. In vivo efficacy studies revealed that a single low dose of IL-2 or IL-4-bearing CYT-IVAC is superior at providing protection against lethal influenza challenge in a mouse model and provides a more balanced Th1/Th2 humoral immune response, similar to live virus infections. Conclusion We have validated the protective efficacy of CYT-IVACs in a mammalian model of influenza virus infection. This technology has broad applications in current influenza virus vaccine development and may prove particularly useful in boosting immune responses in the elderly, where current vaccines are minimally effective.

  10. Activation of the Arabidopsis membrane-bound transcription factor bZIP28 is mediated by site-2 protease, but not site-1 protease.

    Science.gov (United States)

    Iwata, Yuji; Ashida, Makoto; Hasegawa, Chisa; Tabara, Kazuki; Mishiba, Kei-Ichiro; Koizumi, Nozomu

    2017-08-01

    The unfolded protein response (UPR) is a homeostatic cellular response conserved in eukaryotic cells to alleviate the accumulation of unfolded proteins in the endoplasmic reticulum (ER). Arabidopsis bZIP28 is a membrane-bound transcription factor activated by proteolytic cleavage in response to ER stress, thereby releasing its cytosolic portion containing the bZIP domain from the membrane to translocate into the nucleus where it induces the transcription of genes encoding ER-resident molecular chaperones and folding enzymes. It has been widely recognized that the proteolytic activation of bZIP28 is mediated by the sequential cleavage of site-1 protease (S1P) and site-2 protease (S2P). In the present study we provide evidence that bZIP28 protein is cleaved by S2P, but not by S1P. We demonstrated that wild-type and s1p mutant plants produce the active, nuclear form of bZIP28 in response to the ER stress inducer tunicamycin. In contrast, tunicamycin-treated s2p mutants do not accumulate the active, nuclear form of bZIP28. Consistent with these observations, s2p mutants, but not s1p mutants, exhibited a defective transcriptional response of ER stress-responsive genes and significantly higher sensitivity to tunicamycin. Interestingly, s2p mutants accumulate two membrane-bound bZIP28 fragments with a shorter ER lumen-facing C-terminal domain. Importantly, the predicted cleavage sites are located far from the canonical S1P recognition motif previously described. We propose that ER stress-induced proteolytic activation of bZIP28 is mediated by the sequential actions of as-yet-unidentified protease(s) and S2P, and does not require S1P. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  11. Occlusion of /sup 22/Na+ and /sup 86/Rb+ in membrane-bound and soluble protomeric alpha beta-units of Na,K-ATPase

    Energy Technology Data Exchange (ETDEWEB)

    Vilsen, B.; Andersen, J.P.; Petersen, J.; Jorgensen, P.L.

    1987-08-05

    In this work, we examined occlusion of /sup 22/Na+ and /sup 86/Rb+ in membranous and detergent-solubilized Na,K-ATPase from outer renal medulla. Optimum conditions for occlusion of /sup 22/Na+ were provided by formation of the phosphorylated complex from the beta,gamma-bidentate complex of chromium (III) with ATP (CrATP). Release of occluded cations occurred at equally slow rates in soluble and membrane-bound Na,K-ATPase. Values of /sup 22/Na+ occlusion as high as 11 nmol/mg of protein were measured, corresponding to 1.8-2.7 mol of Na+/mol of phosphorylated Na,K-ATPase as determined by /sup 32/P incorporation from (gamma-/sup 32/P)CrATP. Maximum capacity for phosphorylation from (gamma-/sup 32/P)CrATP was 6 nmol/mg of protein and equal to capacities for binding of (48V)vanadate and (/sup 3/H)ouabain. The stoichiometry for occlusion of Rb+ was close to 2 Rb+ ions/phosphorylation site. In an analytical ultracentrifuge, the soluble Na+- or Rb+-occluded complexes showed sedimentation velocities (S20,w = 6.8-7.4) consistent with monomeric alpha beta-units. The data show that soluble monomeric alpha beta-units of Na,K-ATPase can occlude Rb+ or Na+ with the same stoichiometry as the membrane-bound enzyme. The structural basis for occlusion of cations in Na,K-ATPase is suggested to be the formation of a cavity inside a monomeric alpha beta-unit constituting the minimum protein unit required for active Na,K-transport.

  12. ATPaseTb2, a unique membrane-bound FoF1-ATPase component, is essential in bloodstream and dyskinetoplastic trypanosomes.

    Directory of Open Access Journals (Sweden)

    Karolína Šubrtová

    2015-02-01

    Full Text Available In the infectious stage of Trypanosoma brucei, an important parasite of humans and livestock, the mitochondrial (mt membrane potential (Δψm is uniquely maintained by the ATP hydrolytic activity and subsequent proton pumping of the essential FoF1-ATPase. Intriguingly, this multiprotein complex contains several trypanosome-specific subunits of unknown function. Here, we demonstrate that one of the largest novel subunits, ATPaseTb2, is membrane-bound and localizes with monomeric and multimeric assemblies of the FoF1-ATPase. Moreover, RNAi silencing of ATPaseTb2 quickly leads to a significant decrease of the Δψm that manifests as a decreased growth phenotype, indicating that the FoF1-ATPase is impaired. To further explore the function of this protein, we employed a trypanosoma strain that lacks mtDNA (dyskinetoplastic, Dk and thus subunit a, an essential component of the proton pore in the membrane Fo-moiety. These Dk cells generate the Δψm by combining the hydrolytic activity of the matrix-facing F1-ATPase and the electrogenic exchange of ATP4- for ADP3- by the ATP/ADP carrier (AAC. Surprisingly, in addition to the expected presence of F1-ATPase, the monomeric and multimeric FoF1-ATPase complexes were identified. In fact, the immunoprecipitation of a F1-ATPase subunit demonstrated that ATPaseTb2 was a component of these complexes. Furthermore, RNAi studies established that the membrane-bound ATPaseTb2 subunit is essential for maintaining normal growth and the Δψm of Dk cells. Thus, even in the absence of subunit a, a portion of the FoF1-ATPase is assembled in Dk cells.

  13. Determination Hypoiodous Acid (HIO) By Peroxidase System Using Peroxidase Enzyme

    Science.gov (United States)

    Al-Baarri, A. N.; Legowo, A. M.; Widayat; Abduh, S. B. M.; Hadipernata, M.; Wisnubroto; Ardianti, D. K.; Susanto, M. N.; Yusuf, M.; Demasta, E. K.

    2018-02-01

    It has been understood that peroxidase enzyme including peroxidase serves as catalyzer to enzymatic reaction among hydrogen peroxide and halides, therefore this research was done for generating hypoiodous acid (HIO) from peroxidase system using peroxidase enzyme. Hydrogen peroxide, potassium iodide, and peroxidase enzyme were used to produce HIO. Determination the amount of formed HIO was done using 2,2'-azino-bis(3- ethylbenzothiazoline-6-sulphonic acid) or ABTS as substrate through the colorimetric measurement of hydrogen peroxide residue during reaction process using at 412 nm. The result indicated that residual hydrogen peroxide showed the minimum concentration after 60 minutes reaction time. Because the reaction started at the beginning time of mixing, hydrogen peroxide was unable to be eliminated totally to produce HIO. The reaction of peroxidase system was able to determine the beginning of mixing process but the reaction process could not eliminate the initial concentration of hydrogen peroxide indicating the maximum amount of production of HIO could be determined. In conclusion, the less of H2O2, higher HIO obtained and peroxidase enzymes can accelerate the formation of HIO.

  14. Real-Time Sensing of Hydrogen Peroxide by ITO/MWCNT/Horseradish Peroxidase Enzyme Electrode

    Directory of Open Access Journals (Sweden)

    Melinda Magyar

    2016-01-01

    Full Text Available The accurate and sensitive determination of H2O2 is very important in many cases because it is a product of reactions catalysed by several oxidase enzymes in living cells and it is essential in environmental and pharmaceutical analyses. The fabrication of enzyme protein activity based biosensors is a very promising way for this purpose because the function of biological molecules is very specific, sensitive, and selective. Horseradish peroxidase (HRP is the most commonly used enzyme for H2O2 detection because it can oxidize hydrogen atoms and, for example, xenobiotics in the presence of H2O2. In order to define the limit of detection (LOD of H2O2 we made calibrations with guaiacol and amplex red (AR, which are hydrogen donors of HRP. The accumulation of the reaction products, tetraguaiacol, and resorufin, respectively, then can be easily detected by absorption or emission (fluorescence spectroscopy. In our experiments an enzyme electrode was fabricated from ITO (indium tin oxide, functionalized multiwalled carbon nanotubes (f-MWCNTs, and HRP. Although the enzyme activity was smaller by about two orders of magnitude when the enzyme was bound to the f-MWCNTs (ca. 10−2 M H2O2/(M HRP·sec compared to ca. 2 M H2O2/(M HRP·sec and 5 M H2O2/(M HRP·sec with AR and guaiacol in buffer solution, LOD of the H2O2 decomposition was about 6 pM H2O2/sec and 10 pM H2O2/sec in the case of AR and guaiacol, respectively.

  15. Thyroid peroxidase activity is inhibited by amino acids

    Directory of Open Access Journals (Sweden)

    D.P. Carvalho

    2000-03-01

    Full Text Available Normal in vitro thyroid peroxidase (TPO iodide oxidation activity was completely inhibited by a hydrolyzed TPO preparation (0.15 mg/ml or hydrolyzed bovine serum albumin (BSA, 0.2 mg/ml. A pancreatic hydrolysate of casein (trypticase peptone, 0.1 mg/ml and some amino acids (cysteine, tryptophan and methionine, 50 µM each also inhibited the TPO iodide oxidation reaction completely, whereas casamino acids (0.1 mg/ml, and tyrosine, phenylalanine and histidine (50 µM each inhibited the TPO reaction by 54% or less. A pancreatic digest of gelatin (0.1 mg/ml or any other amino acid (50 µM tested did not significantly decrease TPO activity. The amino acids that impair iodide oxidation also inhibit the TPO albumin iodination activity. The inhibitory amino acids contain side chains with either sulfur atoms (cysteine and methionine or aromatic rings (tyrosine, tryptophan, histidine and phenylalanine. Among the amino acids tested, only cysteine affected the TPO guaiacol oxidation reaction, producing a transient inhibition at 25 or 50 µM. The iodide oxidation inhibitory activity of cysteine, methionine and tryptophan was reversed by increasing iodide concentrations from 12 to 18 mM, while no such effect was observed when the cofactor (H2O2 concentration was increased. The inhibitory substances might interfere with the enzyme activity by competing with its normal substrates for their binding sites, binding to the free substrates or reducing their oxidized form.

  16. Secondary organic aerosol (trans)formation through aqueous phase guaiacol photonitration: a kinetic study

    Science.gov (United States)

    Kroflič, Ana; Grgić, Irena

    2014-05-01

    It is well known that atmospheric aerosols play a crucial role in the Earth's climate and public health (Pöschl 2005). Despite a great effort invested in the studies of secondary organic aerosol (SOA) budget, composition, and its formation mechanisms, there is still a gap between field observations and atmospheric model predictions (Heald et al. 2005, Hallquist et al. 2009, and Lim et al. 2010). The insisting uncertainties surrounding SOA formation and aging thus gained an increasing interest in atmospheric aqueous phase chemistry; they call for more complex and time consuming studies at the environmentally relevant conditions allowing confident extrapolation to desired ambient conditions. In addition to the adverse health effects of atmospheric particulate matter (PM) as such, toxicity is also attributed to nitro-aromatic and other organic compounds which have already been detected in real aerosol samples (Traversi et al. 2009). Moreover, low-volatility aromatic derivatives are believed to form at least partly in the aerosol aqueous phase and not only in the gas phase from where they partition into water droplets (Ervens et al. 2011). Two nitro derivatives of biomass burning tracer guaiacol have recently been found in winter PM10 samples from the city of Ljubljana, Slovenia, and aqueous photonitration reaction was proposed as their possible production pathway (Kitanovski et al. 2012). In this study the kinetics of guaiacol nitration in aqueous solution was investigated in the presence of H2O2 and NO2¯ upon simulated solar irradiation (Xenon lamp, 300 W). During the experiment the DURAN® flask with the reaction mixture was held in the thermostated bath and thoroughly mixed. The reaction was monitored for 44 hours at different temperatures. Guaiacol and its main nitro-products (4-nitroguaiacol, 4-NG; 6-nitroguaiacol, 6-NG; and 4,6-dinitroguaiacol, 4,6-DNG) were quantified in every aliquot, taken from the reaction mixture, by use of high pressure liquid

  17. Aggressive re-warming at 38.5 degrees C following deep hypothermia at 21 degrees C increases neutrophil membrane bound elastase activity and pro-inflammatory factor release

    NARCIS (Netherlands)

    Tang, Min; Zhao, Xiao-gang; He, Yi; Gu, Yan; Mei, Ju

    2016-01-01

    Background: Cardiopulmonary bypass (CPB) is often performed under hypothermic condition. The effects of hypothermia and re-warming on neutrophil activity are unclear. This study aimed to compare the effects of different hypothermia and re-warming regimens on neutrophil membrane bound elastase (MBE)

  18. Thermal stability, antioxidant, and anti-inflammatory activity of curcumin and its degradation product 4-vinyl guaiacol.

    Science.gov (United States)

    Esatbeyoglu, Tuba; Ulbrich, Katrin; Rehberg, Clemens; Rohn, Sascha; Rimbach, Gerald

    2015-03-01

    Curcumin is a secondary plant metabolite present in Curcuma longa L. Since curcumin is widely used as a food colorant in thermally processed food it may undergo substantial chemical changes which in turn could affect its biological activity. In the current study, curcumin was roasted at 180 °C up to 70 minutes and its kinetic of degradation was analyzed by means of HPLC-PDA and LC-MS, respectively. Roasting of curcumin resulted in the formation of the degradation products vanillin, ferulic acid, and 4-vinyl guaiacol. In cultured hepatocytes roasted curcumin as well as 4-vinyl guaiacol enhanced the transactivation of the redox-regulated transcription factor Nrf2, known to be centrally involved in cellular stress response and antioxidant defense mechanisms. The antioxidant enzyme paraoxonase 1 was induced by roasted curcumin and 4-vinyl guaiacol. Furthermore, roasted curcumin and 4-vinyl guaiacol decreased interleukin-6 gene expression in lipopolysaccharide stimulated murine macrophages. Current data suggest that curcumin undergoes degradation due to roasting and its degradation product exhibit significant biological activity in cultured cells.

  19. Continuous Catalytic Hydrodeoxygenation of Guaiacol over Pt/SiO2 and Pt/H-MFI-90

    DEFF Research Database (Denmark)

    Hellinger, Melanie; Baier, Sina; Mortensen, Peter Mølgaard

    2015-01-01

    Hydrodeoxygenation of guaiacol in the presence of 1-octanol was studied in a fixed-bed reactor under mild conditions (50–250 °C) over platinum particles supported on silica (Pt/SiO2) and a zeolite with framework type MFI at a Si/Al-ratio of 45 (Pt/H-MFI-90). The deoxygenation selectivity strongly...

  20. Pathogen-expanded CD11b+ invariant NKT cells feedback inhibit T cell proliferation via membrane-bound TGF-β1.

    Science.gov (United States)

    Han, Yanmei; Jiang, Zhengping; Chen, Zhubo; Gu, Yan; Liu, Yanfang; Zhang, Xiang; Cao, Xuetao

    2015-04-01

    Natural killer T cells (NKT cells) are effector cells, but also regulator of immune response, which either promote or suppress immune response through production of different cytokines. However, the subsets of NKT cells with definite phenotype and regulatory function need to be further identified. Furthermore, the mechanisms for NKT cells to regulate immune response remain to be fully elucidated. Here we identified CD11b(+) invariant NKT (CD11b(+) iNKT) cells as a new subset of regulatory NKT cells in mouse models with infection. αGalCer:CD1d complex(+)TCRβ(+)NK1.1(+) NKT cells could be categorized to CD11b(+) and CD11b(-) subsets. NKT cells are enriched in liver. During Listeria monocytogenes infection, hepatic CD11b(+) iNKT cells were significantly induced and expanded, with peak expansion on day 8. CD11b(+) iNKT cells were also expanded significantly in spleen and mesenteric lymph nodes. As compared to CD11b(-) iNKT cells, CD11b(+) iNKT cells expressed higher levels of CD27, FasL, B7H1, CD69, and particularly higher level of membrane-bound TGF-β1 (mTGF-β1), but produced less IFN-γ, IL-4, IL-10 and TGF-β1. Hepatic CD11b(+) iNKT cells suppressed antigen-nonspecific and OVA-specific CD4 and CD8 T cell proliferation through mTGF-β1 both in vitro and in vivo, meanwhile, they did not interfere with activation of CD4 T cells and cytotoxicity of the activated CD8 T cells. Thus, we have identified a new subset of pathogen-expanded CD11b(+) invariant NKT cells which can feedback inhibit T cell response through cell-to-cell contact via cell surface (membrane-bound) TGF-β1, especially at the late stage of immune response against infection. CD11b(+) regulatory iNKT cells may contribute to protect host from pathological injure by preventing immune overactivation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. Extração e caracterização parcial de peroxidase de folhas de Copaifera langsdorffii Desf. Extraction and partial characterization of peroxidase from Copaifera langsdorffii Desf. leaves

    Directory of Open Access Journals (Sweden)

    Hermelinda Penha Freire Maciel

    2007-06-01

    Full Text Available Em recentes publicações têm sido descritos vários processos para obtenção de peroxidases. O propósito deste trabalho foi extrair peroxidase de folhas de Copaifera langsdorffii e caracterizar parcialmente a enzima usando planejamento experimental e teste univariado, para confirmação dos resultados obtidos por planejamento experimental. A atividade da peroxidase foi medida usando sistema guaiacol: peróxido de hidrogênio. A peroxidase isolada apresentou 81,6% da atividade da horseradish peroxidase e é de fácil obtenção, a partir de folhas de uma árvore abundante em todo o país. A peroxidase semi-purificada (COP foi obtida pela precipitação do extrato bruto com acetona 65% (v.v-1, produzindo o pó cetônico. A COP apresentou atividade ótima na faixa de pH 5,0 a 7,0 e temperatura de 5 a 45 °C, com atividade máxima em pH 6,0 e 35 °C. A enzima mostrou-se estável em temperaturas inferiores a 50 °C e pH entre 4,5 e 9,0, por até 24 horas. A peroxidase foi inativada após 4 horas a 80 °C e após 3 minutos a 96 °C. Esta enzima demonstra possibilidade para ser usada como reagente para diagnósticos, construção de biossensores e outros métodos analíticos em vários campos da ciência.In the literature, several processes have been described to obtain peroxidases. The purpose of this work was to obtain peroxidase from Copaifera langsdorffii leaves and characterize it partially using a factorial design of experiments and univaried tests, to confirm the results obtained by the factorial design of experiments. Peroxidase activity was measured using the guaiacol: hydrogen peroxide system. The isolated peroxidase presented 81.6% of horseradish peroxidase activity and was easy to obtain from leaves of an abundant tree distributed all over the country. Semi-purified peroxidase (COP was precipitated with acetone 65% (v.v-1 of the crude extract, obtaining the acetone powder. The COP optimum reaction pH values were between 5.0-7.0 and the

  2. Evaluation of peroxidases from roots of Cyperus hermaphroditus as enzymatic mechanisms in phenanthrene oxidation

    Energy Technology Data Exchange (ETDEWEB)

    Guerrero Zuniga, A. [Inst. Mexicano del Petroleo, Mexico City (Mexico). Environmental Protection Management Office; Rodriguez Dorantes, A.M. [Lab. Fisiologia Vegetal, Escuela Nacional de Ciencias Biologicas, Mexico City (Mexico). Depto Botanica

    2006-07-01

    Although phenanthrene is not mutagenic or carcinogenic, it has been shown to be toxic to aquatic organisms. This study evaluated in-vitro phenanthrene oxidation by peroxidases from radical extracts of Cyperus hermaphroditus plants. The characterization of oxidation products of phenanthrene related to the induction of root peroxidases was also examined. Concentrated ethanol stock of phenanthrene solution was added to the mineral solution of each plant container. The total radical biomass was placed in 4.5 ml of an ionic solution to analyze the enzymatic activity of the extracellular peroxidases. The total protein for each experiment was quantified by the Bradford method. Extracellular peroxidases activity was measured using the spectrophotometric method. The amount of radical biomass was quantified as high in the 80 and 120 ppm phenanthrene treatments relative to the control plants. It was suggested that the nature of the Cyperaceae roots combined with the high-octanol water coefficient and a low water solubility for phenanthrene may have facilitated the stabilization of the contaminant towards the roots. The ability of Cyperus hermaphroditus to immobilize phenanthrene through its adhesion was encouraged by the conditions of the hydroponic culture system. The adsorption of phenanthrene was increased with the time of exposure to the contaminant due to the greater total root mass. The study also showed the transformation of phenanthrene by radical extracts of Cyperus hermaphroditus containing guaiacol peroxidases with 12 per cent residual phenanthrene in the in vitro assays. The spectrophotometric analysis confirmed that the enzymatic systems are responsible for the phytotransformation of the pollutant. 9 refs., 2 tabs., 5 figs.

  3. Differences in wound-induced changes in cell-wall peroxidase activities and isoform patterns between seedlings of Prosopis tamarugo and Prosopis chilensis.

    Science.gov (United States)

    Lehner, Gabriele; Cardemil, Liliana

    2003-05-01

    We determined changes in cell-wall peroxidase activities and isoform patterns in response to wounding in seedlings of Prosopis tamarugo Phil. (an endemic species of the Atacama Desert) and Prosopis chilensis (Mol.) Stuntz (a native species of central Chile), to assess tolerance to predation. In seedlings of both species, the maximal increase in peroxidase activity occurred 48 h after wounding, reaching three times the control value in P. tamarugo and twice the control value in P. chilensis. The activity of ionically bound cell-wall peroxidases increased only locally in wounded embryonic axes, whereas the activity of soluble peroxidases increased systemically in unwounded cotyledons. Analysis of ionic peroxidases by isoelectrofocusing revealed two groups of peroxidases in the cell walls of both species: four distinct acidic isoforms and a group of basic isoforms. In response to wounding, there was a large increase in activity of the acidic isoforms in P. tamarugo, whereas there was an increase in the activity of the basic isoforms in P. chilensis. In P. chilensis, the wound-induced increase in activity of the basic isoforms corresponded with one of the two isoforms detected in P. tamarugo prior to wounding. Experiments with protein and RNA synthesis inhibitors indicated that a preexisting basic peroxidase is activated in P. chilensis after wounding. Assays of ionically bound peroxidase activity with four different substrates corroborated the differences found in isoform patterns between species. In P. tamarugo, the largest increases in activity were found with ortho-phenylenediamine and ferulic acid as substrates, whereas in P. chilensis the largest increase in activity was found with guaiacol as substrate. Because the same basic cell-wall peroxidase that accumulated after wounding in P. chilensis was present in P. tamarugo prior to wounding, and the activity of acidic cell-wall peroxidases increased after wounding in P. tamarugo but not in P. chilensis, we conclude

  4. Kinetics of the flash-induced P515 response in relation to the H+-permeability of the membrane bound ATPase in spinach chloroplasts

    Energy Technology Data Exchange (ETDEWEB)

    Peters, R.L.; van Kooten, O.; Vredenberg, W.J.

    1985-08-01

    The effect of dicyclohexylcarbodiimide (DCCD) on the kinetics of the flash-induced P515 response and on the activity of the ATPase was investigated in isolated spinach chloroplasts. It was found that after the addition of 5 X 10(-8)mol DCCD the rate of ATP hydrolysis induced by a period of 60 sec illumination was decreased to less than 5% of its original value. At this concentration, hardly any effect, if at all, could be detected on the kinetics of the flash-induced P515 response, neither in dark-adapted nor in light-activated chloroplasts. It was concluded that the presence of concentrations of DCCD, sufficiently high to affect the ATPase activity, does not affect the kinetics of the flash-induced P515 response. Since DCCD decreases the H+ permeability of the membrane-bound ATPase, it was concluded that this permeability coefficient for protons is not an important factor in the regulation of the flash-induced membrane potential and, therefore, does not affect the kinetics of the flash-induced P515 response.

  5. Development of immobilized membrane-based affinity columns for use in the online characterization of membrane bound proteins and for targeted affinity isolations

    International Nuclear Information System (INIS)

    Moaddel, Ruin; Wainer, Irving W.

    2006-01-01

    Membranes obtained from cell lines that express or do not express a target membrane bound protein have been immobilized on a silica-based liquid chromatographic support or on the surface of an activated glass capillary. The resulting chromatographic columns have been placed in liquid chromatographic systems and used to characterize the target proteins and to identify small molecules that bind to the target. Membranes containing ligand gated ion channels, G-protein coupled receptors and drug transporters have been prepared and characterized. If a marker ligand has been identified for the target protein, frontal or zonal displacement chromatographic techniques can be used to determine binding affinities (K d values) and non-linear chromatography can be used to assess the association (k on ) and dissociation (k off ) rate constants and the thermodynamics of the binding process. Membrane-based affinity columns have been created using membranes from a cell line that does not express the target protein (control) and the same cell line that expresses the target protein (experimental) after genomic transfection. The resulting columns can be placed in a parallel chromatography system and the differential retention between the control and experimental columns can be used to identify small molecules and protein that bind to the target protein. These applications will be illustrated using columns created using cellular membranes containing nicotinic acetylcholine receptors and the drug transporter P-glycoprotein

  6. Development of immobilized membrane-based affinity columns for use in the online characterization of membrane bound proteins and for targeted affinity isolations

    Energy Technology Data Exchange (ETDEWEB)

    Moaddel, Ruin [Gerontology Research Center, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224-6825 (United States); Wainer, Irving W. [Gerontology Research Center, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224-6825 (United States)]. E-mail: Wainerir@grc.nia.nih.gov

    2006-03-30

    Membranes obtained from cell lines that express or do not express a target membrane bound protein have been immobilized on a silica-based liquid chromatographic support or on the surface of an activated glass capillary. The resulting chromatographic columns have been placed in liquid chromatographic systems and used to characterize the target proteins and to identify small molecules that bind to the target. Membranes containing ligand gated ion channels, G-protein coupled receptors and drug transporters have been prepared and characterized. If a marker ligand has been identified for the target protein, frontal or zonal displacement chromatographic techniques can be used to determine binding affinities (K {sub d} values) and non-linear chromatography can be used to assess the association (k {sub on}) and dissociation (k {sub off}) rate constants and the thermodynamics of the binding process. Membrane-based affinity columns have been created using membranes from a cell line that does not express the target protein (control) and the same cell line that expresses the target protein (experimental) after genomic transfection. The resulting columns can be placed in a parallel chromatography system and the differential retention between the control and experimental columns can be used to identify small molecules and protein that bind to the target protein. These applications will be illustrated using columns created using cellular membranes containing nicotinic acetylcholine receptors and the drug transporter P-glycoprotein.

  7. Cloning, sequencing, and characterization of the gene encoding the smallest subunit of the three-component membrane-bound alcohol dehydrogenase from Acetobacter pasteurianus.

    Science.gov (United States)

    Kondo, K; Beppu, T; Horinouchi, S

    1995-09-01

    The membrane-bound alcohol dehydrogenase (ADH) of Acetobacter pasteurianus NCI1452 consists of three different subunits, a 78-kDa dehydrogenase subunit, a 48-kDa cytochrome c subunit, and a 20-kDa subunit of unknown function. For elucidation of the function of the smallest subunit, this gene was cloned from this strain by the oligonucleotide-probing method, and its nucleotide sequence was determined. Comparison of the deduced amino acid sequence and the NH2-terminal sequence determined for the purified protein indicated that the smallest subunit contained a typical signal peptide of 28 amino acids, as did the larger two subunits. This gene complemented the ADH activity of a mutant strain which had lost the smallest subunit. Disruption of this gene on the chromosome resulted in loss of ADH activity in Acetobacter aceti, indicating that the smallest subunit was essential for ADH activity. Immunoblot analyses of cell lysates prepared from various ADH mutants suggested that the smallest subunit was concerned with the stability of the 78-kDa subunit and functioned as a molecular coupler of the 78-kDa subunit to the 48-kDa subunit on the cytoplasmic membrane.

  8. The extracellular membrane-proximal domain of membrane-bound IgE restricts B cell activation by limiting B cell antigen receptor surface expression.

    Science.gov (United States)

    Vanshylla, Kanika; Opazo, Felipe; Gronke, Konrad; Wienands, Jürgen; Engels, Niklas

    2018-03-01

    Immunoglobulin E (IgE) antibodies are key mediators of allergic reactions. Due to their potentially harmful anaphylactic properties, their production is tightly regulated. The membrane-bound isoform of IgE (mIgE), which is an integral component of the B cell antigen receptor, has been shown to be critical for the regulation of IgE responses in mice. In primate species including humans, mIgE can be expressed in two isoforms that are produced by alternative splicing of the primary ε Ig heavy chain transcript, and differ in the absence or presence of an extracellular membrane-proximal domain (EMPD) consisting of 52 amino acids. However, the function of the EMPD remains unclear. Here, we demonstrate that the EMPD restricts surface expression of mIgE-containing BCRs in human and murine B cells. The EMPD does not interfere with BCR assembly but acts as an autonomous endoplasmic reticulum retention domain. Limited surface expression of EMPD-containing mIgE-BCRs caused impaired activation of intracellular signaling cascades and hence represents a regulatory mechanism that may control the production of potentially anaphylactic IgE antibodies in primate species. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. A Quantitative Approach to Evaluate the Impact of Fluorescent Labeling on Membrane-Bound HIV-Gag Assembly by Titration of Unlabeled Proteins.

    Directory of Open Access Journals (Sweden)

    Julia Gunzenhäuser

    Full Text Available The assembly process of the human immunodeficiency virus 1 (HIV-1 is driven by the viral polyprotein Gag. Fluorescence imaging of Gag protein fusions is widely performed and has revealed important information on viral assembly. Gag fusion proteins are commonly co-transfected with an unlabeled form of Gag to prevent labeling artifacts such as morphological defects and decreased infectivity. Although viral assembly is widely studied on individual cells, the efficiency of the co-transfection rescue has never been tested at the single cell level. Here, we first develop a methodology to quantify levels of unlabeled to labeled Gag in single cells using a fluorescent reporter protein for unlabeled Gag and fluorescence correlation spectroscopy. Using super-resolution imaging based on photoactivated localization microscopy (PALM combined with molecular counting we then study the nanoscale morphology of Gag clusters as a function of unlabeled to labeled Gag ratios in single cells. We show that for a given co-transfection ratio, individual cells express a wide range of protein ratios, necessitating a quantitative read-out for the expression of unlabeled Gag. Further, we show that monomerically labeled Gag assembles into membrane-bound clusters that are morphologically indistinguishable from mixtures of unlabeled and labeled Gag.

  10. In Situ Proteolysis for Crystallization of Membrane Bound Cytochrome P450 17A1 and 17A2 Proteins from Zebrafish.

    Science.gov (United States)

    Lei, Li; Egli, Martin

    2016-04-01

    Fish and human cytochrome P450 (P450) 17A1 catalyze both steroid 17α-hydroxylation and 17α,20-lyase reactions. Fish P450 17A2 catalyzes only 17α-hydroxylation. Both enzymes are microsomal-type P450s, integral membrane proteins that bind to the membrane through their N-terminal hydrophobic segment, the signal anchor sequence. The presence of this N-terminal region renders expression of full-length proteins challenging or impossible. For some proteins, variable truncation of the signal anchor sequence precludes expression or results in poor expression levels. To crystallize P450 17A1 and 17A2 in order to gain insight into their different activities, we used an alternative N-terminal sequence to boost expression together with in situ proteolysis. Key features of our approach to identify crystallizable P450 fragments were the use of an N-terminal leader sequence, a screen composed of 12 proteases to establish optimal cleavage, variations of protease concentration in combination with an SDS-PAGE assay, and analysis of the resulting fragments using Edman sequencing. Described in this unit are protocols for vector preparation, expression, purification, and in situ proteolytic crystallization of two membrane-bound P450 proteins. Copyright © 2016 John Wiley & Sons, Inc.

  11. PROLYTIC ION EXCHANGES PRODUCED IN HUMAN RED CELLS BY METHANOL, ETHANOL, GUAIACOL, AND RESORCINOL

    Science.gov (United States)

    Ponder, Eric

    1947-01-01

    When the washed red cells of heparinized human blood are exposed at 4°C. to methanol, ethanol, guaiacol, or resorcinol in hypolytic concentrations in isotonic NaCl, the prolytic loss of K at the end of 20 hours varies from about 25 per cent of the initial K content of the cells in the case of 3.1 M methanol to about 55 per cent of the initial K in the case of 0.04 M resorcinol. As in the case of the prolytic losses observed with other lysins, the K loss is rapid at first and then slows down so that what appears to be a new steady state is reached logarithmically. The K lost from the cells during the period of the prolytic loss is replaced by an approximately equivalent amount of Na, derived from the isotonic NaCl in which the cells are suspended. The Na which enters can be replaced by K by washing the cells in isotonic KCl, and this K can again be replaced by Na by washing the cells in isotonic NaCl. The remainder of the cell K., i.e. the K which was not lost during the period of the prolytic loss, is retained in the cell unaffected by these washing procedures. The capacity of red cells for undergoing disk-sphere transformations is scarcely affected by their having been exposed to hypolytic concentrations of methanol, ethanol, guaiacol, or resorcinol in isotonic NaCl, and their resistance to osmotic hemolysis and to lysis by saponin and digitonin is altered only in minor respects even when as much as 50 per cent of the cell K has been exchanged for Na. Some restriction to the movement of K between the cell and its environment is apparently modified irreversibly when the cell is exposed to hypolytic concentrations of lysins, and the modification is such that only a fraction of the cell K is affected, the fraction being a function of the lysin concentration, the duration of its action, and other factors. A modification of some part of the cell structure and of the properties dependent on its integrity is probably involved: K may be lost more readily from some cells

  12. Construction of a plasmid for co-expression of mouse membrane-bound form of IL-15 and RAE-1ε and its biological activity.

    Science.gov (United States)

    Qian, Li; Ji, Ming-Chun; Pan, Xin-Yuan; Gong, Wei-Juan; Tian, Fang; Duan, Qiu-Fang

    2011-05-01

    Interleukin 15 (IL-15) is a pivotal cytokine for the proliferation and activation of a specific group of immune cells such as natural killer (NK), IFN-producing killer dendritic cells (IKDC) and CD8 T cells. RAE-1ε, the ligand for the activating NKG2D receptor, which also play an important role in the proliferation and activation of NK cells and IKDCs. In this study, a membrane-bound form of IL-15 (termed mb15) encoding sequence and RAE-1ε gene were obtained by SOE-PCR or PCR amplification. The amplified mb15 and RAE-1ε gene were then digested and inserted into the multiple cloning site1 (MCS1) and MCS2 of pVITRO2-mcs vector, respectively. A recombinant eukaryotic expression vector for co-expression of mb15 and RAE-1ε was successfully constructed. After it was transfected to BaF3 cells, the expression of IL-15 and RAE-1ε in recombinant BaF3/mb15/RAE-1ε cells were verified by RT-PCR, western blot and FCM analysis. Furthermore, BaF3/mb15/RAE-1ε cells had the ability of promoting NK cells proliferation and IFN-γ secretion. In conclusion, BaF3/mb15/RAE-1ε cells were successfully constructed, which is very useful for further studies, especially for the expansion and activation of certain subsets of immune cells such as NK cells and IKDCs. Copyright © 2011 Elsevier Inc. All rights reserved.

  13. Roles of Agrobacterium tumefaciens membrane-bound ferritin (MbfA) in iron transport and resistance to iron under acidic conditions.

    Science.gov (United States)

    Bhubhanil, Sakkarin; Chamsing, Jareeya; Sittipo, Panida; Chaoprasid, Paweena; Sukchawalit, Rojana; Mongkolsuk, Skorn

    2014-05-01

    Agrobacterium tumefaciens membrane-bound ferritin (MbfA) is a member of the erythrin (Er)-vacuolar iron transport family. The MbfA protein has an Er or ferritin-like domain at its N terminus and has been predicted to have five transmembrane segments in its C-terminal region. Analysis of protein localization using PhoA and LacZ reporter proteins supported the view that the N-terminal di-iron site is located in the cytoplasm whilst the C-terminal end faces the periplasm. An A. tumefaciens mbfA mutant strain had 1.5-fold higher total iron content than the WT strain. Furthermore, multi-copy expression of mbfA reduced total iron content two- and threefold in WT and mbfA mutant backgrounds, respectively. These results suggest that MbfA may function as an iron exporter rather than an iron storage protein. The mbfA mutant showed 10-fold increased sensitivity to the iron-activated antibiotic streptonigrin, implying that the mutant had increased accumulation of intracellular free iron. Growth of the mbfA mutant was reduced in the presence of high iron under acidic conditions. The expression of mbfA was induced highly in cells grown in iron-replete medium at pH 5.5, further supporting the view that mbfA is involved in the response to iron under acidic conditions. A. tumefaciens MbfA may play a protective role against increased free iron in the cytoplasm through iron binding and export, thus preventing iron-induced toxicity via the Fenton reaction.

  14. Unexpected Potency Differences between B-Cell-Activating Factor (BAFF) Antagonist Antibodies against Various Forms of BAFF: Trimer, 60-Mer, and Membrane-Bound.

    Science.gov (United States)

    Nicoletti, Amy M; Kenny, Cynthia Hess; Khalil, Ashraf M; Pan, Qi; Ralph, Kerry L M; Ritchie, Julie; Venkataramani, Sathyadevi; Presky, David H; DeWire, Scott M; Brodeur, Scott R

    2016-10-01

    Therapeutic agents antagonizing B-cell-activating factor/B-lymphocyte stimulator (BAFF/BLyS) are currently in clinical development for autoimmune diseases; belimumab is the first Food and Drug Administration-approved drug in more than 50 years for the treatment of lupus. As a member of the tumor necrosis factor superfamily, BAFF promotes B-cell survival and homeostasis and is overexpressed in patients with systemic lupus erythematosus and other autoimmune diseases. BAFF exists in three recognized forms: membrane-bound and two secreted, soluble forms of either trimeric or 60-mer oligomeric states. To date, most in vitro pharmacology studies of BAFF neglect one or more of these forms. Here, we report a comprehensive in vitro cell-based analysis of BAFF in assay systems that measure all forms of BAFF-mediated activation. We demonstrate the effects of these BAFF forms in both a primary human B-cell proliferation assay and in nuclear factor κB reporter assay systems in Chinese hamster ovary cells expressing BAFF receptors and transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI). In contrast to the mouse system, we find that BAFF trimer activates the human TACI receptor. Further, we profiled the activities of two clinically advanced BAFF antagonist antibodies, belimumab and tabalumab. Unexpectedly, we revealed differences in inhibitory potencies against the various BAFF forms, in particular that belimumab does not potently inhibit BAFF 60-mer. Through this increased understanding of the activity of BAFF antagonists against different forms of BAFF, we hope to influence the discovery of BAFF antagonist antibodies with distinct therapeutic mechanisms for improvement in the treatment of lupus or other related autoimmune pathologies. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  15. In vitro assay of the chlorophyll biosynthetic enzyme Mg-chelatase: Resolution of the activity into soluble and membrane-bound fractions

    Energy Technology Data Exchange (ETDEWEB)

    Walker, C.J.; Weinstein, J.D. (Clemson Univ., SC (United States))

    1991-07-01

    The first committed step in chlorophyll synthesis is the Mg-chelatase-catalyzed insertion of magnesium into protoporphyrin IX. Since iron insertion into protoporphyrin leads to heme formation, Mg-chelatase lies at the branch point of heme and chlorophyll synthesis in chloroplasts. Little is known about the enzymology or regulation of Mg-chelatase, as it has been assayed only in intact cucumber chloroplasts. In this report we describe an in vitro assay for Mg-chelatase. Mg-chelatase activity in intact pea chloroplasts was 3- to 4-fold higher than in cucumber chloroplasts. This activity survived chloroplast lysis and could be fractionated by centrifugation into supernatant and pellet components. Both of these fractions were required to reconstitute Mg-chelatase activity, and both were inactivated by boiling indicating that the enzyme is composed of soluble and membrane-bound protein(s). The product of the reaction was confirmed fluorometrically as the magnesium chelate of the porphyrin substrate. The specific activity of the reconstituted system was typically 1 nmol of Mg-deuteroporphyrin per h per mg of protein, and activity was linear for at least 60 min under our assay conditions. ATP and magnesium were required for Mg-chelatase activity and the enzymen was sensitive to the sulfhydryl reagent N-ethylmaleimide (I{sub 50}, 20 {mu}M). Broken and reconstituted cucumber chloroplasts were unable to maintain Mg-chelatase activity. However, the cucumber supernatant fraction was active when combined with the pellet fraction of peas; the converse was not true, which suggested that the cucumber pellet was the component that lost activity during lysis.

  16. Role of a membrane-bound aldehyde dehydrogenase complex AldFGH in acetic acid fermentation with Acetobacter pasteurianus SKU1108.

    Science.gov (United States)

    Yakushi, Toshiharu; Fukunari, Seiya; Kodama, Tomohiro; Matsutani, Minenosuke; Nina, Shun; Kataoka, Naoya; Theeragool, Gunjana; Matsushita, Kazunobu

    2018-04-03

    Acetic acid fermentation is widely considered a consequence of ethanol oxidation by two membrane-bound enzymes-alcohol dehydrogenase and aldehyde dehydrogenase (ALDH)-of acetic acid bacteria. Here, we used a markerless gene disruption method to construct a mutant of the Acetobacter pasteurianus strain SKU1108 with a deletion in the aldH gene, which encodes the large catalytic subunit of a heterotrimeric ALDH complex (AldFGH), to examine the role of AldFGH in acetic acid fermentation. The ΔaldH strain grew less on ethanol-containing medium, i.e., acetic acid fermentation conditions, than the wild-type strain and significantly accumulated acetaldehyde in the culture medium. Unexpectedly, acetaldehyde oxidase activity levels of the intact ΔaldH cells and the ΔaldH cell membranes were similar to those of the wild-type strain, which might be attributed to an additional ALDH isozyme (AldSLC). The apparent K M values of the wild-type and ΔaldH membranes for acetaldehyde were similar to each other, when the cells were cultured in nonfermentation conditions, where ΔaldH cells grow as well as the wild-type cells. However, the membranes of the wild-type cells grown under fermentation conditions showed a 10-fold lower apparent K M value than those of the cells grown under nonfermentation conditions. Under fermentation conditions, transcriptional levels of a gene for AldSLC were 10-fold lower than those under nonfermentation conditions, whereas aldH transcript levels were not dramatically changed under the two conditions. We suggest that A. pasteurianus SKU1108 has two ALDHs, and the AldFGH complex is indispensable for acetic acid fermentation and is the major enzyme under fermentation conditions.

  17. Matriptase-2, a membrane-bound mosaic serine proteinase predominantly expressed in human liver and showing degrading activity against extracellular matrix proteins.

    Science.gov (United States)

    Velasco, Gloria; Cal, Santiago; Quesada, Victor; Sánchez, Luis M; López-Otín, Carlos

    2002-10-04

    We have identified and cloned a fetal liver cDNA encoding a new serine proteinase that has been called matriptase-2. This protein exhibits a domain organization similar to other members of an emerging family of membrane-bound serine proteinases known as type II transmembrane serine proteinases. Matriptase-2 contains a short cytoplasmic domain, a type II transmembrane sequence, a central region with several modular structural domains including two CUB (complement factor C1s/C1r, urchin embryonic growth factor, bone morphogenetic protein) domains and three low density lipoprotein receptor tandem repeats, and finally, a C-terminal catalytic domain with all typical features of serine proteinases. The human matriptase-2 gene maps to 22q12-q13, a location that differs from all type II transmembrane serine proteinase genes mapped to date. Immunofluorescence and Western blot analysis of COS-7 cells transfected with the isolated cDNA confirmed that matriptase-2 is anchored to the cell surface. Matriptase-2 was expressed in Escherichia coli, and the purified recombinant protein hydrolyzed synthetic substrates used for assaying serine proteinases and endogenous proteins such as type I collagen, fibronectin, and fibrinogen. Matriptase-2 could also activate single-chain urokinase plasminogen activator, albeit with low efficiency. These activities were abolished by inhibitors of serine proteinases but not by inhibitors of other classes of proteolytic enzymes. Northern blot analysis demonstrated that matriptase-2 transcripts are only detected at significant levels in both fetal and adult liver, suggesting that this novel serine proteinase may play a specialized role in matrix remodeling processes taking place in this tissue during development or in adult tissues.

  18. Characterization and modelling of VanT: a novel, membrane-bound, serine racemase from vancomycin-resistant Enterococcus gallinarum BM4174.

    Science.gov (United States)

    Arias, C A; Martín-Martinez, M; Blundell, T L; Arthur, M; Courvalin, P; Reynolds, P E

    1999-03-01

    Sequence determination of a region downstream from the vanXYc gene in Enterococcus gallinarum BM4174 revealed an open reading frame, designated vanT, that encodes a 698-amino-acid polypeptide with an amino-terminal domain containing 10 predicted transmembrane segments. The protein contained a highly conserved pyridoxal phosphate attachment site in the C-terminal domain, typical of alanine racemases. The protein was overexpressed in Escherichia coli, and serine racemase activity was detected in the membrane but not in the cytoplasmic fraction after centrifugation of sonicated cells, whereas alanine racemase activity was located almost exclusively in the cytoplasm. When the protein was overexpressed as a polypeptide lacking the predicted transmembrane domain, serine racemase activity was detected in the cytoplasm. The serine racemase activity was partially (64%) inhibited by D-cycloserine, whereas host alanine racemase activity was almost totally inhibited (97%). Serine racemase activity was also detected in membrane preparations of constitutively vancomycin-resistant E. gallinarum BM4174 but not in BM4175, in which insertional inactivation of the vanC-1 D-Ala:D-Ser ligase gene probably had a polar effect on expression of the vanXYc and vanT genes. Comparative modelling of the deduced C-terminal domain was based on the alignment of VanT with the Air alanine racemase from Bacillus stearothermophilus. The model revealed that almost all critical amino acids in the active site of Air were conserved in VanT, indicating that the C-terminal domain of VanT is likely to adopt a three-dimensional structure similar to that of Air and that the protein could exist as a dimer. These results indicate that the source of D-serine for peptidoglycan synthesis in vancomycin-resistant enterococci expressing the VanC phenotype involves racemization of L- to D-serine by a membrane-bound serine racemase.

  19. Carbon-supported bimetallic Pd–Fe catalysts for vapor-phase hydrodeoxygenation of guaiacol

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Junming; Karim, Ayman M.; Zhang, He; Kovarik, Libor; Li, Xiaohong Shari; Hensley, Alyssa J.; McEwen, Jean-Sabin; Wang, Yong

    2013-10-01

    Abstract Carbon supported metal catalysts (Cu/C, Fe/C, Pd/C, Pt/C, PdFe/C and Ru/C) have been prepared, characterized and tested for vapor-phase hydrodeoxygenation (HDO) of guaiacol (GUA) at atmospheric pressure. Phenol was the major intermediate on all catalysts. Over the noble metal catalysts saturation of the aromatic ring was the major pathway observed at low temperature (250 °C), forming predominantly cyclohexanone and cyclohexanol. Substantial ring opening reaction was observed on Pt/C and Ru/C at higher reaction temperatures (e.g., 350 °C). Base metal catalysts, especially Fe/C, were found to exhibit high HDO activity without ring-saturation or ring-opening with the main products being benzene, phenol along with small amounts of cresol, toluene and trimethylbenzene (TMB). A substantial enhancement in HDO activity was observed on the PdFe/C catalysts. Compared with Fe/C, the yield to oxygen-free aromatic products (i.e., benzene/toluene/TMB) on PdFe/C increased by a factor of four at 350 °C, and by approximately a factor of two (83.2% versus 43.3%) at 450 °C. The enhanced activity of PdFe/C is attributed to the formation of PdFe alloy as evidenced by STEM, EDS and TPR.

  20. Effect of eugenol and guaiacol application on tomato aroma composition determined by headspace stir bar sorptive extraction.

    Science.gov (United States)

    Pardo-García, Ana I; Martínez-Gil, Ana M; López-Córcoles, Horacio; Zalacain, Amaya; Salinas, Rosario

    2013-03-30

    The present work was carried out because there is only a small amount of literature on how the volatile composition of tomatoes can be modified by the effect of exogenous substances in contact with tomato plants. This work studies how eugenol and guaiacol, either by foliar application and/or in the surrounding atmosphere, can affect the volatile composition of this fruit. An important work of this study was also conduced to validate the analytical method [headspace stir bar sorptive extraction-gas chromatography-mass spectrometry (HS-SBSE-GC-MS)] to determine the composition of the volatiles in tomato. Analytical method validation parameters such as linearity, limit of detection, limit of quantification, and recovery proved that this method is suitable for the analysis of tomato volatiles. Their eugenol and guaiacol content changed, with an increase of 200 and 35 times, respectively, when foliar treatment was used, and an increase of 10, in both cases, when plants were in contact with the contaminated atmosphere. As consequence of the treatments other volatile compounds changed considerably. For first time, a HS-SBSE-GC-MS method was successfully validated for the study of volatiles in tomatos. Results suggests that exogenous compounds in contact with the plants, such as eugenol and guaiacol, can be absorbed changing the global volatile composition of fruits, which could produce a negative or positive effect in their aroma. © 2012 Society of Chemical Industry.

  1. Influence of the Chemical Structure on Odor Qualities and Odor Thresholds of Halogenated Guaiacol-Derived Odorants

    Directory of Open Access Journals (Sweden)

    Florian Juhlke

    2017-12-01

    Full Text Available Chlorinated guaiacol derivatives are found in waste water of pulp mills using chlorine in the bleaching process of wood pulp. They can also be detected in fish tissue, possibly causing off-odors. To date, there is no systematic investigation on the odor properties of halogenated guaiacol derivatives. To close this gap, odor thresholds in air and odor qualities of 14 compounds were determined by gas chromatography-olfactometry. Overall, the investigated compounds elicited smells that are characteristic for guaiacol, namely smoky, sweet, vanilla-like, but also medicinal and plaster-like. Their odor thresholds in air were, however, very low, ranging from 0.00072 to 23 ng/Lair. The lowest thresholds were found for 5-chloro- and 5-bromoguaiacol, followed by 4,5-dichloro- and 6-chloroguaiacol. Moreover, some inter-individual differences in odor threshold values could be observed, with the highest variations having been recorded for the individual values of 5-iodo- and 4-bromoguaiacol.

  2. Generation of a novel regulatory NK cell subset from peripheral blood CD34+ progenitors promoted by membrane-bound IL-15.

    Directory of Open Access Journals (Sweden)

    Massimo Giuliani

    Full Text Available BACKGROUND: NK cells have been long time considered as cytotoxic lymphocytes competent in killing virus-infected cells and tumors. However, NK cells may also play essential immuno-regulatory functions. In this context, the real existence of a defined NK subset with negative regulatory properties has been hypothesized but never clearly demonstrated. METHODOLOGY/PRINCIPAL FINDINGS: Herein, we show the in vitro generation from human peripheral blood haematopoietic progenitors (PB-HP, of a novel subset of non-cytolytic NK cells displaying a mature phenotype and remarkable immuno-regulatory functions (NK-ireg. The main functional hallmark of these NK-ireg cells is represented by the surface expression/release of HLA-G, a major immunosuppressive molecule. In addition, NK-ireg cells secrete two powerful immuno-regulatory factors: IL-10 and IL-21. Through these factors, NK-ireg cells act as effectors of the down-regulation of the immune response: reconverting mature myeloid DC (mDC into immature/tolerogenic DC, blocking cytolytic functions on conventional NK cells and inducing HLA-G membrane expression on PB-derived monocytes. The generation of "NK-ireg" cells is obtained, by default, in culture conditions favouring cell-to-cell contacts, and it is strictly dependent on reciprocal trans-presentation of membrane-bound IL-15 forms constitutively and selectively expressed by human CD34(+ PB-HP. Finally, a small subset of NKp46(+ HLA-G(+ IL-10(+ is detected within freshly isolated decidual NK cells, suggesting that these cells could represent an in vivo counterpart of the NK-ireg cells. CONCLUSIONS/SIGNIFICANCE: In conclusion, NK-ireg cells represent a novel truly differentiated non-cytolytic NK subset with a self-sustainable phenotype (CD56(+ CD16(+ NKp30(+ NKp44(+ NKp46(+ CD94(+ CD69(+ CCR7(+ generated from specific pSTAT6(+ GATA3(+ precursors. NK-ireg cells could be employed to develop new immuno-suppressive strategies in autoimmune diseases, transplant

  3. Widespread Occurrence of Expressed Fungal Secretory Peroxidases in Forest Soils

    OpenAIRE

    Kellner, Harald; Luis, Patricia; Pecyna, Marek J.; Barbi, Florian; Kapturska, Danuta; Krüger, Dirk; Zak, Donald R.; Marmeisse, Roland; Vandenbol, Micheline; Hofrichter, Martin

    2014-01-01

    Fungal secretory peroxidases mediate fundamental ecological functions in the conversion and degradation of plant biomass. Many of these enzymes have strong oxidizing activities towards aromatic compounds and are involved in the degradation of plant cell wall (lignin) and humus. They comprise three major groups: class II peroxidases (including lignin peroxidase, manganese peroxidase, versatile peroxidase and generic peroxidase), dye-decolorizing peroxidases, and heme-thiolate peroxidases (e.g....

  4. Zonal Changes in Ascorbate and Hydrogen Peroxide Contents, Peroxidase, and Ascorbate-Related Enzyme Activities in Onion Roots1

    Science.gov (United States)

    del Carmen Córdoba-Pedregosa, María; Córdoba, Francisco; Villalba, José Manuel; González-Reyes, José Antonio

    2003-01-01

    Onion (Allium cepa) roots growing hydroponically show differential zonal values for intra- (symplastic) and extra- (apoplastic) cellular ascorbate (ASC) and dehydroascorbate (DHA) contents and for related enzyme activities. In whole roots, ASC and DHA concentrations were higher in root apex and meristem and gradually decreased toward the root base. Guaiacol peroxidase, ASC peroxidase, monodehydroascorbate oxidoreductase, DHA reductase, catalase, and glutathione reductase activities showed differential activity patterns depending on the zone of the root and their apoplastic or symplastic origin. An in vivo staining of peroxidase activity also revealed a specific distribution pattern along the root axis. Using electron microscopy, hydrogen peroxide was found at different locations depending on the root zone but was mainly located in cell walls from epidermal and meristematic cells and in cells undergoing lignification. A balanced control of all of these molecules seems to exist along the root axis and may be directly related to the mechanisms in which the ASC system is involved, as cell division and elongation. The role of ASC on growth and development in relation to its presence at the different zones of the root is discussed. PMID:12586893

  5. Ultraviolet spectroscopy of fundamental lignin subunits: Guaiacol, 4-methylguaiacol, syringol, and 4-methylsyringol

    Science.gov (United States)

    Dean, Jacob C.; Navotnaya, Polina; Parobek, Alexander P.; Clayton, Rachel M.; Zwier, Timothy S.

    2013-10-01

    Ultraviolet spectroscopy of the G- and S-type lignin subunits, guaiacol (G) and syringol (S), along with their para-methylated derivatives 4-methylguaiacol (4-MG) and 4-methylsyringol (4-MS), has been carried out in the cold, isolated environment of a supersonic jet. The excitation spectra and dispersed fluorescence (DFL) spectra of G and 4-MG show strong S0-S1 origins and Franck-Condon activity involving both the ring modes typical of aromatic derivatives, and the four lowest frequency out-of-plane modes (a″) and lowest in-plane mode (a') involving the OH and OCH3 groups. The four low-frequency out-of-plane modes undergo extensive Duschinsky mixing between the ground and excited state. In 4-MG, combination bands involving methyl rotor levels with out-of-plane modes appeared with surprisingly high intensity, indicating a high degree of hindered rotor-vibration coupling in both S0 and S1. These mixing effects accompany the change in geometry upon π-π* electronic excitation going from a planar ground state to a non-planar excited state. Time-dependent density functional theory (TDDFT M05-2X/6-311++G(d,p)) calculations predict a geometric distortion along the out-of-plane oxygen flapping coordinate, yielding a double minimum potential in S1 with a barrier to planarity of 195 cm-1 in G. The excitation spectrum of S and 4-MS showed a much higher degree of spectral congestion and a larger geometry change evident by a shifted intensity distribution peaking ˜300 cm-1 above the electronic origin. TDDFT calculations predict a larger geometry change in S compared with G, with the OH and H-bonded methoxy groups displaced in opposite directions above/below the ring plane. Dispersed fluorescence from all S1 excited state levels in S/4-MS yield only broad emission peaking far to the red of the excitation wavelength (-4500 cm-1). Several hypotheses regarding the source of this broad, redshifted emission were tested, but the cause remains unclear. p-Methylation was found to

  6. The effect of heavy metals on peroxidase from Jerusalem artichoke ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-07-04

    Jul 4, 2008 ... enzyme had Km values of 0.263 and 1.143 mM for guaiacol and H2O2, respectively. The enzyme had Vmax of 33.3x105 and 0.213x105 EU/mL.min for guaiacol and H2O2, respectively. Also, the in vitro effect of some heavy metals such as iron (Fe2+and Fe3+), cobalt (Co2+), strontium (Sr2)+, zinc (Zn2+), ...

  7. Two unrelated putative membrane-bound progestin receptors, progesterone membrane receptor component 1 (PGMRC1 and membrane progestin receptor (mPR beta, are expressed in the rainbow trout oocyte and exhibit similar ovarian expression patterns

    Directory of Open Access Journals (Sweden)

    Fostier Alexis

    2006-02-01

    Full Text Available Abstract Background In lower vertebrates, steroid-induced oocyte maturation is considered to involve membrane-bound progestin receptors. Two totally distinct classes of putative membrane-bound progestin receptors have been reported in vertebrates. A first class of receptors, now termed progesterone membrane receptor component (PGMRC; subtypes 1 and 2 has been studied since 1996 but never studied in a fish species nor in the oocyte of any animal species. A second class of receptors, termed membrane progestin receptors (mPR; subtypes alpha, beta and gamma, was recently described in vertebrates and implicated in the progestin-initiated induction of oocyte maturation in fish. Methods In the present study, we report the characterization of the full coding sequence of rainbow trout PGMRC1 and mPR beta cDNAs, their tissue distribution, their ovarian expression profiles during oogenesis, their hormonal regulation in the full grown ovary and the in situ localization of PGMRC1 mRNA in the ovary. Results Our results clearly show, for the first time in any animal species, that rainbow trout PGMRC1 mRNA is present in the oocyte and has a strong expression in ovarian tissue. In addition, we show that both mPR beta and PGMRC1, two members of distinct membrane-bound progestin receptor classes, exhibit highly similar ovarian expression profiles during the reproductive cycle with maximum levels during vitellogenesis and a down-expression during late vitellogenesis. In addition, the mRNA abundance of both genes is not increased after in vitro hormonal stimulation of full grown follicles by maturation inducing hormones. Conclusion Together, our findings suggest that PGMRC1 is a new possible participant in the progestin-induced oocyte maturation in fish. However, its participation in the process of oocyte maturation, which remains to be confirmed, would occur at post-transcriptional levels.

  8. Site-specific incorporation of 5-fluorotryptophan as a probe of the structure and function of the membrane-bound D-lactate dehydrogenase of Escherichia coli: A 19F nuclear magnetic resonance study

    International Nuclear Information System (INIS)

    Peersen, O.B.; Pratt, E.A.; Truong, H.T. N.; Ho, C.; Rule, G.S.

    1990-01-01

    The structure and function of the membrane-bound D-lactate dehydrogenase of Escherichia coli have been investigated by fluorine-19 nuclear magnetic resonance spectroscopy of 5-fluorotryptophan-labeled enzyme in conjunction with oligonucleotide-directed, site-specific mutagenesis. 5-Fluorotryptophan has been substituted for nine phenylalanine, tyrosine, and leucine residues in the enzyme molecule without loss of activity. The 19 F signals from these additional tryptophan residues have been used as markers for sensitivity to substrate, exposure to aqueous solvent, and proximity to a lipid-bound spin-label. The nuclear magnetic resonance data show that two mutational sites, at amino acid residues 340 and 361, are near the lipid environment used to stabilize the enzyme. There are a number of amino acid residues on the carboxyl side of this region that are strongly sensitive to the aqueous solvent. The environment of the wide-type tryptophan residue at position 469 changes as a result of two of the substitution mutations, suggesting some amino acid residue-residue interactions. Secondary structure prediction methods indicate a possible binding site for the flavin adenine dinucleotide cofactor in the carboxyl end of the enzyme molecule. These results suggest that the membrane-bound D-lactate dehydrogenase may have the two-domain structure of many cytoplasmic dehydrogenases but with the addition of a membrane-binding domain between the catalytic and cofactor-binding domains. This type of three-domain structure may be of general significance for understanding the structure of membrane-bound proteins which do not traverse the lipid bilayer of membranes

  9. Chromatographic separation of human salivary peroxidases.

    Science.gov (United States)

    Mäkinen, K K; Tenovuo, J

    1976-01-01

    A series of rapid and simple chromatographic purification procedures for peroxidase-like enzymes occurring in the human oral cavity is presented. Samples of whole saliva, parotid saliva, gingival exudate and various bacterial preparations contain peroxidases which were purified using molecular exclusion and ion exchange chromatography, and isoelectric focusing. Salivary lactoperoxidase can be easily separated from bacterial and leucocyte peroxidase activity by the methods presented.

  10. Morphology–dependent electrochemical sensing properties of manganese dioxide–graphene oxide hybrid for guaiacol and vanillin

    International Nuclear Information System (INIS)

    Gan, Tian; Shi, Zhaoxia; Deng, Yaping; Sun, Junyong; Wang, Haibo

    2014-01-01

    Highlights: • MnO 2 with different morphologies were prepared via facile methods. • MnO 2 are loaded on GO via simply grinding which have high solubility and stability. • MnO 2 –GO exhibit high electrocatalytic activities depending on their shapes. • MnO 2 –GO is first used to the determination of guaiacol and vanillin simultaneously. - Abstract: Various morphologies of manganese dioxide (MnO 2 ) electrocatalysts, including nanoflowers, nanorods, nanotubes, nanoplates, nanowires and microspheres were prepared via facile hydrothermal synthesis and precipitation methods. By simply grinding with graphene oxide (GO), MnO 2 could be readily dissolved in water with high solubility and stability. The structures and electrochemical performances of these as–prepared MnO 2 –GO hybrids were fully characterized by various techniques, and the properties were found to be strongly dependent on morphology. As sensing materials for the simultaneous determination of guaiacol and vanillin for the first time, the nanoflowers–like MnO 2 , coupled with GO, exhibited relatively high sensitivity. The enhanced electrocatalytic activity was ascribed to the high purity, good crystallinity, and unique porous microstructure, which were favorable for transfer of electrons. These results may provide valuable insights for the development of nanostructured modified electrodes for next–generation high–performance electrochemical sensors

  11. The aryl ether bond reactions with H-donor solvents: guaiacol and tetralin in the presence of catalysts

    Energy Technology Data Exchange (ETDEWEB)

    Afifi, A.I.; Thring, R.W.; Overend, R.P. [Universite de Sherbrooke, Sherbrooke, PQ (Canada). Dept. de Genie Chimique

    1996-03-01

    The effect of homogenous catalysis by Fe and Ru, on the conversion of guaiacol in tetralin to catechol and phenol has been investigated as a model for the behaviour of the aryl-oxy linkage that is found in wood, peat and younger coals. In the absence of catalyst and at low ratios of guaiacol to tetralin, the primary product is catechol. Kinetic analysis has confirmed that the rate constant for this primary and rate determining step is given by an Arrhenius pre-exponential factor of 10{sup 13.8} s{sup -1} with an activation energy of 215 kJ mol{sup -1}. The activation energy found is in good agreement with those of other investigators and lies between the values proposed for homolytic fission ({gt} 240 kJ mol{sup -1}) and for a concerted or pericyclic reaction (188 kJ mol{sup -1}). In the presence of catalysts the rate is not changed; however, the yield of a secondary product phenol is increased with both Fe and Ru. Separate experiments confirmed that the selectivity of catechol to phenol conversion was markedly increased in the presence of these catalysts. There is strong evidence for the formation of catecholato-iron complexes and this suggests that in pyrolysis and liquefaction of biomass and young coals there may well be a role of homogeneous catalysts in directing the product slate towards useful intermediate chemicals such as phenols. 14 refs., 9 figs., 4 tabs.

  12. Continuous Catalytic Hydrodeoxygenation of Guaiacol over Pt/SiO2 and Pt/H-MFI-90

    Directory of Open Access Journals (Sweden)

    Melanie Hellinger

    2015-07-01

    Full Text Available Hydrodeoxygenation of guaiacol in the presence of 1-octanol was studied in a fixed-bed reactor under mild conditions (50–250 °C over platinum particles supported on silica (Pt/SiO2 and a zeolite with framework type MFI at a Si/Al-ratio of 45 (Pt/H-MFI-90. The deoxygenation selectivity strongly depended on the support and the temperature. Both guaiacol and octanol were rapidly deoxygenated in the presence of hydrogen over Pt/H-MFI-90 at 250 °C to cyclohexane and octane, respectively. In contrast, Pt/SiO2 mostly showed hydrogenation, but hardly any deoxygenation activity. The acidic sites of the MFI-90 support lead to improved deoxygenation performance at the mild temperature conditions of this study. Significant conversions under reaction conditions applied already occurred at temperatures of 200 °C. However, during long-term stability tests, the Pt/H-MFI-90 catalyst deactivated after more than 30 h, probably due to carbon deposition, whereas Pt/SiO2 was more stable. The catalytic activity of the zeolite catalyst could only partly be regained by calcination in air, as some of the acidic sites were lost.

  13. Unraveling Pathways of Guaiacol Nitration in Atmospheric Waters: Nitrite, A Source of Reactive Nitronium Ion in the Atmosphere.

    Science.gov (United States)

    Kroflič, Ana; Grilc, Miha; Grgić, Irena

    2015-08-04

    The tropospheric aqueous-phase aging of guaiacol (2-methoxyphenol, GUA), a lignocellulosic biomass burning pollutant, is addressed in this work. Pathways of GUA nitration in aqueous solution under atmospherically relevant conditions are proposed and critically discussed. The influence of NaNO2 and H2O2, hydroxyl radical scavenger, and sunlight was assessed by an experimental-modeling approach. In the presence of the urban pollutant, nitrite, GUA is preferentially nitrated to yield 4- and 6-nitroguaiacol. After a short lag-time, 4,6-dinitroguaiacol is also formed. Its production accelerates after guaiacol is completely consumed, which is nicely described by the model function accounting for NO2(•) and NO2(+) as nitrating agents. Although the estimated second-order kinetic rate constants of methoxyphenol nitration with NO2(•) are substantially higher than the corresponding rate constants of nitration with NO2(+), nitration rates are competitive under nighttime and liquid atmospheric aerosol-like conditions. In contrast to concentrations of radicals, which are governed by the interplay between diffusion-controlled reactions and are therefore mostly constant, concentrations of electrophiles are very much dependent on the ratio of NO2(-) to activated aromatics in solution. These results contribute substantially to the understanding of methoxyphenol aging in the atmospheric waters and underscore the importance of including electrophilic aromatic substitution reactions in atmospheric models.

  14. Modulatory Effect of Taurine on 7,12-Dimethylbenz(a)Anthracene-Induced Alterations in Detoxification Enzyme System, Membrane Bound Enzymes, Glycoprotein Profile and Proliferative Cell Nuclear Antigen in Rat Breast Tissue.

    Science.gov (United States)

    Vanitha, Manickam Kalappan; Baskaran, Kuppusamy; Periyasamy, Kuppusamy; Selvaraj, Sundaramoorthy; Ilakkia, Aruldoss; Saravanan, Dhiravidamani; Venkateswari, Ramachandran; Revathi Mani, Balasundaram; Anandakumar, Pandi; Sakthisekaran, Dhanapal

    2016-08-01

    The modulatory effect of taurine on 7,12-dimethylbenz(a)anthracene (DMBA)-induced breast cancer in rats was studied. DMBA (25 mg/kg body weight) was administered to induce breast cancer in rats. Protein carbonyl levels, activities of membrane bound enzymes (Na(+) /K(+) ATPase, Ca(2+) ATPase, and Mg(2+) ATPase), phase I drug metabolizing enzymes (cytochrome P450, cytochrome b5, NADPH cytochrome c reductase), phase II drug metabolizing enzymes (glutathione-S-transferase and UDP-glucuronyl transferase), glycoprotein levels, and proliferative cell nuclear antigen (PCNA) were studied. DMBA-induced breast tumor bearing rats showed abnormal alterations in the levels of protein carbonyls, activities of membrane bound enzymes, drug metabolizing enzymes, glycoprotein levels, and PCNA protein expression levels. Taurine treatment (100 mg/kg body weight) appreciably counteracted all the above changes induced by DMBA. Histological examination of breast tissue further supported our biochemical findings. The results of the present study clearly demonstrated the chemotherapeutic effect of taurine in DMBA-induced breast cancer. © 2016 Wiley Periodicals, Inc.

  15. Role of fungal peroxidases in biological ligninolysis

    Science.gov (United States)

    Kenneth E. Hammel; Dan Cullen

    2008-01-01

    The degradation of lignin by filamentous fungi is a major route for the recycling of photosynthetically fixed carbon, and the oxidative mechanisms employed have potential biotechnological applications. The lignin peroxidases (LiPs), manganese peroxidases (MnPs), and closely related enzymes of white rot basidiomycetes are likely contributors to fungal ligninolysis. Many...

  16. Vapor-Phase Hydrodeoxygenation of Guaiacol to Aromatics over Pt/HBeta: Identification of the Role of Acid Sites and Metal Sites on the Reaction Pathway

    Energy Technology Data Exchange (ETDEWEB)

    Nie, Lei [Collaborative Innovation Center of Chemical Science and Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072 P.R. China; Institute for Integrated Catalysis, Pacific Northwest National Laboratory, P.O. Box 999 Richland WA 99352 USA; Peng, Bo [Institute for Integrated Catalysis, Pacific Northwest National Laboratory, P.O. Box 999 Richland WA 99352 USA; Zhu, Xinli [Collaborative Innovation Center of Chemical Science and Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072 P.R. China

    2018-02-05

    Hydrodeoxygenation of guaiacol, a phenolic compound derived from lignin fraction of biomass, over a Pt/HBeta catalyst at 350 °C and atmospheric pressure produces benzene, toluene, xylenes, and C9+ aromatics with yield of 42%, 29%, 12%, and 5%, respectively. Reaction pathways for conversion of two functional groups (hydroxyl and methoxyl) over the bifunctional catalyst were studied. Both guaiacol and intermediate products (catechol and cyclopentanone) were fed onto zeolite HBeta and Pt/SiO2 to identify the individual role of acid site and metal site. Acid sites (mainly Brønsted acid site, BAS) catalyze transalkylation and dehydroxylation reactions in sequence, producing phenol, cresols and xylenols as the major products at high conversion. Pt sites catalyze demethylation reaction resulting in catechol as the primary product, which can either be deoxygenated to phenol followed by phenol to benzene, or decarbonylated to cyclopentanone and further to butane. The close proximity of Pt and BAS in bifunctional Pt/HBeta enables both transalkylation and deoxygenation reactions with inhibited demethylation and decarbonylation reactions, producing aromatics as major final products with a total yield > 85%. Both activity and stability of bifunctional Pt/HBeta during hydrodeoxygenation of guaiacol is improved compared to HBeta and Pt/SiO2. The addition of water to the feed further improves the activity and stability via hydrolysis of O-CH3 bond of guaiacol on BAS and removing coke around Pt.

  17. Glycosylation and thermodynamic versus kinetic stability of horseradish peroxidase

    DEFF Research Database (Denmark)

    Tams, J.W.; Welinder, Karen G.

    1998-01-01

    Glycoprotein stability, glycoprotein unfolding, horseradish peroxidase, thermodynamic stability, kinetik stability......Glycoprotein stability, glycoprotein unfolding, horseradish peroxidase, thermodynamic stability, kinetik stability...

  18. Purification and characterization of cell suspensions peroxidase from cotton (Gossypium hirsutum L.).

    Science.gov (United States)

    Kouakou, Tanoh Hilaire; Dué, Edmond Ahipo; Kouadio, N'guessan Eugène Jean Parfait; Niamké, Sébastien; Kouadio, Yatty Justin; Mérillon, Jean-Michel

    2009-06-01

    Two peroxidases, cPOD-I and rPOD-II, have been isolated and purified from cotton cell suspension and their biochemical characteristics studied. rPOD-II from R405-2000, a non-embryogenic cultivar, has higher activity than cPOD-I derived from Coker 312, which developed an embryogenic structure. The cPOD-I and rPOD-II had molecular mass of 39.1 and 64 kDa respectively, as determined by SDS-PAGE. Both enzymes showed high efficiency of interaction with the guaiacol at 25 mM. The optimal pH for cPOD-I and rPOD-II activity was 5.0 and 6.0, respectively. The enzyme had an optimum temperature of 25 degrees C and was relatively stable at 20-30 degrees C. The isoenzymes were highly inhibited by ascorbic acid, dithiothreitol, sodium metabisulfite, and beta-mercaptoethanol. Their activities were highly enhanced by Al(3+), Fe(3+), Ca(2+), and Ni(2+), but they were moderately inhibited by Mn(2+) and K(+). The enzyme lost 50% to 62% of its activity in the presence of Zn(2+) and Hg(2+).

  19. Purification and characterization of a wound-inducible cell wall cationic peroxidase from carrot roots.

    Science.gov (United States)

    Nair, A R; Showalter, A M

    1996-09-04

    We have isolated a novel cell wall, cationic peroxidase (pI > 9.3) from roots of the carrot plant, Daucus carota. The purified isozyme, referred to as CP > 9.3, has a molecular mass of 45 kilodaltons and an Reinheitzahl value of 2.3. Amino-acid composition analysis and N-terminal sequencing have been performed with CP > 9.3. The N-terminal sequence shows no homology to any sequence in the protein and nucleic acid data banks. CP > 9.3 activity is induced by wounding in carrot leaves and petioles; this activity is also present in carrot roots but is unaltered by wounding. Enhanced CP > 9.3 activity is seen at 12 hr post-wounding and continues for at least 60 hr in leaves and petioles. Based on studies using cycloheximide, early activation of CP > 9.3 is not due to de novo protein synthesis, but rather to enzyme activation. Temperature and pH optima for CP > 9.3 using guaiacol as a substrate have been determined to be 32 degrees C and 4.9.

  20. Functional Analysis of Thyroid Peroxidase Gene Mutations Detected in Patients with Thyroid Dyshormonogenesis

    Directory of Open Access Journals (Sweden)

    Srikanta Guria

    2014-01-01

    Full Text Available Thyroid peroxidase (TPO is the key enzyme in the biosynthesis of thyroid hormones. We aimed to identify the spectrum of mutations in the TPO gene leading to hypothyroidism in the population of West Bengal to establish the genetic etiology of the disease. 200 hypothyroid patients (case and their corresponding sex and age matched 200 normal individuals (control were screened depending on their clinical manifestations. Genomic DNA was isolated from peripheral blood samples and TPO gene (Exon 7 to Exon 14 was amplified by PCR. The PCR products were subjected to sequencing to identify mutations. Single nucleotide changes such as Glu 641 Lys, Asp 668 Asn, Thr 725 Pro, Asp 620 Asn, Ser 398 Thr, and Ala 373 Ser were found. Changes in the TPO were assayed in vitro to compare mutant and wild-type activities. Five mutants were enzymatically inactive in the guaiacol and iodide assays. This is a strong indication that the mutations are present at crucial positions of the TPO gene, resulting in inactivated TPO. The results of this study may help to develop a genetic screening protocol for goiter and hypothyroidism in the population of West Bengal.

  1. Assessment of different fixation protocols on the presence of membrane-bound vesicles in Caco-2 cells: a multidimensional view by means of correlative light and 3-D transmission electron microscopy.

    Science.gov (United States)

    Shami, Gerald; Cheng, Delfine; Henriquez, Jeffrey; Braet, Filip

    2014-12-01

    Herein, we present a comparative analysis of a variety of chemical and physical fixation protocols for the specific visualisation of the membrane-bound vesicles (MBVs) in the Caco-2 colorectal cancer (CRC) cell line. In so doing, we validated the applicability of specific specimen preparation protocols for the preservation and contrasting of membrane-associated vesicles. Next, by employing the best respective chemical (GOT) and physical (SHPF) fixation methods for the application of transmission electron tomography and modelling we were able to characterise MBVs in three-dimensions and at the nanometer scale. In the second part of this study, we employ a correlative light and electron microscopy (CLEM) approach in order to determine which vesicular compartments are implicated in the uptake of FITC-BSA as a model protein drug. In so doing, we provide a solid foundation for future studies investigating chemotherapeutic drug uptake, transport and fate in cancer cell lines. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. A microassay for the determination of soluble and membrane-bound glutamate decarboxylase activity--influences of cations, lipid composition, and pyridoxal 5'-phosphate on the glutamate decarboxylase binding to liposomes

    International Nuclear Information System (INIS)

    Hagel, C.; Fleissner, A.; Seifert, R.

    1989-01-01

    A radiochemical microassay for soluble and membrane-bound glutamate decarboxylase (GAD) is described. Up to 180 samples can be determined per day with a variation coefficient of 2%. The method detects newly synthesized gamma-amino-n-butyric acid in the picomole range and can easily be applied to other enzymes whose substrate and product differ by charge. In an aqueous homogenate of brain (1 + 10; w/v) about 15% of the total GAD activity are spun down by centrifugation (1 h, 100,000g) increasing to 35% of the total GAD activity in solutions with 8 mM calcium chloride or 100 mM potassium acetate. There is similar dependence on the cation concentration when GAD binds to phospholipid vesicles (liposomes) as well as dependence on lipid concentration and lipid composition. The coenzyme pyridoxal 5'-phosphate has no influence on GAD binding to liposomes

  3. Genetics Home Reference: eosinophil peroxidase deficiency

    Science.gov (United States)

    ... navigation Home Page Search Home Health Conditions Genes Chromosomes & mtDNA Resources Help Me Understand Genetics Share: Email ... EPXD peroxidase and phospholipid deficiency in eosinophils Presentey anomaly Related Information How are genetic conditions and genes ...

  4. ACTIVE PRODUCERS OF PEROXIDASE BASIDIOMYCETES STRAINS SCREENING

    Directory of Open Access Journals (Sweden)

    T. E. Voloshko

    2013-10-01

    Full Text Available The paper is devoted to the analysis of the research data peroxidase activity of the strains of xylotrophic basidiomycetes in the dynamics of the growth. The objects of study were 57 strains, 5 of which belongs to 5 species of the order Polyporales, and 52 of which belongs to 7 species of the order Agaricales. In order to search for active producers of peroxidase the strains were cultured by the surface method in a liquid glucosepeptone medium. The accumulation of oven-dry biomass was determined by the weight method. The content of soluble protein and peroxidase activity were determined by the spectrophotometry. The studies set the level of accumulation of oven-dry biomass and peroxidase activity of the strains in 9 and 12 days of growth. The results allowed selecting the strains, which are characterized by high levels of peroxidase activity in mycelium and in the culture filtrate, including Agrocybe cylindracea 167, Pleurotus ostreatus Р-кл, Agrocybe cylindracea 960 and 218. These strains which are active producers of peroxidase may be used in the enzyme preparations obtaining technology.

  5. Purification, characterization and stability of barley grain peroxidase BP1, a new type of plant peroxidase

    DEFF Research Database (Denmark)

    Rasmussen, Christine B; Henriksen, Anette; Abelskov, A. Katrine

    1997-01-01

    The major peroxidase of barley grain (BP 1) has enzymatic and spectroscopic properties that are very differeant from those of other known plant peroxidases (EC 1.11.1.7) and can therefore contribute to the understanding of the many physiological functions ascribed to these enzymes. To study...

  6. HDO of guaiacol over NiMo catalyst supported on activated carbon derived from castor de-oiled cake

    Directory of Open Access Journals (Sweden)

    Viviana Ospina

    2015-05-01

    Full Text Available Physical and chemical activation methods were used to prepare two different activated carbons (ACs from castor de-oiled cake. H2O/CO2 mixture was used as the physical activating agent, and for chemical activation potassium carbonate (K2CO3 was used. For both materials, textural and chemical properties were characterized by N2 adsorption–desorption isotherms, thermogravimetric analysis (TGA, Fourier Transform Infrared Spectroscopy (FTIR, thermal programmed reduction (TPR, X-ray fluorescence (XRF, and scanning electron microscopy (SEM. The ACs were used as supports for NiMo sulfide catalysts, which were prepared by wetness impregnation and in-situ sulfided for the hydrodeoxygenation (HDO of guaiacol (GUA as a model compound of bio-oil. The HDO reaction was carried out in a typical batch reactor at 5 MPa of H2 and 350 °C. Under the same test conditions, commercial catalysts were also tested in the reaction. Although the commercial catalysts displayed higher GUA conversion, the prepared catalysts showed higher activity and non-oxygenated and saturated products yield.

  7. Optimization of laccase and manganese peroxidase production in ...

    African Journals Online (AJOL)

    The effect of temperature, pH, carbon, nitrogen, Cu2+, 2,5- xylidine, ferulic acid, Mn2+ and immobilization using Luffa cylindrica sponges in submerged culture fermentations were investigated for maximum enzymes production. After 7 days of incubation, 83 to 100% oxidation of RBBR, ABTS and guaiacol was observed.

  8. Occurrence and properties of Petunia peroxidase a

    NARCIS (Netherlands)

    Hendriks, T.

    1989-01-01

    Peroxidases are probably the most extensively studied enzymes in higher plants. Various isoenzymes occur as soluble proteins in the apoplast and in the vacuole, or are bound to membranes and cell walls. Their occurrence is often organ-specific and developmentally controlled, and there is

  9. Ficus sycomorus latex: A thermostable peroxidase

    African Journals Online (AJOL)

    ajl yemi

    2011-11-30

    Nov 30, 2011 ... Peroxidase from sycamore fig Ficus sycomorus latex (POLI) was purified by heat treatment, anion exchange chromatography and molecular exclusion chromatography. The purity was determined from high specific activity (9166 units/mg protein), purification fold (28), RZ value 3.1 and a single band in.

  10. Inhibition of Heme Peroxidases by Melamine

    Directory of Open Access Journals (Sweden)

    Pattaraporn Vanachayangkul

    2012-01-01

    Full Text Available In 2008 melamine-contaminated infant formula and dairy products in China led to over 50,000 hospitalizations of children due to renal injuries. In North America during 2007 and in Asia during 2004, melamine-contaminated pet food products resulted in numerous pet deaths due to renal failure. Animal studies have confirmed the potent renal toxicity of melamine combined with cyanuric acid. We showed previously that the solubility of melamine cyanurate is low at physiologic pH and ionic strength, provoking us to speculate how toxic levels of these compounds could be transported through the circulation without crystallizing until passing into the renal filtrate. We hypothesized that melamine might be sequestered by heme proteins, which could interfere with heme enzyme activity. Four heme peroxidase enzymes were selected for study: horseradish peroxidase (HRP, lactoperoxidase (LPO, and cyclooxygenase-1 and -2 (COX-1 and -2. Melamine exhibited noncompetitive inhibition of HRP (9.5±0.7mM, and LPO showed a mixed model of inhibition (14.5±4.7mM. The inhibition of HRP and LPO was confirmed using a chemiluminescent peroxidase assay. Melamine also exhibited COX-1 inhibition, but inhibition of COX-2 was not detected. Thus, our results demonstrate that melamine inhibits the activity of three heme peroxidases.

  11. Peroxidase-like activity of magnetoferritin

    Czech Academy of Sciences Publication Activity Database

    Melníková, V.; Pospíšková, K.; Mitróová, Z.; Kopčanský, P.; Šafařík, Ivo

    2014-01-01

    Roč. 181, 3-4 (2014), s. 295-301 ISSN 0026-3672 R&D Projects: GA MŠk(CZ) LD13021 Institutional support: RVO:67179843 Keywords : magnetoferritin * magnetic nanoparticles * peroxidase-like activity * hydrogen peroxide * oxidative stress Subject RIV: CE - Biochemistry Impact factor: 3.741, year: 2014

  12. Thyroid peroxidase autoantibodies in euthyroid subjects

    NARCIS (Netherlands)

    Prummel, Mark F.; Wiersinga, Wilmar M.

    2005-01-01

    Thyroid peroxidase (TPO) is a key enzyme in the formation of thyroid hormones and a major autoantigen in autoimmune thyroid diseases. Titers of TPO antibodies also correlate with the degree of lymphocytic infiltration in euthyroid subjects, and they are frequently present in euthyroid subjects

  13. Ficus sycomorus latex: A thermostable peroxidase | Mohamed ...

    African Journals Online (AJOL)

    Peroxidase from sycamore fig Ficus sycomorus latex (POLI) was purified by heat treatment, anion exchange chromatography and molecular exclusion chromatography. The purity was determined from high specific activity (9166 units/mg protein), purification fold (28), RZ value 3.1 and a single band in native polyacrylamide ...

  14. Partial purification and characterization of ascorbate peroxidase ...

    African Journals Online (AJOL)

    Ascorbate peroxidase (EC 1.11.1.11; APX) was purified from ripe ber (Ziziphus mauritiana L.) fruits var. Illaichi using conventional techniques of ammonium sulphate fractionation, gel filtration through Sephadex G-100 and ion-exchange chromatography on DEAE-cellulose. The enzyme was purified about 47.4 fold with ...

  15. Widespread occurrence of expressed fungal secretory peroxidases in forest soils.

    Science.gov (United States)

    Kellner, Harald; Luis, Patricia; Pecyna, Marek J; Barbi, Florian; Kapturska, Danuta; Krüger, Dirk; Zak, Donald R; Marmeisse, Roland; Vandenbol, Micheline; Hofrichter, Martin

    2014-01-01

    Fungal secretory peroxidases mediate fundamental ecological functions in the conversion and degradation of plant biomass. Many of these enzymes have strong oxidizing activities towards aromatic compounds and are involved in the degradation of plant cell wall (lignin) and humus. They comprise three major groups: class II peroxidases (including lignin peroxidase, manganese peroxidase, versatile peroxidase and generic peroxidase), dye-decolorizing peroxidases, and heme-thiolate peroxidases (e.g. unspecific/aromatic peroxygenase, chloroperoxidase). Here, we have repeatedly observed a widespread expression of all major peroxidase groups in leaf and needle litter across a range of forest ecosystems (e.g. Fagus, Picea, Acer, Quercus, and Populus spp.), which are widespread in Europe and North America. Manganese peroxidases and unspecific peroxygenases were found expressed in all nine investigated forest sites, and dye-decolorizing peroxidases were observed in five of the nine sites, thereby indicating biological significance of these enzymes for fungal physiology and ecosystem processes. Transcripts of selected secretory peroxidase genes were also analyzed in pure cultures of several litter-decomposing species and other fungi. Using this information, we were able to match, in environmental litter samples, two manganese peroxidase sequences to Mycena galopus and Mycena epipterygia and one unspecific peroxygenase transcript to Mycena galopus, suggesting an important role of this litter- and coarse woody debris-dwelling genus in the disintegration and transformation of litter aromatics and organic matter formation.

  16. Widespread occurrence of expressed fungal secretory peroxidases in forest soils.

    Directory of Open Access Journals (Sweden)

    Harald Kellner

    Full Text Available Fungal secretory peroxidases mediate fundamental ecological functions in the conversion and degradation of plant biomass. Many of these enzymes have strong oxidizing activities towards aromatic compounds and are involved in the degradation of plant cell wall (lignin and humus. They comprise three major groups: class II peroxidases (including lignin peroxidase, manganese peroxidase, versatile peroxidase and generic peroxidase, dye-decolorizing peroxidases, and heme-thiolate peroxidases (e.g. unspecific/aromatic peroxygenase, chloroperoxidase. Here, we have repeatedly observed a widespread expression of all major peroxidase groups in leaf and needle litter across a range of forest ecosystems (e.g. Fagus, Picea, Acer, Quercus, and Populus spp., which are widespread in Europe and North America. Manganese peroxidases and unspecific peroxygenases were found expressed in all nine investigated forest sites, and dye-decolorizing peroxidases were observed in five of the nine sites, thereby indicating biological significance of these enzymes for fungal physiology and ecosystem processes. Transcripts of selected secretory peroxidase genes were also analyzed in pure cultures of several litter-decomposing species and other fungi. Using this information, we were able to match, in environmental litter samples, two manganese peroxidase sequences to Mycena galopus and Mycena epipterygia and one unspecific peroxygenase transcript to Mycena galopus, suggesting an important role of this litter- and coarse woody debris-dwelling genus in the disintegration and transformation of litter aromatics and organic matter formation.

  17. Purification, characterization and stability of barley grain peroxidase BP1, a new type of plant peroxidase

    DEFF Research Database (Denmark)

    Rasmussen, Christine B; Henriksen, Anette; Abelskov, A. Katrine

    1997-01-01

    -protoporphyrin IX, which is characteristic of plant peroxidases. BP 1 is stable from pH 3 to 11, indicating that its unusual spectral characteristics do not result from enzyme instability. The thermostability is also normal with a melting temperature of 75 degrees C at pH 6.6, and 67 degrees C at pH 4.0 and 8......The major peroxidase of barley grain (BP 1) has enzymatic and spectroscopic properties that are very differeant from those of other known plant peroxidases (EC 1.11.1.7) and can therefore contribute to the understanding of the many physiological functions ascribed to these enzymes. To study.......3. It is clear that the unusual properties of BP 1 are genuine, and reflect a novel regulation of plant peroxidase function....

  18. The study of ascorbate peroxidase, catalase and peroxidase during in vitro regeneration of Argyrolobium roseum.

    Science.gov (United States)

    Habib, Darima; Chaudhary, Muhammad Fayyaz; Zia, Muhammad

    2014-01-01

    Here, we demonstrate the micropropagation protocol of Argyrolobium roseum (Camb.), an endangered herb exhibiting anti-diabetic and immune-suppressant properties, and antioxidant enzymes pattern is evaluated. Maximum callogenic response (60 %) was observed from leaf explant at 1.0 mg L(-1) 1-nephthalene acetic acid (NAA) and 0.5 mg L(-1) 6-benzyl aminopurine (BA) in Murashige and Skoog (MS) medium using hypocotyl and root explants (48 % each). Addition of AgNO3 and PVP in the culture medium led to an increase in callogenic response up to 86 % from leaf explant and 72 % from hypocotyl and root explants. The best shooting response was observed in the presence of NAA, while maximum shoot length and number of shoots were achieved based on BA-supplemented MS medium. The regenerated shoots were rooted and successfully acclimatized under greenhouse conditions. Catalase and peroxidase enzymes showed ascending pattern during in vitro plant development from seed while ascorbate peroxidase showed descending pattern. Totally reverse response of these enzymes was observed during callus induction from three different explants. During shoot induction, catalase and peroxidase increased at high rate while there was a mild reduction in ascorbate peroxidase activity. Catalase and peroxidase continuously increased; on the other hand, ascorbate peroxidase activity decreased during root development and acclimatization states. The protocol described here can be employed for the mass propagation and genetic transformation of this rare herb. This study also highlights the importance and role of ascorbate peroxidase, catalase, and peroxidase in the establishment of A. roseum in vitro culture through callogenesis and organogenesis.

  19. Peroxidase isozyme profiles in some sweet cherry rootstocks and ...

    African Journals Online (AJOL)

    PERS

    2012-01-10

    , 2005). Santamour (1980) defined role of peroxidase in graft compatibility as; 1) lignification is essential for a strong and permanent graft union; 2) peroxidase isoenzymes mediate the polymeri- zation of cinnamic alcohols to ...

  20. 21 CFR 864.7675 - Leukocyte peroxidase test.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Leukocyte peroxidase test. 864.7675 Section 864...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7675 Leukocyte peroxidase test. (a) Identification. A leukocyte peroxidase test is a device used to distinguish certain...

  1. Comparative study of peroxidase purification from apple and orange ...

    African Journals Online (AJOL)

    This paper reports the isolation and purification of peroxidase from low cost material; moreover, no significant work has been done on the isolation and purification of peroxidase from such cost effective sources (apple and orange seeds). Peroxidases had attracted considerable interest in recent years because of their ...

  2. Effect of Vitamin C on Glutathione Peroxidase Activities in Pregnant ...

    African Journals Online (AJOL)

    Glutathione peroxidase is one of the most important antioxidant enzymes in humans. We studied the relationship between serum glutathione peroxidase activity and vitamin C ingestion during normal pregnancy in women attending antenatal clinic in the University of Ilorin Teaching Hospital, Ilorin. Glutathione peroxidase ...

  3. Characterization of plasma membrane bound inorganic ...

    African Journals Online (AJOL)

    ... N-ethylmaliemide (NEM), phenylarsineoxide, ABC superfamily transport modulator verapamil and was also by F1Fo-ATPase inhibitor quercetin. Conclusion: We conclude that there are significant differences within promastigote, amastigote and mammalian host in cytosolic pH homeostasis to merit the inclusion of PPase ...

  4. A membrane-bound vertebrate globin.

    Directory of Open Access Journals (Sweden)

    Miriam Blank

    Full Text Available The family of vertebrate globins includes hemoglobin, myoglobin, and other O(2-binding proteins of yet unclear functions. Among these, globin X is restricted to fish and amphibians. Zebrafish (Danio rerio globin X is expressed at low levels in neurons of the central nervous system and appears to be associated with the sensory system. The protein harbors a unique N-terminal extension with putative N-myristoylation and S-palmitoylation sites, suggesting membrane-association. Intracellular localization and transport of globin X was studied in 3T3 cells employing green fluorescence protein fusion constructs. Both myristoylation and palmitoylation sites are required for correct targeting and membrane localization of globin X. To the best of our knowledge, this is the first time that a vertebrate globin has been identified as component of the cell membrane. Globin X has a hexacoordinate binding scheme and displays cooperative O(2 binding with a variable affinity (P(50∼1.3-12.5 torr, depending on buffer conditions. A respiratory function of globin X is unlikely, but analogous to some prokaryotic membrane-globins it may either protect the lipids in cell membrane from oxidation or may act as a redox-sensing or signaling protein.

  5. Metals content of Glossoscolex paulistus extracellular hemoglobin: Its peroxidase activity and the importance of these ions in the protein stability.

    Science.gov (United States)

    Caruso, Celia S; Biazin, Ezer; Carvalho, Francisco A O; Tabak, Marcel; Bachega, José F R

    2016-08-01

    In this work we investigate the presence of divalent cations bound to the Glossoscolex paulistus (HbGp) hemoglobin and their effect over the protein stability and the peroxidase (POD) activity. Atomic absorption studies show that the HbGp iron content is consistent with the presence of 144 ions per protein. Moreover, using iron as a reference, the content of calcium was estimated as 30±4 ions per protein, independently of the EDTA pre-treatment or not prior to the acidic treatment performed in the protein digestion. The zinc content was 14±2 ions in the absence of EDTA pre-treatment, and 3±1 ions per protein in the presence of EDTA pre-treatment, implying the presence of one zinc ion per protomer (1/12 of the whole molecule). Finally, the copper concentration is negligible. Different from the vertebrate hemoglobins, where the effectors are usually organic anions, the hexagonal bilayer hemoglobins have as effectors inorganic cations that increase the oxygen affinity and stabilize the structure. Previous studies have suggested that the presence of divalent cations, such as copper and zinc, is related to the different types of antioxidant enzymatic activities as the superoxide dismutase (SOD) activity shown by giant hemoglobin from Lumbricus terrestris (HbLt). Recently, studies on HbGp crystal structure have confirmed the presence of Zn(2+) and Ca(2+) binding sites. The Ca(2+) sites are similar as observed in the HbLt crystal structure. Otherwise, the Zn(2+) sites have no relation with those observed in Cu/Zn SODs. Our peroxidase assays with guaiacol confirm the POD activity and the effect of the zinc ions for HbGp. Our present results on HbGp metal content and their stability effects is the first step to understand the role of these cations in HbGp function in the future. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. NADH peroxidase: kinetic mechanism and nucleotide specificity

    Energy Technology Data Exchange (ETDEWEB)

    Stoll, V.S.; Blanchard, J.S.

    1987-05-01

    NADH peroxidase is a flavoprotein reductase isolated from Streptococcus faecalis which catalyzes the pyridine nucleotide dependent reduction of hydrogen peroxide to water. Initial velocity, product and dead-end inhibition studies have been performed and all support a ping-pong kinetic mechanism. Further support for the ping-pong nature of the kinetic mechanism are the hydrogen peroxide independent transhydrogenase activity of the enzyme, measured either with thio-NAD or with radiolabeled NAD (isotope exchange studies). Kinetic parameters will be presented for a number of reduced pyridine nucleotide analogs. Analogs which have been modified in the adenine ring exhibit much higher K/sub m/'s relative to their adenine analogs. NADH peroxidase catalyzes the stereo-specific removal of the 4S hydrogen of NADH and primary deuterium kinetic isotope effects have been determined for a number of these substrates with 4S-deuterated molecules. There is a strong correlation between their steady-state K/sub m/ and /sup D/V/K. Small values for /sup D/V are interpreted as supporting rate-limitation in the oxidative half-reaction. These data will be discussed in terms of a kinetic and chemical mechanism proposed for NADH peroxidase.

  7. NADH peroxidase: kinetic mechanism and nucleotide specificity

    International Nuclear Information System (INIS)

    Stoll, V.S.; Blanchard, J.S.

    1987-01-01

    NADH peroxidase is a flavoprotein reductase isolated from Streptococcus faecalis which catalyzes the pyridine nucleotide dependent reduction of hydrogen peroxide to water. Initial velocity, product and dead-end inhibition studies have been performed and all support a ping-pong kinetic mechanism. Further support for the ping-pong nature of the kinetic mechanism are the hydrogen peroxide independent transhydrogenase activity of the enzyme, measured either with thio-NAD or with radiolabeled NAD (isotope exchange studies). Kinetic parameters will be presented for a number of reduced pyridine nucleotide analogs. Analogs which have been modified in the adenine ring exhibit much higher K/sub m/'s relative to their adenine analogs. NADH peroxidase catalyzes the stereo-specific removal of the 4S hydrogen of NADH and primary deuterium kinetic isotope effects have been determined for a number of these substrates with 4S-deuterated molecules. There is a strong correlation between their steady-state K/sub m/ and /sup D/V/K. Small values for /sup D/V are interpreted as supporting rate-limitation in the oxidative half-reaction. These data will be discussed in terms of a kinetic and chemical mechanism proposed for NADH peroxidase

  8. Evolution and expression of class III peroxidases.

    Science.gov (United States)

    Mathé, Catherine; Barre, Annick; Jourda, Cyril; Dunand, Christophe

    2010-08-01

    Class III peroxidases are members of a large multigenic family, only detected in the plant kingdom and absent from green algae sensu stricto (chlorophyte algae or Chlorophyta). Their evolution is thought to be related to the emergence of the land plants. However class III peroxidases are present in a lower copy number in some basal Streptophytes (Charapyceae), which predate land colonization. Gene structures are variable among organisms and within species with respect to the number of introns, but their positions are highly conserved. Their high copy number, as well as their conservation could be related to plant complexity and adaptation to increasing stresses. No specific function has been assigned to respective isoforms, but in large multigenic families, particular structure-function relations can be expected. Plant peroxidase sequences contain highly conserved residues and motifs, variable domains surrounded by conserved residues and present a low identity level among their promoter regions, further suggesting the existence of sub-functionalization of the different isoforms. 2010 Elsevier Inc. All rights reserved.

  9. Early Cold-Induced Peroxidases and Aquaporins Are Associated With High Cold Tolerance in Dajiao (Musa spp. ‘Dajiao’

    Directory of Open Access Journals (Sweden)

    Wei-Di He

    2018-03-01

    Full Text Available Banana is an important tropical fruit with high economic value. One of the main cultivars (‘Cavendish’ is susceptible to low temperatures, while another closely related specie (‘Dajiao’ has considerably higher cold tolerance. We previously reported that some membrane proteins appear to be involved in the cold tolerance of Dajiao bananas via an antioxidation mechanism. To investigate the early cold stress response of Dajiao, here we applied comparative membrane proteomics analysis for both cold-sensitive Cavendish and cold-tolerant Dajiao bananas subjected to cold stress at 10°C for 0, 3, and 6 h. A total of 2,333 and 1,834 proteins were identified in Cavendish and Dajiao, respectively. Subsequent bioinformatics analyses showed that 692 Cavendish proteins and 524 Dajiao proteins were predicted to be membrane proteins, of which 82 and 137 differentially abundant membrane proteins (DAMPs were found in Cavendish and Dajiao, respectively. Interestingly, the number of DAMPs with increased abundance following 3 h of cold treatment in Dajiao (80 was seven times more than that in Cavendish (11. Gene ontology molecular function analysis of DAMPs for Cavendish and Dajiao indicated that they belong to eight categories including hydrolase activity, binding, transporter activity, antioxidant activity, etc., but the number in Dajiao is twice that in Cavendish. Strikingly, we found peroxidases (PODs and aquaporins among the protein groups whose abundance was significantly increased after 3 h of cold treatment in Dajiao. Some of the PODs and aquaporins were verified by reverse-transcription PCR, multiple reaction monitoring, and green fluorescent protein-based subcellular localization analysis, demonstrating that the global membrane proteomics data are reliable. By combining the physiological and biochemical data, we found that membrane-bound Peroxidase 52 and Peroxidase P7, and aquaporins (MaPIP1;1, MaPIP1;2, MaPIP2;4, MaPIP2;6, MaTIP1;3 are mainly

  10. Enzyme Technology of Peroxidases: Immobilization, Chemical and Genetic Modification

    Science.gov (United States)

    Longoria, Adriana; Tinoco, Raunel; Torres, Eduardo

    An overview of enzyme technology applied to peroxidases is made. Immobilization on organic, inorganic, and hybrid supports; chemical modification of amino acids and heme group; and genetic modification by site-directed and random mutagenesis are included. Different strategies that were carried out to improve peroxidase performance in terms of stability, selectivity, and catalytic activity are analyzed. Immobilization of peroxidases on inorganic and organic materials enhances the tolerance of peroxidases toward the conditions normally found in many industrial processes, such as the presence of an organic solvent and high temperature. In addition, it is shown that immobilization helps to increase the Total Turnover Number at levels high enough to justify the use of a peroxidase-based biocatalyst in a synthesis process. Chemical modification of peroxidases produces modified enzymes with higher thermostability and wider substrate variability. Finally, through mutagenesis approaches, it is possible to produce modified peroxidases capable of oxidizing nonnatural substrates with high catalytic activity and affinity.

  11. Structural motifs of syringyl peroxidases are conserved during angiosperm evolution.

    Science.gov (United States)

    Gómez Ros, Laura V; Aznar-Asensio, Ginés J; Hernandez, Jose A; Bernal, Maria A; Núñez-Flores, María J L; Cuello, Juan; Ros Barceló, Alfonso

    2007-05-16

    The most distinctive variation in the monomer composition of lignins in vascular land plants is that between the two main groups of seed plants. Thus, whereas gymnosperm (softwood) lignins are typically composed of guaiacyl (G) units, angiosperm (hardwood) lignins are largely composed of similar levels of G and syringyl (S) units. However, there are some studies that suggest that certain angiosperm peroxidases are unable to oxidize sinapyl alcohol, and a coniferyl alcohol shuttle has been proposed for oxidizing S units during the biosynthesis of lignins. With this in mind, a screening of the presence of S peroxidases in angiosperms (including woody species and forages) was performed. Contrarily to what might be expected, the intercellular washing fluids from lignifying tissues of 25 woody, herbaceous, and shrub species, belonging to both monocots and dicotyledons, all showed both S peroxidase activities and basic peroxidase isoenzymes analogous, with regard the isoelectric point, to the Zinnia elegans basic peroxidase isoenzyme, the only S peroxidase that has been fully characterized. These results led to the protein database in the search for homologies between angiosperm peroxidases and a true eudicot S peroxidase, the Z. elegans peroxidase. The findings showed that certain structural motifs of S peroxidases are conserved within the first 15 million years of angiosperm history, because they are found in peroxidases from the two major lineages of flowering plants, eumagnoliids and eudicotyledons, of note being the presence of these peroxidases in Amborella and Nymphaeales, which represent the first stages of angiosperm evolution. These phylogenetic studies also suggest that guaiacyl peroxidases apparently constitute the most "evolved state" of the plant peroxidase family evolution.

  12. Infrared spectroscopy of secondary organic aerosol precursors and investigation of the hygroscopicity of SOA formed from the OH reaction with guaiacol and syringol.

    Science.gov (United States)

    Ahmad, Waed; Coeur, Cecile; Tomas, Alexandre; Fagniez, Thomas; Brubach, Jean-Blaise; Cuisset, Arnaud

    2017-04-10

    Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) synchrotron analyses supplemented by density functional theory (DFT) anharmonic calculations have been undertaken to study the fundamental vibrational signatures of guaiacol and syringol, two methoxyphenol compounds found at the highest concentrations in fresh wood smoke and precursors of secondary organic aerosols (SOA) affecting the radiative balance and chemistry of the atmosphere. Nitroderivatives of these two compounds have also been studied experimentally for nitroguaiacol and theoretically for nitrosyringol. All the active fundamental vibrational bands have been assigned and compared to available gas phase measurements, providing a vibrational database of the main precursors for the analysis of SOA produced by atmospheric oxidation of methoxyphenols. In addition, the SOA formed in an atmospheric simulation chamber from the OH reaction with guaiacol and syringol were analyzed using the ATR-FTIR synchrotron spectroscopy and their hygroscopic properties were also investigated. The vibrational study confirms that nitroguaiacol and nitrosyringol are the main oxidation products of methoxyphenols by OH and are key intermediates in SOA production. The hydration experiments highlight the hydrophilic and hydrophobic characters of nitrosyringol and nitroguaiacol, respectively.

  13. Immobilization of Peroxidase onto Magnetite Modified Polyaniline

    Directory of Open Access Journals (Sweden)

    Eduardo Fernandes Barbosa

    2012-01-01

    Full Text Available The present study describes the immobilization of horseradish peroxidase (HRP on magnetite-modified polyaniline (PANImG activated with glutaraldehyde. After the optimization of the methodology, the immobilization of HRP on PANImG produced the same yield (25% obtained for PANIG with an efficiency of 100% (active protein. The optimum pH for immobilization was displaced by the effect of the partition of protons produced in the microenvironment by the magnetite. The tests of repeated use have shown that PANImG-HRP can be used for 13 cycles with maintenance of 50% of the initial activity.

  14. Immobilization of Peroxidase onto Magnetite Modified Polyaniline

    Science.gov (United States)

    Barbosa, Eduardo Fernandes; Molina, Fernando Javier; Lopes, Flavio Marques; García-Ruíz, Pedro Antonio; Caramori, Samantha Salomão; Fernandes, Kátia Flávia

    2012-01-01

    The present study describes the immobilization of horseradish peroxidase (HRP) on magnetite-modified polyaniline (PANImG) activated with glutaraldehyde. After the optimization of the methodology, the immobilization of HRP on PANImG produced the same yield (25%) obtained for PANIG with an efficiency of 100% (active protein). The optimum pH for immobilization was displaced by the effect of the partition of protons produced in the microenvironment by the magnetite. The tests of repeated use have shown that PANImG-HRP can be used for 13 cycles with maintenance of 50% of the initial activity. PMID:22489198

  15. 3D structure prediction of lignolytic enzymes lignin peroxidase and manganese peroxidase based on homology modelling

    Directory of Open Access Journals (Sweden)

    SWAPNIL K. KALE

    2016-04-01

    Full Text Available Lignolytic enzymes have great biotechnological value in biopulping, biobleaching, and bioremediation. Manganese peroxidase (EC 1:11:1:13 and lignin peroxidase (EC 1:11:1:14 are extracellular and hem-containing peroxidases that catalyze H2O2-dependent oxidation of lignin. Because of their ability to catalyse oxidation of a wide range of organic compounds and even some inorganic compounds, they got tremendous industrial importance. In this study, 3D structure of lignin and manganese peroxidase has been predicted on the basis of homology modeling using Swiss PDB workspace. The physicochemical properties like molecular weight, isoelectric point, Grand average of hydropathy, instability and aliphatic index of the target enzymes were performed using Protparam. The predicted secondary structure of MnP has 18 helices and 6 strands, while LiP has 20 helices and 4 strands. Generated 3D structure was visualized in Pymol. The generated model for MnP and LiP has Z-score Qmean of 0.01 and -0.71, respectively. The predicted models were validated through Ramachandran Plot, which indicated that 96.1 and 95.5% of the residues are in most favored regions for MnP and LiP respectively. The quality of predicted models were assessed and confirmed by VERIFY 3D, PROCHECK and ERRAT. The modeled structure of MnP and LiP were submitted to the Protein Model Database.

  16. Two oxidation sites for low redox potential substrates: a directed mutagenesis, kinetic, and crystallographic study on Pleurotus eryngii versatile peroxidase.

    Science.gov (United States)

    Morales, María; Mate, María J; Romero, Antonio; Martínez, María Jesús; Martínez, Ángel T; Ruiz-Dueñas, Francisco J

    2012-11-30

    Versatile peroxidase shares with manganese peroxidase and lignin peroxidase the ability to oxidize Mn(2+) and high redox potential aromatic compounds, respectively. Moreover, it is also able to oxidize phenols (and low redox potential dyes) at two catalytic sites, as shown by biphasic kinetics. A high efficiency site (with 2,6-dimethoxyphenol and p-hydroquinone catalytic efficiencies of ∼70 and ∼700 s(-1) mM(-1), respectively) was localized at the same exposed Trp-164 responsible for high redox potential substrate oxidation (as shown by activity loss in the W164S variant). The second site, characterized by low catalytic efficiency (∼3 and ∼50 s(-1) mM(-1) for 2,6-dimethoxyphenol and p-hydroquinone, respectively) was localized at the main heme access channel. Steady-state and transient-state kinetics for oxidation of phenols and dyes at the latter site were improved when side chains of residues forming the heme channel edge were removed in single and multiple variants. Among them, the E140G/K176G, E140G/P141G/K176G, and E140G/W164S/K176G variants attained catalytic efficiencies for oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) at the heme channel similar to those of the exposed tryptophan site. The heme channel enlargement shown by x-ray diffraction of the E140G, P141G, K176G, and E140G/K176G variants would allow a better substrate accommodation near the heme, as revealed by the up to 26-fold lower K(m) values (compared with native VP). The resulting interactions were shown by the x-ray structure of the E140G-guaiacol complex, which includes two H-bonds of the substrate with Arg-43 and Pro-139 in the distal heme pocket (at the end of the heme channel) and several hydrophobic interactions with other residues and the heme cofactor.

  17. Salacia campestris root bark extract: peroxidase inhibition, antioxidant and antiradical profile

    Directory of Open Access Journals (Sweden)

    José Carlos Rebuglio Vellosa

    2009-03-01

    Full Text Available Reactive oxygen species (ROS and free radical species have been implicated in initiating or accompanying many diseases in living organisms; there is thus, a continual need for antioxidants molecules to inactivate ROS/free radicals. Many studies of plants crude extracts have demonstrated free-radical scavenging and antioxidant action. Salacia species have long been used, in several countries, as traditional medicines against certain diseases and for their anti-inflammatory properties. In this study, Salacia campestris Walp (Hippocrateaceae root bark ethanol extract (ScEtOH was assessed for its ability to scavenge free radicals and reactive oxygen species; the results were expressed as percentage inhibition of the active species. ScEtOH was efficient against studied species: DPPH radical (obtained inhibition = 30%, ABTS•+ (IC50 = 1.8±0.8 μg/mL, HOCl (IC50 = 1.7 ± 0.1 μg/mL, O2•- (obtained inhibition = 32%, and NO• (obtained inhibition = 18 %. Peroxidase activity inhibition was evaluated through the guaiacol oxidation reaction catalyzed by hemin, HRP and myeloperoxidase (MPO; data showed that ScEtOH at 10 μg/mL led to 54 and 51% of inhibition, respectively, for the hemin and HRP systems. In the MPO system, ScEtOH promoted a 50% inhibition at 8.9 μg/mL, whereas quercetin, a powerful MPO inhibitor, inhibited this system at 1.35 μg/mL.Espécies reativas do oxigênio (ERO e radicais livres estão relacionados ao início ou à exacerbação de muitas doenças em organismos vivos; existindo portanto uma necessidade contínua por moléculas antioxidantes que inativem as ERO e radicais livres. Muitos estudos com extratos brutos de plantas têm demonstrado propriedades antioxidantes e seqüestradoras de radicais livres. Espécies de Salacia são utilizadas, em muitos países, como remédio tradicional contra certas doenças e por suas propriedades antiinflamatórias. Neste estudo, o extrato bruto etanólico da casca da raiz da Salacia

  18. Structure of soybean seed coat peroxidase: a plant peroxidase with unusual stability and haem-apoprotein interactions

    DEFF Research Database (Denmark)

    Henriksen, A; Mirza, O; Indiani, C

    2001-01-01

    Soybean seed coat peroxidase (SBP) is a peroxidase with extraordinary stability and catalytic properties. It belongs to the family of class III plant peroxidases that can oxidize a wide variety of organic and inorganic substrates using hydrogen peroxide. Because the plant enzyme is a heterogeneous...... be of functional importance. SBP has one of the most solvent accessible delta-meso haem edge (the site of electron transfer from reducing substrates to the enzymatic intermediates compound I and II) so far described for a plant peroxidase and structural alignment suggests that the volume of Ile74 is a factor...

  19. [Equilibrium and kinetic parameters of interaction between peroxidase conjugates of strophanthin and anti-peroxidase antibodies].

    Science.gov (United States)

    Tarun, E I; Karaseva, E I; Metelitsa, D I

    1997-01-01

    Interactions of three horseradish peroxidase (HRP)-strophanthin conjugates containing one, two, or three glycoside molecules (HRP-Str1, 2, or 3, respectively) with polyclonal anti-HRP antibodies were studied by homogeneous enzyme immunoassay. The total peroxidase activity of free conjugates and their immune complexes was estimated from the oxidation of o-phenylenediamine. The dissociation constants of the immune complexes and the rate constants of their dissociation and formation were determined. The equilibrium and kinetic parameters were determined for the interactions of the HRP-Str2 immune complex with anti-strophanthin and anti-HRP antibodies. The determined equilibrium and kinetic parameters of the HRP-Str interactions with anti-HRP antibodies depended on the molecular weights, sizes, and structures of the antigens studied.

  20. Production of lignin peroxidase by Ganoderma leucidum using solid ...

    African Journals Online (AJOL)

    The main objectives of this study were to optimize the culture conditions for the production of lignin peroxidase by Ganoderma leucidum, economic utilization of waste corn cobs as inducers substrate by pollution free fermentation technology and to optimize the solid state fermentation (SSF) process for lignin peroxidase ...

  1. Expression, purification and characterization of a peroxidase from ...

    African Journals Online (AJOL)

    Peroxidase is one of the key enzymes of the cellular antioxidant defense system, which is mostly involved in the reduction of hydrogen peroxide. Here, a peroxidase gene, named ThPOD1 was isolated from a cDNA library, which was generated from root tissue of Tamarix hispida that was exposed to 0.4 M NaCl. The cDNA ...

  2. Heat stable peroxidases from Vigna species (V) | Mbassi | African ...

    African Journals Online (AJOL)

    Shoots of three landraces of a Vigna species from two climatic areas of Cameroon were evaluated for their content of heat-resistant peroxidases. The peroxidase activity in the three landraces was detected with a greater catalytic efficiency for oxidation of O-dianisidine relative to ABTS (2, 2'-azino-bis-(3- ...

  3. Apple and quince peroxidase activity in response to essential oils ...

    African Journals Online (AJOL)

    Jane

    2011-09-28

    Sep 28, 2011 ... activity was affected by in vivo 50 µl/100 ml concentrations of pure rosemary essential oil (reductions in peroxidase activity by 90%) when the enzyme was obtained from quince. Key words: Peroxidase, essential oil, antioxidant, apple, quince. INTRODUCTION. Enzymatic browning in fruits and vegetables ...

  4. Cloning and analysis of the ascorbate peroxidase gene promoter ...

    African Journals Online (AJOL)

    enoh

    2012-03-22

    Mar 22, 2012 ... Ascorbate peroxidase (APX) is known to catalyze the reduction of H2O2 to water and enhance plants' tolerance in stress environment. An ascorbate peroxidase protein (BnAPX) was previously isolated from Brassica napus in our laboratory and it was located in the chloroplast. In order to clarify the.

  5. Apple and quince peroxidase activity in response to essential oils ...

    African Journals Online (AJOL)

    Enzymatic browning arises by peroxidase in fruits. However, essential oils are recognized as natural antioxidant agents. So in this study, the effect of thyme, coriander and rosemary essential oils were evaluated on the reduction of peroxidase activity in apples (Malus domestica Mill. cv Golden delicious), (M. domestica Mill.

  6. Cytochrome c as a peroxidase : tuning of heme reactivity

    NARCIS (Netherlands)

    Diederix, Rutger Ernest Michiel

    2003-01-01

    This thesis describes the peroxidase activity of the electron-transfer protein cytochrome c, and how it is controlled by the protein matrix. It is shown that unfolding cytochrome c has the effect to significantly enhance its peroxidase activity of (up to several thousand-fold). This can be achieved

  7. Peroxidase-like catalytic activities of ionic metalloporphyrins ...

    Indian Academy of Sciences (India)

    The efficiency order for the various PS-MTPPS was seen to be Co>Mn>Fe, with CoTPPS showing efficiency comparable to that of horseradish peroxidase. The catalytic efficiency was found to be increasing with temperature for all the catalysts. The re-usability of these PS-MTPPS systems for peroxidase-like activity was also ...

  8. Cloning and analysis of the ascorbate peroxidase gene promoter ...

    African Journals Online (AJOL)

    Ascorbate peroxidase (APX) is known to catalyze the reduction of H2O2 to water and enhance plants' tolerance in stress environment. An ascorbate peroxidase protein (BnAPX) was previously isolated from Brassica napus in our laboratory and it was located in the chloroplast. In order to clarify the physiological function of ...

  9. Expression, purification and characterization of a peroxidase from ...

    African Journals Online (AJOL)

    Yomi

    2012-01-24

    Jan 24, 2012 ... Peroxidase is one of the key enzymes of the cellular antioxidant defense system, which is mostly ... Key words: Peroxidase, prokaryotic expression, abiotic stress, Tamarix hispida. .... empty vector pET28a(+) without IPTG induction; lane 3 (L2), whole cell lysate of non-induced Rosetta gami E. coli cells.

  10. Purification and partial characterization of peroxidase from lettuce ...

    African Journals Online (AJOL)

    Peroxidase (EC1.11.1.7) was purified to homogeneity from lettuce (Lactuca sativa L.) stems by means of 40 to 80% ammonium sulfate precipitation, Sephadex G-100 gel filtration and affinity chromatography with concanavalin A. Peroxidase was purified 17.92-fold with 2.67% recovery and its molecular mass was 35 kDa on ...

  11. The relationship between lignin peroxidase and manganese peroxidase production capacities and cultivation periods of mushrooms.

    Science.gov (United States)

    Xu, Jian Z; Zhang, Jun L; Hu, Kai H; Zhang, Wei G

    2013-05-01

    Mushrooms are able to secrete lignin peroxidase (LiP) and manganese peroxidase (MnP), and able to use the cellulose as sources of carbon. This article focuses on the relation between peroxidase-secreting capacity and cultivation period of mushrooms with non-laccase activity. Methylene blue and methyl catechol qualitative assay and spectrophotometry quantitative assay show LiP secreting unvaryingly accompanies the MnP secreting in mushroom strains. The growth rates of hyphae are detected by detecting the dry hyphal mass. We link the peroxidase activities to growth rate of mushrooms and then probe into the relationship between them. The results show that there are close relationships between LiP- and/or MnP-secretory capacities and the cultivation periods of mushrooms. The strains with high LiP and MnP activities have short cultivation periods. However, those strains have long cultivation periods because of the low levels of secreted LiP and/or MnP, even no detectable LiP and/or MnP activity. This study provides the first evidence on the imitate relation between the level of secreted LiP and MnP activities and cultivation periods of mushrooms with non-laccase activity. Our study has significantly increased the understanding of the role of LiP and MnP in the growth and development of mushrooms with non-laccase activity. © 2012 The Authors. Microbial Biotechnology © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

  12. The Quantum Mixed-Spin Heme State of Barley Peroxidase: A Paradigm for Class III Peroxidases

    Energy Technology Data Exchange (ETDEWEB)

    Howes, B.D.; Ma, J.; Marzocchi, M.P.; Schiodt, C.B.; Shelnutt, J.A.; Smulevich, G.; Welinder, K.G.; Zhang, J.

    1999-03-23

    Electronic absorption and resonance Raman (RR) spectra of the ferric form of barley grain peroxidase (BP 1) at various pH values both at room temperature and 20 K are . reported, together with EPR spectra at 10 K. The ferrous forms and the ferric complex with fluoride have also been studied. A quantum mechanically mixed-spin (QS) state has been identified. The QS heme species co-exists with 6- and 5-cHS heroes; the relative populations of these three spin states are found to be dependent on pH and temperature. However, the QS species remains in all cases the dominant heme spin species. Barley peroxidase appears to be further characterized by a splitting of the two vinyl stretching modes, indicating that the vinyl groups are differently conjugated with the porphyrin. An analysis of the presently available spectroscopic data for proteins from all three peroxidase classes suggests that the simultaneous occurrence of the QS heme state as well as the splitting of the two vinyl stretching modes is confined to class III enzymes. The former point is discussed in terms of the possible influences of heme deformations on heme spin state. It is found that moderate saddling alone is probably not enough to cause the QS state, although some saddling maybe necessary for the QS state.

  13. Tc-cAPX, a cytosolic ascorbate peroxidase of Theobroma cacao L. engaged in the interaction with Moniliophthora perniciosa, the causing agent of witches' broom disease.

    Science.gov (United States)

    Camillo, Luciana Rodrigues; Filadelfo, Ciro Ribeiro; Monzani, Paulo Sérgio; Corrêa, Ronan Xavier; Gramacho, Karina Peres; Micheli, Fabienne; Pirovani, Carlos Priminho

    2013-12-01

    The level of hydrogen peroxide (H2O2) in plants signalizes the induction of several genes, including that of ascorbate peroxidase (APX-EC 1.11.1.11). APX isoenzymes play a central role in the elimination of intracellular H2O2 and contribute to plant responses to diverse stresses. During the infection process in Theobroma cacao by Moniliophthora perniciosa oxidative stress is generated and the APX action recruited from the plant. The present work aimed to characterize the T. cacao APX involved in the molecular interaction of T. cacao-M. perniciosa. The peroxidase activity was analyzed in protein extracts from cocoa plants infected by M. perniciosa and showed the induction of peroxidases like APX in resistant cocoa plants. The cytosolic protein of T. cacao (GenBank: ABR68691.2) was phylogenetically analyzed in relation to other peroxidases from the cocoa genome and eight genes encoding APX proteins with conserved domains were also analyzed. The cDNA from cytosolic APX was cloned in pET28a and the recombinant protein expressed and purified (rTc-cAPX). The secondary structure of the protein was analyzed by Circular Dichroism (CD) displaying high proportion of α-helices when folded. The enzymatic assay shows stable activity using ascorbate and guaiacol as an electron donor for H2O2 reduction. The pH 7.5 is the optimum for enzyme activity. Chromatographic analysis suggests that rTc-cAPX is a homodimer in solution. Results indicate that the rTc-cAPX is correctly folded, stable and biochemically active. The purified rTc-cAPX presented biotechnological potential and is adequate for future structural and functional studies. Copyright © 2013 The Authors. Published by Elsevier Masson SAS.. All rights reserved.

  14. Applications and Prospective of Peroxidase Biocatalysis in the Environmental Field

    Science.gov (United States)

    Torres-Duarte, Cristina; Vazquez-Duhalt, Rafael

    Environmental protection is, doubtless, one of the most important challenges for the human kind. The huge amount of pollutants derived from industrial activities represents a threat for the environment and ecologic equilibrium. Phenols and halogenated phenols, polycyclic aromatic hydrocarbons, endocrine disruptive chemicals, pesticides, dioxins, polychlorinated biphenyls, industrial dyes, and other xenobiotics are among the most important pollutants. A large variety of these xenobiotics are substrates for peroxidases and thus susceptible to enzymatic transformation. The literature reports mainly the use of horseradish peroxidase, manganese peroxidase, lignin peroxidase, and chloroperoxidase on the transformation of these pollutants. Peroxidases are enzymes able to transform a variety of compounds following a free radical mechanism, giving oxidized or polymerized products. The peroxidase transformation of these pollutants is accompanied by a reduction in their toxicity, due to a biological activity loss, a reduction in the bioavailability or due to the removal from aqueous phase, especially when the pollutant is found in water. In addition, when the pollutants are present in soil, peroxidases catalyze a covalent binding to soil organic matter. In most of cases, oxidized products are less toxic and easily biodegradable than the parent compounds. In spite of their versatility and potential use in environmental processes, peroxidases are not applied at large scale yet. Diverse challenges, such as stability, redox potential, and the production of large amounts, should be solved in order to apply peroxidases in the pollutant transformation. In this chapter, we critically review the transformation of different xenobiotics by peroxidases, with special attention on the identified transformation products, the probable reaction mechanisms, and the toxicity reports. Finally, the design and development of an environmental biocatalyst is discussed. The design challenges are

  15. Formation of a tyrosine adduct involved in lignin degradation by Trametopsis cervina lignin peroxidase: a novel peroxidase activation mechanism

    Science.gov (United States)

    Yuta Miki; Rebecca Pogni; Sandra Acebes; Fatima Lucas; Elena Fernandez-Fueyo; Maria Camilla Baratto; Maria I. Fernandez; Vivian De Los Rios; Francisco J. Ruiz-duenas; Adalgisa Sinicropi; Riccardo Basosi; Kenneth E. Hammel; Victor Guallar; Angel T. Martinez

    2013-01-01

    LiP (lignin peroxidase) from Trametopsis cervina has an exposed catalytic tyrosine residue (Tyr181) instead of the tryptophan conserved in other lignin-degrading peroxidases. Pristine LiP showed a lag period in VA (veratryl alcohol) oxidation. However, VA-LiP (LiP after treatment with H2O2...

  16. The molecular characterization of the lignin-forming peroxidase

    Energy Technology Data Exchange (ETDEWEB)

    Lagrimini, L.M.

    1992-01-01

    This laboratory is committed to understanding the function of plant peroxidases via a multi-disciplinary approach. We have chosen the lignin-forming peroxidase from tobacco as the first isoenzyme to be subjected to this comprehensive approach. The goals which were set out upon the initiation of this project were as follows: (1) utilize a cDNA clone to the tobacco anionic peroxidase to generate transgenic plants which either over-produced this isoenzyme or specifically under-produced this isoenzyme via antisense RNA, (2) describe any phenotypic changes resulting from altered peroxidase expression, (3) perform morphological, physiological, and biochemical analysis of the above mentioned plants to help in determining the in planta function for this enzyme, and (4) clone and characterize the gene for the tobacco anionic peroxidase. A summary of progress thus far which includes both published and unpublished work will be presented in three sections: generation and characterization of transgenic plants, description of phenotypes, and biochemical and physiological analysis of peroxidase function, and cloning and characterization of the tobacco anionic peroxidase gene.

  17. Production and Purification of Peroxidase from Aspergillus niger.

    Directory of Open Access Journals (Sweden)

    Mohammed A. Jebor

    2017-02-01

    Full Text Available This study was conducted in the laboratories of Biology Department, College of Science, which deals with isolation and purification of peroxidase and optimization of process parameters to achieve maximum yield of peroxidase by Aspergillus niger. Solid-state fermentation of Aspergillus niger was carried out for enhanced production of peroxidase using hydrogen peroxide as the substrate of enzyme maximum activity of the enzyme was achieved under optimum growth conditions. The optimum conditions were the isolated of Aspergillus niger from soil and growth in synthetic medium, it gave high titer of peroxidase activity, the fructose as carbon source, peptone as nitrogen source, after 12 days of incubation, incubation temperature 25 °C and pH = 6.5. Peroxidase purified in four purification steps; precipitation with 70% saturation of ammonium sulfate, step of dialysis, the third by ion exchange chromatography using DEAE-Cellulose and fourth by gel filtration throughout Sephadex G-100. The specific activity of the purified enzyme was 150U/mg with 7.75 folds. The peroxidase was shown to have molecular weight of 40kDa in SDS-PAGA and about 40kDa in gel filtration.The optimum pH and temperature for peroxidase activity 7 and 35 C0 respectively.

  18. Redundancy among manganese peroxidases in Pleurotus ostreatus.

    Science.gov (United States)

    Salame, Tomer M; Knop, Doriv; Levinson, Dana; Yarden, Oded; Hadar, Yitzhak

    2013-04-01

    Manganese peroxidases (MnPs) are key players in the ligninolytic system of white rot fungi. In Pleurotus ostreatus (the oyster mushroom) these enzymes are encoded by a gene family comprising nine members, mnp1 to -9 (mnp genes). Mn(2+) amendment to P. ostreatus cultures results in enhanced degradation of recalcitrant compounds (such as the azo dye orange II) and lignin. In Mn(2+)-amended glucose-peptone medium, mnp3, mnp4, and mnp9 were the most highly expressed mnp genes. After 7 days of incubation, the time point at which the greatest capacity for orange II decolorization was observed, mnp3 expression and the presence of MnP3 in the extracellular culture fluids were predominant. To determine the significance of MnP3 for ligninolytic functionality in Mn(2+)-sufficient cultures, mnp3 was inactivated via the Δku80 strain-based P. ostreatus gene-targeting system. In Mn(2+)-sufficient medium, inactivation of mnp3 did not significantly affect expression of nontargeted MnPs or their genes, nor did it considerably diminish the fungal Mn(2+)-mediated orange II decolorization capacity, despite the significant reduction in total MnP activity. Similarly, inactivation of either mnp4 or mnp9 did not affect orange II decolorization ability. These results indicate functional redundancy within the P. ostreatus MnP gene family, enabling compensation upon deficiency of one of its members.

  19. Carbon Nanodots as Peroxidase Nanozymes for Biosensing

    Directory of Open Access Journals (Sweden)

    Bhaskar Garg

    2016-12-01

    Full Text Available ‘Nanozymes’, a term coined by Scrimin, Pasquato, and co-workers to describe nanomaterials with enzyme-like characteristics, represent an exciting and emerging research area in the field of artificial enzymes. Indubitably, the last decade has witnessed substantial advancements in the design of a variety of functional nanoscale materials, including metal oxides and carbon-based nanomaterials, which mimic the structures and functions of naturally occurring enzymes. Among these, carbon nanodots (C-dots or carbon quantum dots (CQDs offer huge potential due to their unique properties as compared to natural enzymes and/or classical artificial enzymes. In this mini review, we discuss the peroxidase-like catalytic activities of C-dots and their applications in biosensing. The scope intends to cover not only the C-dots but also graphene quantum dots (GQDs, doped C-dots/GQDs, carbon nitride dots, and C-dots/GQDs nanocomposites. Nevertheless, this mini review is designed to be illustrative, not comprehensive.

  20. Molecular characterization of the lignin-forming peroxidase: Role in growth, development and response to stress

    Energy Technology Data Exchange (ETDEWEB)

    Lagrimini, L.M.

    1993-01-01

    This laboratory has continued its comprehensive study of the structure and function of plant peroxidases and their genes. Specifically, we are characterizing the anionic peroxidase of tobacco. During the past year we have completed the nucleotide sequence of the tobacco anionic peroxidase gene, joined the anionic peroxidase promoter to [Beta]-glucuronidase and demonstrated expression in transformed plants, measured lignin, auxin, and ethylene levels in transgenic tobacco plants over-expressing the anionic peroxidase, developed chimeric peroxidase genes to over-or under-express the anionic peroxidase in tissue specific manner in transgenic plants, and over-expressed the tobacco anionic peroxidase in transgenic tomato and sweetgum plants.

  1. Ionically Bound Peroxidase from Peach Fruit

    Directory of Open Access Journals (Sweden)

    Neves Valdir Augusto

    2002-01-01

    Full Text Available Soluble, ionically bound peroxidase (POD and polyphenoloxidase (PPO were extracted from the pulp of peach fruit during ripening at 20°C. Ionically bound form was purified 6.1-fold by DEAE-cellulose and Sephadex G-100 chromatography. The purified enzyme showed only one peak of activity on Sephadex G-100 and PAGE revealed that the enzyme was purified by the procedures adopted. The purified enzyme showed a molecular weight of 29000 Da, maximum activity at pH 5.0 and at 40ºC. The calculated apparent activation energy (Ea for the reaction was10.04 kcal/mol. The enzyme was heat-labile in the temperature range of 60 to 75ºC with a fast inactivation at 75ºC. Measurement of residual activity showed a stabilizing effect of sucrose at various temperature/sugar concentrations (0, 10, 20 %, w/w, with an activation energy (Ea for inactivation increasing with sucrose concentration from 0 to 20% (w/w. The Km and Vmax values were 9.35 and 15.38 mM for 0-dianisidine and H2O2, respectively. The bound enzyme was inhibited competitively by ferulic, caffeic and protocatechuic acids with different values of Ki,. L-cysteine, p-coumaric and indolacetic acid and Fe++ also inhibited the enzyme but at a lower grade. N-ethylmaleimide and p-CMB were not effective to inhibit the enzyme demonstrating the non-essentiality of SH groups.

  2. Cell wall bound anionic peroxidases from asparagus byproducts.

    Science.gov (United States)

    Jaramillo-Carmona, Sara; López, Sergio; Vazquez-Castilla, Sara; Jimenez-Araujo, Ana; Rodriguez-Arcos, Rocio; Guillen-Bejarano, Rafael

    2014-10-08

    Asparagus byproducts are a good source of cationic soluble peroxidases (CAP) useful for the bioremediation of phenol-contaminated wastewaters. In this study, cell wall bound peroxidases (POD) from the same byproducts have been purified and characterized. The covalent forms of POD represent >90% of the total cell wall bound POD. Isoelectric focusing showed that whereas the covalent fraction is constituted primarily by anionic isoenzymes, the ionic fraction is a mixture of anionic, neutral, and cationic isoenzymes. Covalently bound peroxidases were purified by means of ion exchange chromatography and affinity chromatography. In vitro detoxification studies showed that although CAP are more effective for the removal of 4-CP and 2,4-DCP, anionic asparagus peroxidase (AAP) is a better option for the removal of hydroxytyrosol (HT), the main phenol present in olive mill wastewaters.

  3. Partial characterization of lignin peroxidase expressed by bacterial ...

    African Journals Online (AJOL)

    Lignin peroxidase producing microorganisms were isolated from the gut of Macrotermes nigeriense (Soldier and worker termite). The microorganisms isolated were Staphylococcus aureus, Staphylococcus epidermis, Bacillus subtilis, Micrococcus luteus, Epidermophyton flocussum, Microsporum distortum, Trichophyton ...

  4. Purification and characterization of a peroxidase present in ...

    African Journals Online (AJOL)

    Purification and characterization of a peroxidase present in xilopodium exsudates of umbu plants (Spondias tuberosa A.) M dos Santos Teixeira Pinto, JM Ribeiro, FP de Araujo, NF de Melo, KVS Fernandes ...

  5. Altered phenotypes in plants transformed with chimeric tobacco peroxidase genes

    Energy Technology Data Exchange (ETDEWEB)

    Lagrimini, L.M.

    1990-12-31

    Peroxidases have been implicated in a variety of secondary metabolic reactions including lignification, cross-linking of cell wall polysaccharides, oxidation of indole-3-acetic acid, regulation of cell elongation, wound-healing, phenol oxidation, and pathogen defense. However, due to the many different isoenzymes and even more potential substrates, it has proven difficult to verify actual physiological roles for peroxidase. We are studying the molecular biology of the tobacco peroxidase genes, and have utilized genetic engineering techniques to produce transgenic plants which differ only in their expression of an individual peroxidase isoenzyme. Many of the in planta functions for any individual isoenzyme may be predicted through the morphological and physiological analysis of transformed plants.

  6. Altered phenotypes in plants transformed with chimeric tobacco peroxidase genes

    Energy Technology Data Exchange (ETDEWEB)

    Lagrimini, L.M.

    1990-01-01

    Peroxidases have been implicated in a variety of secondary metabolic reactions including lignification, cross-linking of cell wall polysaccharides, oxidation of indole-3-acetic acid, regulation of cell elongation, wound-healing, phenol oxidation, and pathogen defense. However, due to the many different isoenzymes and even more potential substrates, it has proven difficult to verify actual physiological roles for peroxidase. We are studying the molecular biology of the tobacco peroxidase genes, and have utilized genetic engineering techniques to produce transgenic plants which differ only in their expression of an individual peroxidase isoenzyme. Many of the in planta functions for any individual isoenzyme may be predicted through the morphological and physiological analysis of transformed plants.

  7. The Daily Dynamics of Phenolic Compounds Content and Guaiacol-Peroksidase Activity in Leaves and Flowers of Alchemilla subcrenata and Veronica chamaedrys

    Directory of Open Access Journals (Sweden)

    Zhivetev M.A.

    2015-12-01

    Full Text Available The daily dynamics of peroxidase activity and the total content of phenolic compounds in leaves and inflorescences of Alchemilla subcrenata Buser and Veronica chamaedrys L. were studied. Significant changes in the content of phenolic compounds and activity peroksidase depending on the time of day was shown.

  8. Platelet crossmatch tests using radiolabelled staphylococcal protein A or peroxidase anti-peroxidase in alloimmunised patients

    International Nuclear Information System (INIS)

    Yam, P.; Petz, L.D.; Scott, E.P.; Santos, S.

    1984-01-01

    Refractoriness to random-donor platelets as a result of alloimmunization remains a major problem in long-term platelet transfusion therapy despite the use of HLA-matched platelets. A study has been made of two methods for detection of platelet associated IgG as platelet crossmatch tests for the selection of platelet donors. These methods use radiolabelled staphylococcal protein A( 125 I-SPA) and peroxidase anti-peroxidase (PAP), respectively. One hundred and ten crossmatch tests using 125 I-SPA were performed retrospectively in 18 alloimmunized patients. The results indicated that the predictive value of a positive or a negative test was 87%; the sensitivity was 73% and the specificity was 95%. Results with the PAP test were similar. The HLA types were known for 48 donor-recipient pairs. With few exceptions, there was a correlation between the results of the platelet crossmatch tests and the effectiveness of platelet transfusion regardless of the degree of HLA match. These results indicate that platelet crossmatch tests may be valuable even when closely HLA matched donors are not available. A large-scale prospective study is warranted, particularly in highly immunized patients. (author)

  9. Optimization of lignin peroxidase, manganese peroxidase, and Lac production from Ganoderma lucidum under solid state fermentation of pineapple leaf

    OpenAIRE

    Sudha Hariharan; Padma Nambisan

    2013-01-01

    This study was undertaken to isolate ligninase-producing white-rot fungi for use in the extraction of fibre from pineapple leaf agriwaste. Fifteen fungal strains were isolated from dead tree trunks and leaf litter. Ligninolytic enzymes (lignin peroxidase (LiP), manganese peroxidase (MnP), and laccase (Lac)), were produced by solid-state fermentation (SSF) using pineapple leaves as the substrate. Of the isolated strains, the one showing maximum production of ligninolytic enzymes was identified...

  10. Engineering a fungal peroxidase that degrades lignin at very acidic pH

    NARCIS (Netherlands)

    Fernandez-Fueyo, E.; Ruiz-Duenas, F.J.; Martinez, A.T.

    2014-01-01

    Background Ligninolytic peroxidases are divided into three families: manganese peroxidases (MnPs), lignin peroxidases (LiPs), and versatile peroxidases (VPs). The latter two are able to degrade intact lignins, as shown using nonphenolic lignin model compounds, with VP oxidizing the widest range of

  11. Peroxidase extraction from jicama skin peels for phenol removal

    Science.gov (United States)

    Chiong, T.; Lau, S. Y.; Khor, E. H.; Danquah, M. K.

    2016-06-01

    Phenol and its derivatives exist in various types of industrial effluents, and are known to be harmful to aquatic lives even at low concentrations. Conventional treatment technologies for phenol removal are challenged with long retention time, high energy consumption and process cost. Enzymatic treatment has emerged as an alternative technology for phenol removal from wastewater. These enzymes interact with aromatic compounds including phenols in the presence of hydrogen peroxide, forming free radicals which polymerize spontaneously to produce insoluble phenolic polymers. This work aims to extract peroxidase from agricultural wastes materials and establish its application for phenol removal. Peroxidase was extracted from jicama skin peels under varying extraction conditions of pH, sample-to-buffer ratio (w/v %) and temperature. Experimental results showed that extraction process conducted at pH 10, 40% w/v and 25oC demonstrated a peroxidase activity of 0.79 U/mL. Elevated temperatures slightly enhanced the peroxidase activities. Jicama peroxidase extracted at optimum extraction conditions demonstrated a phenol removal efficiency of 87.5% at pH 7. Phenol removal efficiency was ∼ 97% in the range of 30 - 40oC, and H2O2 dosage has to be kept below 100 mM for maximum removal under phenol concentration tested.

  12. Purification, crystallization and preliminary crystallographic analysis of banyan peroxidase

    International Nuclear Information System (INIS)

    Sharma, Anurag; Palm, Gottfried J.; Kumari, Moni; Panjikar, Santosh; Jagannadham, M. V.; Hinrichs, Winfried

    2012-01-01

    Crystals of a new peroxidase, named banyan peroxidase, from the latex of F. benghalensis belonged to the trigonal space group P3 2 21 and provided X-ray diffraction data to a resolution of 1.66 Å. The anomalous signal of the intrinsic iron and calcium ions was sufficent for phasing by SAD. Plant peroxidases are extensively used in a wide range of biotechnological applications owing to their high environmental and thermal stability. A new peroxidase, named banyan peroxidase, was purified from the latex of Ficus benghalensis and crystallized. X-ray diffraction data were collected from native crystals and from bromide and xenon derivatives to resolutions of up to 1.66 Å in the trigonal space group P3 2 21, with unit-cell parameters a = b = 73.1, c = 164.6 Å. The anomalous signal of the intrinsic iron and calcium ions was sufficient for structure solution by SAD, although the sequence is not yet known

  13. Inorganic chemistry of defensive peroxidases in the human oral cavity.

    Science.gov (United States)

    Ashby, M T

    2008-10-01

    The innate host response system is comprised of various mechanisms for orchestrating host response to microbial infection of the oral cavity. The heterogeneity of the oral cavity and the associated microenvironments that are produced give rise to different chemistries that affect the innate defense system. One focus of this review is on how these spatial differences influence the two major defensive peroxidases of the oral cavity, salivary peroxidase (SPO) and myeloperoxidase (MPO). With hydrogen peroxide (H(2)O(2)) as an oxidant, the defensive peroxidases use inorganic ions to produce antimicrobials that are generally more effective than H(2)O(2) itself. The concentrations of the inorganic substrates are different in saliva vs. gingival crevicular fluid (GCF). Thus, in the supragingival regime, SPO and MPO work in unison for the exclusive production of hypothiocyanite (OSCN(-), a reactive inorganic species), which constantly bathes nascent plaques. In contrast, MPO is introduced to the GCF during inflammatory response, and in that environment it is capable of producing hypochlorite (OCl(-)), a chemically more powerful oxidant that is implicated in host tissue damage. A second focus of this review is on inter-person variation that may contribute to different peroxidase function. Many of these differences are attributed to dietary or smoking practices that alter the concentrations of relevant inorganic species in the oral cavity (e.g.: fluoride, F(-); cyanide, CN(-); cyanate, OCN(-); thiocyanate, SCN(-); and nitrate, NO(3)(-)). Because of the complexity of the host and microflora biology and the associated chemistry, it is difficult to establish the significance of the human peroxidase systems during the pathogenesis of oral diseases. The problem is particularly complex with respect to the gingival sulcus and periodontal pockets (where the very different defensive stratagems of GCF and saliva co-mingle). Despite this complexity, intriguing in vitro and in vivo

  14. Resonance Raman spectroscopy of cytochrome c peroxidase variants that mimic manganese peroxidase.

    Science.gov (United States)

    Feng, Manliang; Tachikawa, Hiroyasu; Wang, Xiaotang; Pfister, Thomas D; Gengenbach, Alan J; Lu, Yi

    2003-09-01

    Cytochrome c peroxidase (C cP) variants with an engineered Mn(II) binding site, including MnC cP [C cP(MI, G41E, V45E, H181D)], MnC cP(W191F), and MnC cP(W191F, W51F), that mimic manganese peroxidase (MnP), have been characterized by resonance Raman (RR) spectroscopy. Analysis of the Raman bands in the 200-700 cm(-1) and 1300-1650 cm(-1) regions indicates that both the coordination and spin state of the heme iron in the variants differ from that of C cP(MI), the recombinant yeast C cP containing additional Met-Ile residues at the N-terminus. At neutral pH the frequencies of the nu(3) mode indicate that a pure five-coordinate heme iron exists in C cP(MI) whereas a six-coordinate low-spin iron is the dominant species in the C cP variants with the engineered Mn(II) binding site. The H181D mutation, which weakens the proximal linkage to the heme iron, may be responsible for these spectral and structural changes. Raman spectra of the variants C cP(MI, W191F) and C cP(MI, W191F, W51F) were also obtained to clarify the structural and functional roles of mutations at two tryptophan sites. The W51F mutation was found to disrupt H-bonding to the distal water molecules and the resulting variants tended to form transitional or mixed coordination states that possess spectral and structural features similar to that of MnP. Such structural features, with a loosened distal water, may facilitate the binding of H(2)O(2) and increase the rate constant for compound I formation. This effect, in addition to the elimination of an H-bond to ferryl oxygen by the same mutation, accounts for the increased MnP specific activity of MnC cP(W191F, W51F).

  15. Peroxidase activity in Spondias dulcis = Atividade da peroxidase em Spondias dulcis

    Directory of Open Access Journals (Sweden)

    Lúcio Cardozo-Filho

    2010-10-01

    Full Text Available In this study, the best conditions to obtain crude extracts showingPeroxidase activity from Spondia dulcis (caja-mango were evaluated. Fresh fruits (25 g were blended in different sodium phosphate buffer (0.05 to 0.2 M with a pH varying from 3.0 to 9.0. The muddy material was centrifuged for 20 minutes. In order to improve POD activity, the crude extract was submitted to precipitation with ammonium sulfate at 90% saturation. This precipitated was re-suspended in sodium phosphate buffer 0.2 M pH 6.5 and then, optimum pH for activity assay (pH varying from 5.0 to 9.0 and thermal stability (exposure to different temperatures varying from 30 to 75ºC for periods between 0 to 15 minutes were determined. The best conditions for activity assay were in phosphate buffer 0.2 M at pH7.0. The results obtained for thermal inactivation study suggest that the heating at 75ºCfor 15 minutes inactivated 95% of initial POD activity.Foram avaliadas, neste trabalho, algumas condições para a obtenção de extratos brutos com atividade peroxidase de Spondias dulcis (cajá-manga. Frutas frescas (25 g foram trituradas com tampão fosfato de sódio (0,05 a 0,2 M em pHs diferentes (3,0 a 9,0. O material obtido foi centrifugado por 20 min. O extrato bruto foi submetido à precipitação com sulfato de amônio até 90% de saturação. Este precipitado foi ressuspenso em tampão fosfato de sódio 0,2 M pH 6,5 e, assim, o pH ótimo para o ensaio de atividade (pH que varia de 5,0 a 9,0 e a estabilidade térmica (exposição a temperaturas de 30, 60, 65, 70 e 75ºC por um período de 0 a 15 min. deste foram determinados. As melhores condições encontradas para o ensaio de atividade foram em tampão fosfato 0,2 M pH 7,0. Os resultados para a inativação térmica sugerem que o aquecimento a 75ºC por 15 mininativa 95% da atividade de POD inicial.

  16. Self-Assembled Complexes of Horseradish Peroxidase with Magnetic Nanoparticles Showing Enhanced Peroxidase Activity

    KAUST Repository

    Corgié, Stéphane C.

    2012-02-15

    Bio-nanocatalysts (BNCs) consisting of horseradish peroxidase (HRP) self-assembled with magnetic nanoparticles (MNPs) enhance enzymatic activity due to the faster turnover and lower inhibition of the enzyme. The size and magnetization of the MNPs affect the formation of the BNCs, and ultimately control the activity of the bound enzymes. Smaller MNPs form small clusters with a low affinity for the HRP. While the turnover for the bound fraction is drastically increased, there is no difference in the H 2O 2 inhibitory concentration. Larger MNPs with a higher magnetization aggregate in larger clusters and have a higher affinity for the enzyme and a lower substrate inhibition. All of the BNCs are more active than the free enzyme or the MNPs (BNCs > HRP ≤laquo; MNPs). Since the BNCs show surprising resilience in various reaction conditions, they may pave the way towards new hybrid biocatalysts with increased activities and unique catalytic properties for magnetosensitive enzymatic reactions. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Towards uncovering the roles of switchgrass peroxidases in plant processes

    Directory of Open Access Journals (Sweden)

    Aaron eSaathoff

    2013-06-01

    Full Text Available Herbaceous perennial plants selected as potential biofuel feedstocks had been understudied at the genomic and functional genomic levels. Recent investments, primarily by the U.S. Department of Energy, have led to the development of a number of molecular resources for bioenergy grasses, such as the partially annotated genome for switchgrass (Panicum virgatum L., and some related diploid species. In its current version, the switchgrass genome contains 65,878 gene models arising from the A and B genomes of this tetraploid grass. The availability of these gene sequences provides a framework to exploit transcriptomic data obtained from next generation sequencing platforms to address questions of biological importance. One such question pertains to discovery of genes and proteins important for biotic and abiotic stress responses, and how these components might affect biomass quality and stress response in plants engineered for a specific end purpose. It can be expected that production of switchgrass on marginal lands will expose plants to diverse stresses, including herbivory by insects. Class III plant peroxidases have been implicated in many developmental responses such as lignification and in the adaptive responses of plants to insect feeding. Here, we have analyzed the class III peroxidases encoded by the switchgrass genome, and have mined available transcriptomic datasets to develop a first understanding of the expression profiles of the class III peroxidases in different plant tissues. Lastly, we have identified switchgrass peroxidases that appear to be orthologs of enzymes shown to play key roles in lignification and plant defense responses to hemipterans.

  18. Decolourization of Direct Blue 2 by peroxidases obtained from an ...

    African Journals Online (AJOL)

    Also, an increase in toxicity, determined by Vibrio fisheri, was observed after the enzymatic oxidation of the dye. Results suggest that the oxidation of DB2 with peroxidases can be recommended as a pretreatment step before a conventional treatment process. Keywords: decolourization, Direct Blue 2, industrial waste, ...

  19. Effect of industrial wastewater ontotal protein and the peroxidase ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-10-19

    Oct 19, 2009 ... The aim of this study is to investigate the effects of industrial wastewaters on protein and the peroxidase activity in Lycopersicon esculentum Mill., Capsicum annuum L., Phaseolus vulgaris L. and. Vicia faba L. Industrial wastewaters were taken from Dardanel Fisheries Company, Tekel alcoholic.

  20. Decolourization of Direct Blue 2 by peroxidases obtained from an ...

    African Journals Online (AJOL)

    2018-04-02

    Apr 2, 2018 ... Effects of different parameters like pH, temperature, reaction time, H2O2 dosage and dye concentration were tested in order to optimize the dye degradation. .... fischeri (Microtox Azure Ambiental). The reagent is a ... The impact of reaction time on the decolourization of DB2 in presence of peroxidase ...

  1. Peroxidase isozyme profiles in some sweet cherry rootstocks and ...

    African Journals Online (AJOL)

    PERS

    2012-01-10

    Jan 10, 2012 ... study in pear (Davarynejad et al., 2008 ). They proposed that presence of A band in the graft union was an indication of compatibility of Beurre Hardy and Passa with. QA, whereas the absence of this band was related to incompatibility of Dargazi, Shahmivah and Torsh pear cultivars. Similarly a peroxidase ...

  2. 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 ...

  3. Suicide inactivation of horseradish peroxidase by excess hydrogen ...

    African Journals Online (AJOL)

    In reactions carried out in sodium acetate buffer, higher inactivation rates were observed when the buffer ion concentration was increased, an indication that peroxidase might be generating reactive radicals from the buffer molecules. Promethazine exerted a modest protective effect against inactivation; however, higher ...

  4. Purification and characterization of a peroxidase present in ...

    African Journals Online (AJOL)

    Marcio Pinto

    2015-02-22

    Feb 22, 2015 ... be related to oxidation of phenols by poliphenoloxidase or peroxidase, a common trouble viewed in food processing. (Mousavizadeh and Sedaghathoor, 2011). Due to the formation of xylopodium and other characteristics, umbu plants are highly resistant to drought (Silva et al., 2009). A unique umbu tree is ...

  5. Ascorbate peroxidase gene from Brassica napus enhances salt and ...

    African Journals Online (AJOL)

    A full-length cDNA clone, BnAPX (GenBank: FJ965556.1) encoding ascorbate peroxidase and isolated from Brassica napus, was successfully introduced into Arabidopsis thaliana. Investigation into the function of BnAPX demonstrated that BnAPX transgenic plants grew better than wild type under NaCl stress, and also had ...

  6. Hepatic and erythrocytic glutathione peroxidase activity in liver diseases.

    Science.gov (United States)

    Cordero, R; Ortiz, A; Hernández, R; López, V; Gómez, M M; Mena, P

    1996-09-01

    Hepatic and erythrocytic glutathione peroxidase activity, together with malondialdehyde levels, were determined as indicators of peroxidation in 83 patients from whom liver biopsies had been taken for diagnostic purposes. On histological study, the patients were classified into groups as minimal changes (including normal liver), steatosis, alcoholic hepatitis, hepatic cirrhosis, light to moderately active chronic hepatitis, and severe chronic active hepatitis. The glutathione peroxidase activity in erythrocytes showed no significant changes in any liver disease group. In the hepatic study, an increased activity was observed in steatosis with respect to the minimal changes group, this increased activity induced by the toxic agent in the initial stages of the alcoholic hepatic disease declining as the hepatic damage progressed. There was a negative correlation between the levels of hepatic malondialdehyde and hepatic glutathione peroxidase in subjects with minimal changes. This suggested the existence of an oxidative equilibrium in this group. This equilibrium is broken in the liver disease groups as was manifest in a positive correlation between malondialdehyde and glutathione peroxidase activity.

  7. Efficient production of Arthromyces ramosus peroxidase by Aspergillus awamori

    NARCIS (Netherlands)

    Lokman, B.C.; Joosten, V.; Hovenkamp, J.; Gouka, R.J.; Verrips, C.T.; Hondel, C.A.M.J.J. van den

    2003-01-01

    The heterologous production of Arthromyces ramosus peroxidase (ARP) was analysed in the filamentous fungus Aspergillus awamori under control of the inducible endoxylanase promoter. Secretion of active ARP was achieved up to 800 mg l-1 in shake flask cultures. Western blot analysis showed that an

  8. Bioepoxidation of isosafrol catalyzed by radish and turnip peroxidases

    African Journals Online (AJOL)

    Peroxidases (PODs) from radish (Raphanus sativus L.) and turnip (Brassica napus L.) were extracted and precipitated with ammonium sulfate using a simple, low cost and quick method. The activities of all ... The products of the reaction were analyzed by high resolution gas chromatography (GC) and mass spectrometry.

  9. Effect of industrial wastewater ontotal protein and the peroxidase ...

    African Journals Online (AJOL)

    The aim of this study is to investigate the effects of industrial wastewaters on protein and the peroxidase activity in Lycopersicon esculentum Mill., Capsicum annuum L., Phaseolus vulgaris L. and Vicia faba L. Industrial wastewaters were taken from Dardanel Fisheries Company, Tekel alcoholic drinks companies' ...

  10. Cloning and characterization of an ascorbate peroxidase gene ...

    African Journals Online (AJOL)

    DR. NJ TONUKARI

    2012-05-29

    May 29, 2012 ... An ascorbate peroxidase (APX) cDNA, designated MaAPX1, was isolated from banana fruit by suppression subtractive hybridization (SSH). MaAPX1 shares an extensive sequence identity (79 to. 83%) with other plant APX homologues. Southern blot analysis revealed only two copies of the APX gene in ...

  11. Changes in activities of polyphenol oxidase, ascorbate, peroxidase ...

    African Journals Online (AJOL)

    Activities of peroxidase (POD), Polyphenol oxidase (PPO), hydroperoxide and lipid contents were investigated during desiccation of cotyledonary tissues of Irvingia gabonensis at ambient temperature (26OC - 30OC), 35OC and 20OC. Activities of POD and PPO increased initially but declined in the latter desiccation period.

  12. Cross Reactivities of Rabbit Anti-Chicken Horse Radish Peroxidase ...

    African Journals Online (AJOL)

    Dr Olaleye

    (BSA) was used as blocking agent. Sera of other avian species and mammals did not react with the conjugate. It is concluded that rabbit anti chicken Horse radish peroxidase could be used to detect antibodies in chickens as well as Turkey and that BSA and NRS could be used as blocking agent without loss of reactivities.

  13. Evaluation of Crude Oil Biodegradation Efficiency and Peroxidase ...

    African Journals Online (AJOL)

    ADOWIE PERE

    biomass enhanced degradation efficiency above 80 % after 10 days for all concentration of crude oil studied. Peroxidase production increased maximally during the log phase of ... mixer with equal volume of toluene to extract hydrocarbons from the samples. The extracted crude oil was detected at 420 nm. A blank was.

  14. KINETICS OF QUERCETIN NITRATIO N BY HORSERADISH PEROXIDASE

    Directory of Open Access Journals (Sweden)

    Andrija Šmelcerović

    2013-03-01

    Full Text Available In this study we investigated the kinetics of the nitration of quercetin by horseradish peroxidase. Quercetin nitration reaction was followed by recording the spectral changes over the time at 380 nm. The reaction rate increases with increasing of the quercetin concentration and follows the Michaelis-Menten type kinetics. Kinetic parameters of the studied enzymatic reaction were determined.

  15. Optimisation of soybean peroxidase treatment of 2, 4-dichlorophenol ...

    African Journals Online (AJOL)

    In the presence of hydrogen peroxide (H2O2), peroxidase enzymes (PE) catalyse the oxidation of various chlorinated phenols to free radicals, which then combine to form insoluble polymers that precipitate out of solution. This study systematically characterises the treatment of 2, 4-dichlorophenol (2, 4-DCP) using soybean ...

  16. Optimisation of soybean peroxidase treatment of 2,4-dichlorophenol

    African Journals Online (AJOL)

    drinie

    2002-04-02

    Apr 2, 2002 ... In the presence of hydrogen peroxide (H2O2), peroxidase enzymes (PE) catalyse the oxidation of various chlorinated phenols to free radicals, which then combine to form insoluble polymers that precipitate out of solution. This study systematically characterises the treatment of 2,4-dichlorophenol (2,4-DCP) ...

  17. Kinetics of the reaction of compound III of horseradish peroxidase ...

    African Journals Online (AJOL)

    Each reaction was first order with respect to the concentration of horseradish peroxidase. The observed rate constants were ionic strength dependent within the range of 0.06 – 0.30 M. The logarithmic values of the rate constants against the square root of the ionic strength showed that both NADPH and Compound III of ...

  18. Production of lignin peroxidase by Ganoderma leucidum using solid ...

    African Journals Online (AJOL)

    Jane

    2011-08-29

    Aug 29, 2011 ... The main objectives of this study were to optimize the culture conditions for the production of lignin peroxidase by Ganoderma leucidum, economic utilization of waste corn cobs as inducers substrate by pollution free fermentation technology and to optimize the solid state fermentation (SSF) process for.

  19. Molecular cloning and characterization of a new peroxidase gene ...

    African Journals Online (AJOL)

    length cDNA of O.violaceus peroxidase gene (OvRCI, GenBank. Acc. No. AY428037) was 1220 bp and contained an 1128 bp open reading frame encoding a protein of 375 amino acids. Homology analysis and molecular modeling revealed that ...

  20. Frequency of anti thyroid peroxidase antibody in patients of vitiligo

    International Nuclear Information System (INIS)

    Zhokhar, A.; Shaikh, Z.I.

    2013-01-01

    Objective: The objective of this study was to compare the frequency of anti thyroid peroxidase antibody in patients suffering from vitiligo with healthy control group. Type of Study: Case control study. Settings: Dermatology Department, Military Hospital, Rawalpindi, from 20th March 2010 to 20th July 2011. Material and Methods: Fifty clinically diagnosed patients of vitiligo, age = 18 yrs and both genders with no history of thyroid disease, past or current use of drugs for thyroid disorder or thyroid surgery were included as cases (Group A). Fifty healthy individuals with no evidence of vitiligo or thyroid disorder on history and physical examination and with no family history of vitiligo, matched for age and gender with cases, were included as control (Group B). Serum anti thyroid peroxidase (anti TPO) antibodies were measured using enzyme linked immunosorbent assay (ELISA) in both cases and control. Results: Eight (16%) patients in Group A were anti-thyroid peroxidase antibody positive and forty two (84%) patients were negative while one (2%) patient was anti-thyroid peroxidase antibody positive in Group B and forty nine (98%) patients were negative (p = 0.001). Conclusion: Anti TPO antibody is significantly more common in patients of vitiligo as compared to general population. (author)

  1. Selenite supplementation in euthyroid subjects with thyroid peroxidase antibodies

    NARCIS (Netherlands)

    Eskes, Silvia A.; Endert, Erik; Fliers, Eric; Birnie, Erwin; Hollenbach, Birgit; Schomburg, Lutz; Köhrle, Josef; Wiersinga, Wilmar M.

    2014-01-01

    Euthyroid thyroid peroxidase (TPO-Ab)-positive subjects are at risk for progression to subclinical and overt autoimmune hypothyroidism. Previous studies have shown a decrease in TPO-Ab and improvement of quality-of-life (QoL) in L-T4-treated hypothyroid patients upon selenium supplementation. To

  2. Cross reactivities of rabbit anti-chicken horse radish peroxidase ...

    African Journals Online (AJOL)

    The cross reactivities of rabbit anti chicken horse radish peroxidase (conjugate) was tested with sera of Chicken, Ducks, Geese, Guinea fowl, Hawks, Pigeons and Turkeys in indirect enzyme linked immunosorbent assay (ELISA) technique. Sera from mammalian species (Bat, Equine and swine) were used as negative ...

  3. Polyamines, peroxidase and proteins involved in the senescence ...

    African Journals Online (AJOL)

    Senescence is the natural aging process at the cellular level or range of phenomena associated with this process. The objective of this review was to show the involvement of substances that may be related to senescence in plants, such as polyamines, peroxidase and proteins. These substances were related with the ...

  4. Thylakoid-bound ascorbate peroxidase increases resistance to salt ...

    African Journals Online (AJOL)

    Reactive oxygen species (ROS) are cellular indicators of stress. In plants, they function as secondary messengers in response to environmental stress. Ascorbate peroxidase (APX) is an important enzyme directly involved in the scavenging of ROS. In this study, we aimed at identifying the function of the Brassica napus ...

  5. Peroxidase-like catalytic activities of ionic metalloporphyrins ...

    Indian Academy of Sciences (India)

    Unknown

    present in high concentration in the root of the horseradish plant. Others of interest are cytochrome c-peroxidase, chloroperoxidase, myeloperoxidase, ... uniformly spreading the fine paste obtained on a strip of Whatmann 41 filter paper. The reference strip contained paste made from the porphyrin-free polymer support. 3.

  6. Candida albicans biofilm on titanium: effect of peroxidase precoating

    Directory of Open Access Journals (Sweden)

    Mohamed Ahariz

    2010-08-01

    Full Text Available Mohamed Ahariz1, Philippe Courtois1,21Laboratory of Experimental Hormonology, Université Libre de Bruxelles, Brussels, 2UER de Biologie Médicale, Haute Ecole Francisco Ferrer, Brussels, BelgiumAbstract: The present study aimed to document Candida albicans biofilm development on titanium and its modulation by a peroxidase-precoated material which can generate antimicrobials, such as hypoiodite or hypothiocyanite, from hydrogen peroxide, iodide, or thiocyanate. For this purpose, titanium (powder or foil was suspended in Sabouraud liquid medium inoculated with C. albicans ATCC10231. After continuous stirring for 2–21 days at room temperature, the supernatant was monitored by turbidimetry at 600 nm and titanium washed three times in sterile Sabouraud broth. Using the tetrazolium salt MTT-formazan assay, the titanium-adherent fungal biomass was measured as 7.50 ± 0.60 × 106 blastoconidia per gram of titanium powder (n = 30 and 0.50 ± 0.04 × 106 blastoconidia per cm² of titanium foil (n = 12. The presence of yeast on the surface of titanium was confirmed by microscopy both on fresh preparations and after calcofluor white staining. However, in the presence of peroxidase systems (lactoperoxidase with substrates such as hydrogen peroxide donor, iodide, or thiocyanate, Candida growth in both planktonic and attached phases appeared to be inhibited. Moreover, this study demonstrates the possible partition of peroxidase systems between titanium material (peroxidase-precoated and liquid environment (containing peroxidase substrates to limit C. albicans biofilm formation.Keywords: adhesion, material, oral, yeast

  7. Phenol removal by peroxidases extracted from Chinese cabbage root

    Energy Technology Data Exchange (ETDEWEB)

    Rhee, H.I.; Jeong, Y.H. [Kangwon National Univ., Chuncheon (Korea, Republic of). Dept. of Applied Biology and Technology

    1995-12-31

    More than four million tons of Chinese cabbages are produced in Korea. Most of them are used as raw materials for Kimchi, but root parts of them are discarded as agricultural wastes. A trial for the application of agricultural waste to industrial waste water treatment was made as an effort to the efficient use of natural resources and to reduce water pollution problem simultaneously. Peroxidases of both solid and liquid phases were obtained from Chinese cabbage roots by using commercial juicer. The differences in peroxidase activity among the various cultivars of Chinese cabbages in Korea were little and electrophoretic patterns of various peroxidases will be discussed. The optimum pH and temperature for enzyme activity will be discussed also. Since peroxidases are distributed into 66% in liquid (juice) and 34% in solid phase (pulp), enzymes from both phases were applied to investigate the enzymatic removal of phenol from waste water. After phenol solution at 150 ppm being reacted with liquid phase enzyme (1,800 unit/1) for 3 hours in a batch stirred reactor, 96% of phenol could be removed through polymerization and precipitation. Also, phenol could be removed from initial 120 ppm to final 5 ppm by applying solid phase enzyme in an air lift reactor (600 unit/1). Almost equivalent efficiencies of phenol removal were observed between two systems, even though only one third of the enzymes in batch stirred reactor was applied in air lift reactor. The possible reason for this phenomenon is because peroxidases exist as immobilized forms in solid phase.

  8. Induction of Laccase, Lignin Peroxidase and Manganese Peroxidase Activities in White-Rot Fungi Using Copper Complexes

    Directory of Open Access Journals (Sweden)

    Martina Vrsanska

    2016-11-01

    Full Text Available Ligninolytic enzymes, such as laccase, lignin peroxidase and manganese peroxidase, are biotechnologically-important enzymes. The ability of five white-rot fungal strains Daedaleopsis confragosa, Fomes fomentarius, Trametes gibbosa, Trametes suaveolens and Trametes versicolor to produce these enzymes has been studied. Three different copper(II complexes have been prepared ((Him[Cu(im4(H2O2](btc·3H2O, where im = imidazole, H3btc = 1,3,5-benzenetricarboxylic acid, [Cu3(pmdien3(btc](ClO43·6H2O and [Cu3(mdpta3(btc](ClO43·4H2O, where pmdien = N,N,N′,N′′,N′′-pentamethyl-diethylenetriamine and mdpta = N,N-bis-(3-aminopropylmethyl- amine, and their potential application for laccase and peroxidases induction have been tested. The enzyme-inducing activities of the complexes were compared with that of copper sulfate, and it has been found that all of the complexes are suitable for the induction of laccase and peroxidase activities in white-rot fungi; however, the newly-synthesized complex M1 showed the greatest potential for the induction. With respect to the different copper inducers, this parameter seems to be important for enzyme activity, which depends also on the fungal strains.

  9. Ligninolytic enzymes of the fungus Irpex lacteus (Polyporus tulipiferae): isolation and characterization of lignin peroxidase

    Czech Academy of Sciences Publication Activity Database

    Rothschild, N.; Novotný, Čeněk; Šašek, Václav; Dosoretz, C. G.

    2002-01-01

    Roč. 31, - (2002), s. 627-633 ISSN 0141-0229 Institutional research plan: CEZ:AV0Z5020903 Keywords : lignin * peroxidase * heme peroxidase Subject RIV: EE - Microbiology, Virology Impact factor: 1.773, year: 2002

  10. Lignin-degrading Peroxidases from Genome of Selective Ligninolytic Fungus Ceriporiopsis subverispora

    Science.gov (United States)

    Elena Fernandez-Fueyo; Francisco J. Ruiz-Duenas; Yuta Miki; Marta Jesus Martinez; Kenneth E. Hammel; Angel T. Martinez

    2012-01-01

    Background: The first genome of a selective lignin degrader is available. Results: Its screening shows 26 peroxidase genes, and 5 genes were heterologously expressed and the catalytic properties investigated. Conclusion: Two new peroxidases oxidize simple and dimeric lignin models and efficiently depolymerize lignin. Significance: Although lignin peroxidase and...

  11. Purification and characterization of an intracellular peroxidase from Streptomyces cyaneus.

    OpenAIRE

    Mliki, A; Zimmermann, W

    1992-01-01

    An intracellular peroxidase (EC 1.11.1.7) from Streptomyces cyaneus was purified to homogeneity. The enzyme had a molecular weight of 185,000 and was composed of two subunits of equal size. It had an isoelectric point of 6.1. The enzyme had a peroxidase activity toward o-dianisidine with a Km of 17.8 microM and a pH optimum of 5.0. It also showed catalase activity with a Km of 2.07 mM H2O2 and a pH optimum of 8.0. The purified enzyme did not catalyze C alpha-C beta bond cleavage of 1,3-dihydr...

  12. Peroxidase-like catalytic activities of ionic metalloporphyrins ...

    Indian Academy of Sciences (India)

    Unknown

    The ideal pH was seen to be in the 8⋅0–8⋅5 range, with maximum effect at 8⋅2. The efficiency order for the various PS-MTPPS was seen to be Co>Mn>Fe, with CoTPPS showing efficiency comparable to that of horseradish peroxidase. The catalytic efficiency was found to be increasing with temperature for all the catalysts.

  13. Potential Applications of Peroxidases in the Fine Chemical Industries

    Science.gov (United States)

    Casella, Luigi; Monzani, Enrico; Nicolis, Stefania

    A description of selected types of reactions catalyzed by heme peroxidases is given. In particular, the discussion is focused mainly on those of potential interest for fine chemical synthesis. The division into subsections has been done fromthe point of view of the enzyme action, i.e., giving emphasis to themechanismof the enzymatic reaction, and from that of the substrate, i.e., analyzing the type of transformation promoted by the enzyme. These two approaches have several points in common.

  14. Polyphenoloxidase and peroxidase in avocado pulp (Persea americana Mill.)

    OpenAIRE

    Vanini,Lucimara Salvat; Kwiatkowski,Angela; Clemente,Edmar

    2010-01-01

    The aim of the present investigation was to evaluate the enzymatic activity of polyphenoloxidase and peroxidase in avocado pulps, from the Northwest area of Paraná-Brazil, in order to compare the varieties on their enzymatic activity for both, minimum and industrial processing. Enzymatic extracts were prepared from avocado pulp of Choquete, Fortuna and Quintal varieties, in green and ripe maturation stage. Thermal treatment was applied with temperatures 60, 65, 70, 75 and 80 °C. The enzymatic...

  15. Kinetic mechanism and nucleotide specificity of NADH peroxidase

    Energy Technology Data Exchange (ETDEWEB)

    Stoll, V.S.; Blanchard, J.S.

    1988-02-01

    NADH peroxidase is a flavoprotein isolated from Streptococcus faecalis which catalyzes the pyridine nucleotide-dependent reduction of hydrogen peroxide to water. Initial velocity, product, and dead-end inhibition studies have been performed at pH 7.5 and support a ping-pong kinetic mechanism. In the absence of hydrogen peroxide, both transhydrogenation between NADH and thioNAD, and isotope exchange between (/sup 14/C)NADH and NAD, have been demonstrated, although in both these experiments, the maximal velocity of nucleotide exchange was less than 1.5% the maximal velocity of the peroxidatic reaction. We propose that NADH binds tightly to both oxidized and two-electron reduced enzyme. NADH oxidation proceeds stereospecifically with the transfer of the 4S hydrogen to enzyme, and then, via exchange, to water. No primary tritium kinetic isotope effect was observed, and no statistically significant primary deuterium kinetic isotope effects on V/K were determined, although primary deuterium kinetic isotope effects on V were observed in the presence and absence of sodium acetate. NADH peroxidase thus shares with other flavoprotein reductases striking kinetic, spectroscopic, and stereochemical similarities. On this basis, we propose a chemical mechanism for the peroxide cleaving reaction catalyzed by NADH peroxidase which involves the obligate formation of a flavinperoxide, and peroxo bond cleavage by nucleophilic attack by enzymatic dithiols.

  16. Kinetic mechanism and nucleotide specificity of NADH peroxidase

    International Nuclear Information System (INIS)

    Stoll, V.S.; Blanchard, J.S.

    1988-01-01

    NADH peroxidase is a flavoprotein isolated from Streptococcus faecalis which catalyzes the pyridine nucleotide-dependent reduction of hydrogen peroxide to water. Initial velocity, product, and dead-end inhibition studies have been performed at pH 7.5 and support a ping-pong kinetic mechanism. In the absence of hydrogen peroxide, both transhydrogenation between NADH and thioNAD, and isotope exchange between [ 14 C]NADH and NAD, have been demonstrated, although in both these experiments, the maximal velocity of nucleotide exchange was less than 1.5% the maximal velocity of the peroxidatic reaction. We propose that NADH binds tightly to both oxidized and two-electron reduced enzyme. NADH oxidation proceeds stereospecifically with the transfer of the 4S hydrogen to enzyme, and then, via exchange, to water. No primary tritium kinetic isotope effect was observed, and no statistically significant primary deuterium kinetic isotope effects on V/K were determined, although primary deuterium kinetic isotope effects on V were observed in the presence and absence of sodium acetate. NADH peroxidase thus shares with other flavoprotein reductases striking kinetic, spectroscopic, and stereochemical similarities. On this basis, we propose a chemical mechanism for the peroxide cleaving reaction catalyzed by NADH peroxidase which involves the obligate formation of a flavinperoxide, and peroxo bond cleavage by nucleophilic attack by enzymatic dithiols

  17. Chemiluminescent detection systems of horseradish peroxidase employing nucleophilic acylation catalysts.

    Science.gov (United States)

    Marzocchi, Ettore; Grilli, Stefano; Della Ciana, Leopoldo; Prodi, Luca; Mirasoli, Mara; Roda, Aldo

    2008-06-15

    The light output of the peroxidase-catalyzed luminol chemiluminescent oxidation reaction can be greatly increased by incorporating different enhancers. Such an increase is attributed to the preferential oxidation of the enhancer by peroxidase intermediates and the rapid formation of enhancer radicals that, in turn, quickly oxidize luminol to its radical anion. These enhancers, which include substituted phenols, substituted boronic acids, indophenols, and N-alkyl phenothiazines, behave as electron transfer mediators. A further, very significant increase in light output was also observed by the addition of nucleophilic acylation catalyst to the enhancer/luminol/oxidant substrate. The effect of the new component is general and applicable to many of the known enhancers but is much more remarkable in association with phenothiazine enhancers (up to 10-fold light output). The addition of a nucleophilic acylation catalyst to these substrates lowered the limit of detection for horseradish peroxidase from 50 to 8 amol. Similar improvements were observed in "sandwich" enzyme-linked immunosorbent assays and Western blot assays.

  18. Cloning and Characterization of a cDNA Encoding a Novel Extracellular Peroxidase from Trametes versicolor

    Science.gov (United States)

    Collins, Patrick J.; O’Brien, Margaret M.; Dobson, Alan D. W.

    1999-01-01

    The white rot basidiomycete Trametes versicolor secretes a large number of peroxidases which are believed to be involved in the degradation of polymeric lignin. These peroxidases have been classified previously as lignin peroxidases or manganese peroxidases (MnP). We have isolated a novel extracellular peroxidase-encoding cDNA sequence from T. versicolor CU1, the transcript levels of which are repressed by low concentrations of Mn2+ and induced by nitrogen and carbon but not induced in response to a range of stresses which have been reported to induce MnP expression. PMID:10049906

  19. DYNAMICS OF LEAF PEROXIDASE ACTIVITY DURING ONTOGENY OF HEMP PLANTS, IN RELATION TO SEXUAL PHENOTYPE

    Directory of Open Access Journals (Sweden)

    Elena Truta

    2005-08-01

    Full Text Available During vegetation of female and male hemp plants (Cannabis sativa L., five quantitative determinations of peroxidase activities were made (40 days, 55 days, 70 days, 85 days, 105 days. Peroxidase activity presented some differences in hemp plants, between females and males, during their vegetation cycle. In female plants, before anthesis were registered peaks of peroxidase activities. The blossoming of male plants was coincident with the increase of catalitic action of peroxidase. Generally, the male plants displayed greater levels of peroxidasic activity.

  20. Arabidopsis ATP A2 peroxidase. Expression and high-resolution structure of a plant peroxidase with implications for lignification

    DEFF Research Database (Denmark)

    Ostergaard, L; Teilum, K; Mirza, O

    2000-01-01

    Lignins are phenolic biopolymers synthesized by terrestrial, vascular plants for mechanical support and in response to pathogen attack. Peroxidases have been proposed to catalyse the dehydrogenative polymerization of monolignols into lignins, although no specific isoenzyme has been shown...... to be involved in lignin biosynthesis. Recently we isolated an extracellular anionic peroxidase, ATP A2, from rapidly lignifying Arabidopsis cell suspension culture and cloned its cDNA. Here we show that the Atp A2 promoter directs GUS reporter gene expression in lignified tissues of transgenic plants. Moreover......, an Arabidopsis mutant with increased lignin levels compared to wild type shows increased levels of ATP A2 mRNA and of a mRNA encoding an enzyme upstream in the lignin biosynthetic pathway. The substrate specificity of ATP A2 was analysed by X-ray crystallography and docking of lignin precursors. The structure...

  1. Production and characterization of monoclonal antibodies to wall-localized peroxidases from corn seedlings

    Science.gov (United States)

    Kim, S. H.; Terry, M. E.; Hoops, P.; Dauwalder, M.; Roux, S. J.

    1988-01-01

    A library of 22 hybridomas, which make antibodies to soluble wall antigens from the coleoptiles and primary leaves of etiolated corn (Zea mays L.) seedlings, was raised and cloned three times by limit dilution to assure monoclonal growth and stability. Two of these hybridomas made immunoglobulin G antibodies, designated mWP3 and mWP19, which both effectively immunoprecipitated peroxidase activity from crude and partially purified preparations of wall peroxidases. Direct peroxidase-binding assays revealed that both antibodies bound enzymes with peroxidase activity. As judged by immunoblot analyses, mWP3 recognized a Mr 98,000 wall peroxidase with an isoelectric point near 4.2, and mWP19 recognized a Mr 58,000 wall peroxidase. Immunogold localization studies showed both peroxidases are predominately in cell walls.

  2. Mn(II) regulation of lignin peroxidases and manganese-dependent peroxidases from lignin-degrading white rot fungi

    International Nuclear Information System (INIS)

    Bonnarme, P.; Jeffries, T.W.

    1990-01-01

    Two families of peroxidases-lignin peroxidase (LiP) and manganese-dependent lignin peroxidase (MnP)-are formed by the lignin-degrading white rot basidiomycete Phanerochaete chrysosporium and other white rot fungi. Isoenzymes of these enzyme families carry out reactions important to the biodegradation of lignin. This research investigated the regulation of LiP and MnP production by Mn(II). In liquid culture, LiP titers varied as an inverse function of and MnP titers varied as a direct function of the Mn(II) concentration. The extracellular isoenzyme profiles differed radically at low and high Mn(II) levels, whereas other fermentation parameters, including extracellular protein concentrations, the glucose consumption rate, and the accumulation of cell dry weight, did not change significantly with the Mn(II) concentration. In the absence of Mn(II), extracellular LiP isoenzymes predominated, whereas in the presence of Mn(II), MnP isoenzymes were dominant. The release of 14 CO 2 from 14 C-labeled dehydrogenative polymerizate lignin was likewise affected by Mn(II). The rate of 14 CO 2 release increased at low Mn(II) and decreased at high Mn(II) concentrations. This regulatory effect of Mn(II) occurred with five strains of P. chrysosporium, two other species of Phanerochaete, three species of Phlebia, Lentinula edodes, and Phellinus pini

  3. Purification and characterization of peroxidase from sprouted green gram (Vigna radiata) roots and removal of phenol and p-chlorophenol by immobilized peroxidase.

    Science.gov (United States)

    Basha, Shaik Akbar; Prasada Rao, Ummiti Js

    2017-08-01

    Peroxidase activity was increased during germination of green gram and such an increase may have benefits in many physiological processes. The present study aimed to investigate the optimum conditions for the extraction, purification and characterization of peroxidase from the germinated green gram roots and also its application for the removal of phenols in water. Peroxidase activity was increased by 300-fold in 5-day germinated green gram. Because the root was rich in peroxidase activity, peroxidase from roots was isolated and purified to homogeneity. The purified peroxidase showed a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis with a molecular weight of 50 kDa, an optimum pH of 5.5 and a pH stability ranging from 5 to 9. The enzyme had 50% residual activity at 70 °C. It catalyzed the oxidation of a variety of substrates. The K m value of the enzyme was 1.28 mmol L -1 for o-dianisidine and 0.045 mmol L -1 for H 2 O 2 . The enzyme lost 100% activity in the presence of dithiothreitol and cysteine. The addition of copper ion increased the enzyme activity by three-fold. Both soluble and immobilized peroxidases removed more phenol than p-chlorphenol, whereas horseradish peroxidase removed more p-chlorphenol. Thus, the green gram root peroxidase showed good pH and temperature stability, as well as the ability to remove phenolic compounds from effluent. Peroxidase with good thermal and pH stability was purified from germinated green gram roots and has the ability to oxidize phenolic compounds from waste water. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  4. Peroxidase synthesis and activity in the interaction of soybean with Phytophthora megasperma f. sp. glycinea (Pmg)

    International Nuclear Information System (INIS)

    Chibbar, R.N.; Esnault, R.; Lee, D.; van Huystee, R.B.; Ward, E.W.B.

    1986-01-01

    Changes, in peroxidase (EC1.11.1.7) have been reported following infection. However, determinations of biosynthesis of quantities of the peroxidase protein molecule have not been made! In this study hypocotyl of soybean seedlings (Glycine max; cv Harosoy, susceptible; cv Harosoy 63, resistant) were inoculated with zoospores of Pmg. Incorporation of 35 S-methionine (supplied with inoculum) in TCA precipitates was measured. Peroxidase synthesis was measured by immuno precipitation using antibodies against a cationic and an anionic peroxidase derived from peanut cells. Specific peroxidase activity increased rapidly from 5 to 9 h following infection in the resistant reaction but not in the susceptible reaction or the water controls. There was increased synthesis of the anionic peroxidase but not of the cationic peroxidase in the resistant reaction. The anionic peroxidase did not increase in the susceptible until 15 h. The ratio of peroxidase synthesis to total protein synthesis decreased in inoculated tissues compared to control. Peroxidase synthesis is, therefore, a relative minor host response to infection

  5. Isolation and purification of membrane-bound cytochrome c from ...

    African Journals Online (AJOL)

    Administrator

    2007-05-02

    ferrochrome and redox spectra showed the presence of heme-c. Key words: Cytochrome c, respiratory chain and Proteus mirabilis. INTRODUCTION. Proteus mirabilis is facultative anaerobic, rod-shaped, gram negative bacterium.

  6. Investigating membrane-bound Argonaute functions in Arabidopsis

    DEFF Research Database (Denmark)

    Barghetti, Andrea

    by direct cleavage or by inhibition of their translation. Arabidopsis genome encode for 10 different AGO proteins, of which AGO1 is the main effector of post-transcriptional gene silencing mediated by sRNA. Importantly, a fraction of AGO1 population is associated with endomembranes, in particular...

  7. Biogenesis of membrane bound respiratory complexes in Escherichia coli

    OpenAIRE

    Price, Claire E.; Driessen, Arnold J.M.

    2010-01-01

    Escherichia colt is one of the preferred bacteria for studies on the energetics and regulation of respiration Respiratory chains consist of primary dehydrogenases and terminal reductases or oxidases linked by quinones. In order to assemble this complex arrangement of protein complexes, synthesis of the subunits occurs in the cytoplasm followed by assembly in the cytoplasm and/or membrane, the incorporation of metal or organic cofactors and the anchoring of the complex to the membrane In the c...

  8. Biogenesis of membrane bound respiratory complexes in Escherichia coli

    NARCIS (Netherlands)

    Price, Claire E.; Driessen, Arnold J. M.

    Escherichia colt is one of the preferred bacteria for studies on the energetics and regulation of respiration Respiratory chains consist of primary dehydrogenases and terminal reductases or oxidases linked by quinones. In order to assemble this complex arrangement of protein complexes, synthesis of

  9. Isolation and purification of membrane-bound cytochrome c from ...

    African Journals Online (AJOL)

    In the present studies, respiratory chain pathogenic bacterium, Proteus mirabilis, was investigated. In the first phase, growth profile study was performed to optimize the P. mirabilis growth. Maximum bacterial growth could be obtained between 10 – 12 h of culturing time. Down-stream processing was performed by using ...

  10. Calcium Modulation of Plant Plasma Membrane-Bound Atpase Activities

    Science.gov (United States)

    Caldwell, C.

    1983-01-01

    The kinetic properties of barley enzyme are discussed and compared with those of other plants. Possibilities for calcium transport in the plasma membrane by proton pump and ATPase-dependent calcium pumps are explored. Topics covered include the ph phase of the enzyme; high affinity of barley for calcium; temperature dependence, activation enthalpy, and the types of ATPase catalytic sites. Attention is given to lipids which are both screened and bound by calcium. Studies show that barley has a calmodulin activated ATPase that is found in the presence of magnesium and calcium.

  11. Bacillary haemoglobinuria diagnosis by the peroxidase-antiperoxidase (PAP) technique.

    Science.gov (United States)

    Uzal, F A; Belak, K; Rivera, E; Robles, C A; Feinstein, R E

    1992-10-01

    A peroxidase-antiperoxidase (PAP) technique was used to diagnose bacillary haemoglobinuria in formalin-fixed, paraffin-embedded liver tissues of cattle. The PAP method revealed Clostridium haemolyticum in the zone of liver necrosis characteristic of the disease and also in culture smears of this microorganism, but C. novyi type B, C. chauvoei, C. septicum and C. perfringens types B and C remained unstained by the PAP reaction. The PAP technique performed provides a specific, simple and rapid method to diagnose bacillary haemoglobinuria.

  12. Polyphenoloxidase and peroxidase in avocado pulp (Persea americana Mill. Polifenoloxidase e peroxidase na polpa de abacate (Persea americana Mill.

    Directory of Open Access Journals (Sweden)

    Lucimara Salvat Vanini

    2010-06-01

    Full Text Available The aim of the present investigation was to evaluate the enzymatic activity of polyphenoloxidase and peroxidase in avocado pulps, from the Northwest area of Paraná-Brazil, in order to compare the varieties on their enzymatic activity for both, minimum and industrial processing. Enzymatic extracts were prepared from avocado pulp of Choquete, Fortuna and Quintal varieties, in green and ripe maturation stage. Thermal treatment was applied with temperatures 60, 65, 70, 75 and 80 °C. The enzymatic activities were determined by using spectrophotometer. A decline of polyphenoloxidase activity was observed in all of the varieties when both, temperature and time increased. Total inactivation of enzymes was not observed in the largest temperature. Fortuna and Choquete variety showed the lowest polyphenoloxidase activity in the ripe stage. Soluble peroxidase showed activity in the green stage, whereas, ionically bound peroxidase activity increased with the change from green to ripe maturation stage in Choquete variety.O objetivo foi avaliar a atividade enzimática da polifenoloxidase (PPO e da peroxidase (POD em polpas de abacates, da região Noroeste do Paraná, Brasil, visando comparar as variedades e suas atividades enzimáticas para processamento mínimo ou industrial. Extratos enzimáticos foram preparados da polpa de abacate das variedades Choquete, Fortuna e Quintal no estágio de maturação verde e maduro. Foi aplicado tratamento térmico com temperaturas de 60, 65, 70, 75 e 80 °C. As atividades enzimáticas foram determinadas por espectrofotometria. Observou-se declínio da atividade da PPO, à medida que aumentava a temperatura e o tempo em todas as variedades. Não foi observada inativação total das enzimas na maior temperatura. As variedades Fortuna e Choquete apresentaram menor atividade da PPO no estágio maduro. A POD solúvel apresentou menor atividade no estágio verde, e, atividade da POD ionicamente ligada aumentou com a mudan

  13. Versatile peroxidase of Bjerkandera fumosa: substrate and inhibitor specificity.

    Science.gov (United States)

    Pozdnyakova, Natalia; Makarov, Oleg; Chernyshova, Marina; Turkovskaya, Olga; Jarosz-Wilkolazka, Anna

    2013-01-10

    The inhibitor and substrate specificities of versatile peroxidase from Bjerkandera fumosa (VPBF) were studied. Two different effects were found: NaN(3), Tween-80, anthracene, and fluorene decreased the activity of VPBF, but p-aminobenzoic acid increased it. A mixed mechanism of effector influence on the activity of this enzyme was shown. The catalytic properties of VPBF in the oxidation of mono- and polycyclic aromatic compounds were studied also. 2,7-Diaminofluorene, ABTS, veratryl alcohol, and syringaldazine can be oxidized by VPBF in two ways: either directly by the enzyme or by diffusible chelated Mn(3+) as an oxidizing agent. During VPBF oxidation of 2,7-diaminofluorene, both with and without Mn(2+), biphasic kinetics with apparent saturation in both micromolar and millimolar ranges were obtained. In the case of ABTS, inhibition of VPBF activity by an excess of substrate was observed. Direct oxidation of p-aminobenzoic acid by versatile peroxidase was found for the first time. The oxidation of three- and four-ring PAHs by VPBF was investigated, and the oxidation of anthracene, phenanthrene, fluorene, pyrene, chrysene, and fluoranthene was shown. The products of PAH oxidation (9,10-anthraquinone, 9,10-phenanthrenequinone, and 9-fluorenone) catalyzed by VPBF were identified. Copyright © 2012 Elsevier Inc. All rights reserved.

  14. Horseradish peroxidase-modified porous silicon for phenol monitoring

    Energy Technology Data Exchange (ETDEWEB)

    Kermad, A., E-mail: amina_energetique@yahoo.fr [Unité de Recherche Matériaux et Energies Renouvelables (URMER), Département de Physique, Faculté des Sciences, Université Abou Baker Belkaid, B.P. 119, Tlemcen 13000 (Algeria); Sam, S., E-mail: Sabrina.sam@polytechnique.edu [Centre de Recherche en Technologie des Semi-conducteurs pour l’Energétique (CRTSE), 02 Bd. Frantz-Fanon, B.P. 140, Alger-7 merveilles, Algiers (Algeria); Ghellai, N., E-mail: na_ghellai@yahoo.fr [Unité de Recherche Matériaux et Energies Renouvelables (URMER), Département de Physique, Faculté des Sciences, Université Abou Baker Belkaid, B.P. 119, Tlemcen 13000 (Algeria); Khaldi, K., E-mail: Khadidjaphy@yahoo.fr [Unité de Recherche Matériaux et Energies Renouvelables (URMER), Département de Physique, Faculté des Sciences, Université Abou Baker Belkaid, B.P. 119, Tlemcen 13000 (Algeria); Gabouze, N., E-mail: ngabouze@yahoo.fr [Centre de Recherche en Technologie des Semi-conducteurs pour l’Energétique (CRTSE), 02 Bd. Frantz-Fanon, B.P. 140, Alger-7 merveilles, Algiers (Algeria)

    2013-11-01

    Highlights: • Horseradish peroxidase enzyme (HRP) was covalently immobilized on porous silicon (PSi) surface. • Multistep strategy was used allowing the maintaining of the enzymatic activity of the immobilized enzyme. • Direct electron transfer has occurred between the immobilized enzyme and the surface. • Electrochemical measurements showed a response of HRP-modified PSi toward phenol in the presence of H{sub 2}O{sub 2}. -- Abstract: In this study, horseradish peroxidase enzyme (HRP) was covalently immobilized on porous silicon (PSi) surface using multistep strategy. First, acid terminations were generated on hydrogenated PSi surface by thermal hydrosilylation of undecylenic acid. Then, the carboxyl-terminated monolayer was transformed to active ester (succinimidyl ester) using N-hydroxysuccinimide (NHS) in the presence of the coupling agent N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide (EDC). Subsequently, the enzyme was anchored on the surface via an amidation reaction. The structure of the PSi layers was observed by scanning electron microscopy (SEM). Infrared spectroscopy (FTIR) and contact angle measurements confirmed the efficiency of the modification at each step of the functionalization. Cyclic voltammetry was recorded using the HRP-modified PSi as working electrode. The results show that the enzymatic activity of the immobilized HRP is preserved and in the presence of hydrogen peroxide, the enzyme oxidizes phenolic molecules which were subsequently reduced at the modified-PSi electrode.

  15. Horseradish peroxidase-modified porous silicon for phenol monitoring

    International Nuclear Information System (INIS)

    Kermad, A.; Sam, S.; Ghellai, N.; Khaldi, K.; Gabouze, N.

    2013-01-01

    Highlights: • Horseradish peroxidase enzyme (HRP) was covalently immobilized on porous silicon (PSi) surface. • Multistep strategy was used allowing the maintaining of the enzymatic activity of the immobilized enzyme. • Direct electron transfer has occurred between the immobilized enzyme and the surface. • Electrochemical measurements showed a response of HRP-modified PSi toward phenol in the presence of H 2 O 2 . -- Abstract: In this study, horseradish peroxidase enzyme (HRP) was covalently immobilized on porous silicon (PSi) surface using multistep strategy. First, acid terminations were generated on hydrogenated PSi surface by thermal hydrosilylation of undecylenic acid. Then, the carboxyl-terminated monolayer was transformed to active ester (succinimidyl ester) using N-hydroxysuccinimide (NHS) in the presence of the coupling agent N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide (EDC). Subsequently, the enzyme was anchored on the surface via an amidation reaction. The structure of the PSi layers was observed by scanning electron microscopy (SEM). Infrared spectroscopy (FTIR) and contact angle measurements confirmed the efficiency of the modification at each step of the functionalization. Cyclic voltammetry was recorded using the HRP-modified PSi as working electrode. The results show that the enzymatic activity of the immobilized HRP is preserved and in the presence of hydrogen peroxide, the enzyme oxidizes phenolic molecules which were subsequently reduced at the modified-PSi electrode

  16. Is Root Catalase a Bifunctional Catalase-Peroxidase?

    Science.gov (United States)

    Chioti, Vasileia; Zervoudakis, George

    2017-05-25

    Plant catalases exhibit spatial and temporal distribution of their activity. Moreover, except from the typical monofunctional catalase, a bifunctional catalase-peroxidase has been reported. The aim of this study was to investigate whether the leaf and root catalases from six different plant species ( Lactuca sativa , Cichorium endivia , Apium graveolens , Petroselinum crispum, Lycopersicon esculentum , and Solanum melongena ) correspond to the monofunctional or the bifunctional type based on their sensitivity to the inhibitor 3-amino-1,2,4-triazole (3-AT). The leaf catalases from all species seem to be monofunctional since they are very sensitive to 3-AT. On the other hand, the root enzymes from Lactuca sativa , Cichorium endivia , Lycopersicon esculentum , and Solanum melongena seem to be bifunctional catalase-peroxidases, considering that they are relatively insensitive to 3-AT, whereas the catalases from Apium graveolens and Petroselinum crispum display the same monofunctional characteristics as the leaves' enzymes. The leaf catalase activity is usually higher ( Lactuca sativa , Petroselinum crispum , and Solanum melongena ) or similar ( Cichorium endivia and Apium graveolens ) to the root one, except for the enzyme from Lycopersicon esculentum , while in all plant species the leaf protein concentration is significantly higher than the root protein concentration. These results suggest that there are differences between leaf and root catalases-differences that may correspond to their physiological role.

  17. Hierarchical hybrid peroxidase catalysts for remediation of phenol wastewater

    KAUST Repository

    Duan, Xiaonan

    2014-02-20

    We report a new family of hierarchical hybrid catalysts comprised of horseradish peroxidase (HRP)-magnetic nanoparticles for advanced oxidation processes and demonstrate their utility in the removal of phenol from water. The immobilized HRP catalyzes the oxidation of phenols in the presence of H2O2, producing free radicals. The phenoxy radicals react with each other in a non-enzymatic process to form polymers, which can be removed by precipitation with salts or condensation. The hybrid peroxidase catalysts exhibit three times higher activity than free HRP and are able to remove three times more phenol from water compared to free HRP under similar conditions. In addition, the hybrid catalysts reduce substrate inhibition and limit inactivation from reaction products, which are common problems with free or conventionally immobilized enzymes. Reusability is improved when the HRP-magnetic nanoparticle hybrids are supported on micron-scale magnetic particles, and can be retained with a specially designed magnetically driven reactor. The performance of the hybrid catalysts makes them attractive for several industrial and environmental applications and their development might pave the way for practical applications by eliminating most of the limitations that have prevented the use of free or conventionally immobilized enzymes. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Hierarchical hybrid peroxidase catalysts for remediation of phenol wastewater.

    Science.gov (United States)

    Duan, Xiaonan; Corgié, Stéphane C; Aneshansley, Daniel J; Wang, Peng; Walker, Larry P; Giannelis, Emmanuel P

    2014-04-04

    We report a new family of hierarchical hybrid catalysts comprised of horseradish peroxidase (HRP)-magnetic nanoparticles for advanced oxidation processes and demonstrate their utility in the removal of phenol from water. The immobilized HRP catalyzes the oxidation of phenols in the presence of H2 O2 , producing free radicals. The phenoxy radicals react with each other in a non-enzymatic process to form polymers, which can be removed by precipitation with salts or condensation. The hybrid peroxidase catalysts exhibit three times higher activity than free HRP and are able to remove three times more phenol from water compared to free HRP under similar conditions. In addition, the hybrid catalysts reduce substrate inhibition and limit inactivation from reaction products, which are common problems with free or conventionally immobilized enzymes. Reusability is improved when the HRP-magnetic nanoparticle hybrids are supported on micron-scale magnetic particles, and can be retained with a specially designed magnetically driven reactor. The performance of the hybrid catalysts makes them attractive for several industrial and environmental applications and their development might pave the way for practical applications by eliminating most of the limitations that have prevented the use of free or conventionally immobilized enzymes. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. The Effects on Gluten Strength and Bread Volume of Adding Soybean Peroxidase Enzyme to Wheat Flour

    OpenAIRE

    Kirby, Ratia

    2007-01-01

    The Effects on Gluten Strength and Bread Volume of Adding Soybean Peroxidase Enzyme to Wheat Flour Ratia Kirby ABSTRACT Soy peroxidase enzyme obtained from isoelectic precipitation procedures was added to all-purpose flour (APF) to assess its effects on the rheological properties and consumer acceptability of yeast bread. A pH 4.8 isoelectrically precipitated fraction from soybeans was used because it produced the most precipitate and had about the same peroxidase activity as the...

  20. Acquisition of Heat Stable Enzymes from Thermophilic Microorganisms: Peroxidases, Ureases, and Glucose Oxidases.

    Science.gov (United States)

    1992-04-01

    peroxidase production. The addition of methyl viologen ( Paraquat ) at micromolar levels generated a stress that resulted in an increased cellular...130 supematant from 10,000 MW cutoff Horseradish peroxidase has a molecular weight of 40,000 (Glenn). Most other peroxidases from plants , fungi, and...this project resulted in selection of two bacteria producing thermally stable urease. Origin Urease is found in many plants , bacteria, fungi, yeast and

  1. Association of salivary peroxidase activity and concentration with periodontal health: A validity study.

    Science.gov (United States)

    Memarzadeh Zahedani, Maral; Schwahn, Christian; Baguhl, Romy; Kocher, Thomas; Below, Harald; Welk, Alexander

    2017-08-01

    Whereas the relationship between myeloperoxidase and periodontitis has been widely examined that between salivary peroxidase and periodontitis has received little attention. We examined how periodontitis depends on both salivary peroxidase activity and concentration. A full mouth, clinical assessment of probing depth was performed in a sample of 46 participants aged 25-54 years. To minimise bias, these data were corrected by data from the general population (Study of Health in Pomerania). Using five repeated measurements of activity and concentration over 1 day, we assessed daily biological variability and increased the reliability of salivary peroxidase measurements. Salivary peroxidase activity was associated with probing depth (interquartile range effect = -0.48; robust estimates of 95% confidence interval: -0.90 to -0.31; p = .0052), and its effect was not confounded by salivary peroxidase concentration. In turn, the effect of salivary peroxidase concentration was confounded by salivary peroxidase activity, and it was smaller than that of activity. We found an inverse association between salivary peroxidase activity and probing depth. Thus, our results imply that salivary peroxidase activity could be a protective factor against periodontitis. However, large, well-designed studies are needed to explore the causal mechanisms of this association. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  2. The effects of xylitol and sorbitol on lysozyme- and peroxidase-related enzymatic and candidacidal activities.

    Science.gov (United States)

    Kim, Bum-Soo; Chang, Ji-Youn; Kim, Yoon-Young; Kho, Hong-Seop

    2015-07-01

    To investigate whether xylitol and sorbitol affect enzymatic and candidacidal activities of lysozyme, the peroxidase system, and the glucose oxidase-mediated peroxidase system. Xylitol and sorbitol were added to hen egg-white lysozyme, bovine lactoperoxidase, glucose oxidase-mediated peroxidase, and whole saliva in solution and on hydroxyapatite surfaces. The enzymatic activities of lysozyme, peroxidase, and glucose oxidase-mediated peroxidase were determined by the turbidimetric method, the NbsSCN assay, and production of oxidized o-dianisidine, respectively. Candidacidal activities were determined by comparing colony forming units using Candida albicans ATCC strains 10231, 11006, and 18804. While xylitol and sorbitol did not affect the enzymatic activity of hen egg-white lysozyme both in solution and on hydroxyapatite surfaces, they did inhibit the enzymatic activity of salivary lysozyme significantly in solution, but not on the surfaces. Xylitol and sorbitol enhanced the enzymatic activities of both bovine lactoperoxidase and salivary peroxidase significantly in a dose-dependent manner in solution, but not on the surfaces. Sorbitol, but not xylitol, inhibited the enzymatic activity of glucose oxidase-mediated peroxidase significantly. Both xylitol and sorbitol did not affect candidacidal activities of hen egg-white lysozyme, the bovine lactoperoxidase system, or the glucose oxidase-mediated bovine lactoperoxidase system. Xylitol and sorbitol inhibited salivary lysozyme activity, but enhanced both bovine lactoperoxidase and salivary peroxidase activities significantly in solution. Xylitol and sorbitol did not augment lysozyme- and peroxidase-related candidacidal activities. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Red blood cell glutathione peroxidase activity in female nulligravid and pregnant rats

    Directory of Open Access Journals (Sweden)

    Martino Guglielmo

    2009-01-01

    Full Text Available Abstract Background The alterations of the glutathione peroxidase enzyme complex system occur in physiological conditions such as aging and oxidative stress consequent to strenuous exercise. Methods Authors optimize the spectrophotometric method to measure glutathione peroxidase activity in rat red blood cell membranes. Results The optimization, when applied to age paired rats, both nulligravid and pregnant, shows that pregnancy induces, at seventeen d of pregnancy, an increase of both reactive oxygen substance concentration in red blood cells and membrane glutathione peroxidase activity. Conclusion The glutathione peroxidase increase in erythrocyte membranes is induced by systemic oxidative stress long lasting rat pregnancy.

  4. Peroxidase (POD and polyphenoloxidase (PPO in grape (Vitis vinifera L. Peroxidase (POD e polifenoloxidase (PPO em uva (Vitis vinifera L.

    Directory of Open Access Journals (Sweden)

    Estela de Pieri Troiani

    2003-06-01

    Full Text Available The enzimatic activity of peroxidase (POD and polyphenoloxidase (PPO extracted from three grape cultivars (Vitis vinifera L., cultivated in Marialva city, state of Paraná, was evaluated in this study. The enzymatic extracts were prepared starting from the Rubi, Borbon and Benitaka grape cultivars pulp and peel. The activity of the peroxidase was 53.00 units/100 g in the extract from the Rubi cultivar peel, and 327.00 units/100 g from the Benitaka cultivar, these values being superior to those observed in the same cultivars pulp extracts, which were 7.67 units/100 g and 44.00 units/100 g respectively. However, the result was opposite in the Borbon cultivar, with values of 141.11 units/100 g in the pulp and 11.50 units/100 g in the peel being found. The results of the polyphenoloxidase in the Borbon cultivar activity were 100.18 units/100 g in the pulp and 102.60 units/100 g in the peel, and in the Rubi and Benitaka cultivars were 60.40 units/100 g, 48.62 units/100 g in the pulp and 17.40 units/100 g, and 26.20 units/100 g in the peel, respectively. Protein determination was carried out in each extract, and the results found in the pulp and peel, respectively, were 0.56 and 0.64 mg/100 g for cultivar Benitaka, 1.38 and 6.45 mg/100 g for cultivar Rubi, and 21.38 and 5.68 mg/100 g for Borbon. The extracts were submitted to thermal treatments (60°C, 65°C, 70°C and 75°C for a 1 to 10 minutes period to observe the behavior of the peroxidase and polyphenoloxidase enzymatic activity, being verified a continuous decrease of the peroxidase and polyphenoloxidase activities as a result of the thermal treatment. The extracts of the Rubi and Benitaka cultivars were more heat stable than the extract from the Borbon cultivar for both enzymes. However, the temperatures used were not enough for a total inactivation of the enzymes.Neste trabalho, estudou-se a atividade enzimática da peroxidase (POD e da polifenoloxidase (PPO extraídas de três cultivares de

  5. Asparagus byproducts as a new source of peroxidases.

    Science.gov (United States)

    Jaramillo-Carmona, Sara; Lopez, Sergio; Vazquez-Castilla, Sara; Rodriguez-Arcos, Rocio; Jimenez-Araujo, Ana; Guillen-Bejarano, Rafael

    2013-07-03

    Soluble peroxidase (POD) from asparagus byproducts was purified by ion exchange chromatographies, and its kinetic and catalytic properties were studied. The isoelectric point of the purified isoperoxidases was 9.1, and the optimum pH and temperature values were 4.0 and 25 °C, respectively. The cationic asparagus POD (CAP) midpoint inactivation temperature was 57 °C, which favors its use in industrial processes. The Km values of cationic asparagus POD for H₂O₂ and ABTS were 0.318 and 0.634 mM, respectively. The purified CAP is economically obtained from raw materials using a simple protocol and possesses features that make it advantageous for the potential use of this enzyme in a large number of processes with demonstrated requirements of thermostable POD. The results indicate that CAP can be used as a potential candidate for removing phenolic contaminants.

  6. Erythrocytic glutathione peroxidase: Its relationship to plasma selenium in man

    International Nuclear Information System (INIS)

    Perona, G.; Cellerino, R.; Guidi, G.C.; Moschini, G.; Stievano, B.M.; Tregnaghi, C.

    1977-01-01

    Erythrocytic glutathione-peroxidase (GSH-Px) activity and plasma selenium concentrations were measured in 14 patients: 7 with iron deficiency and 7 with raised serum iron levels. The decreased enzymatic activity in iron deficiency was confirmed. Plasma selenium was significantly lower in patients with lower serum iron; furthermore there is a significant correlation between serum iron and plasma selenium concentrations. Another correlation even more significant was found between plasma selenium and enzyme activity in all the cases we studied. These data suggests that the importance of iron for GSH-Px activity may be merely due to its relationship with selenium and that plasma selenium concentration may be of critical importance for enzyme activity. (author)

  7. Computational Modeling of the Catalytic Cycle of Glutathione Peroxidase Nanomimic.

    Science.gov (United States)

    Kheirabadi, Ramesh; Izadyar, Mohammad

    2016-12-29

    To elucidate the role of a derivative of ebselen as a mimic of the antioxidant selenoenzyme glutathione peroxidase, density functional theory and solvent-assisted proton exchange (SAPE) were applied to model the reaction mechanism in a catalytic cycle. This mimic plays the role of glutathione peroxidase through a four-step catalytic cycle. The first step is described as the oxidation of 1 in the presence of hydrogen peroxide, while selenoxide is reduced by methanthiol at the second step. In the third step of the reaction, the reduction of selenenylsulfide occurs by methanthiol, and the selenenic acid is dehydrated at the final step. Based on the kinetic parameters, step 4 is the rate-determining step (RDS) of the reaction. The bond strength of the atoms involved in the RDS is discussed with the quantum theory of atoms in molecules (QTAIM). Low value of electron density, ρ(r), and positive Laplacian values are the evidence for the covalent nature of the hydrogen bonds rupture (O 30 -H 31 , O 33 -H 34 ). A change in the sign of the Laplacian, L(r), from the positive value in the reactant to a negative character at the transition state indicates the depletion of the charge density, confirming the N 5 -H 10 and O 11 -Se 1 bond breaking. The analysis of electron location function (ELF) and localized orbital locator (LOL) of the Se 1 -N 5 and Se 1 -O 11 bonds have been done by multi-WFN program. High values of ELF and LOL at the transition state regions between the Se, N, and O atoms display the bond formation. Finally, the main donor-acceptor interaction energies were analyzed using the natural bond orbital analysis for investigation of their stabilization effects on the critical bonds at the RDS.

  8. Glutathione peroxidase 4 and vitamin E cooperatively prevent hepatocellular degeneration

    Directory of Open Access Journals (Sweden)

    Bradley A. Carlson

    2016-10-01

    Full Text Available The selenoenzyme glutathione peroxidase 4 (Gpx4 is an essential mammalian glutathione peroxidase, which protects cells against detrimental lipid peroxidation and governs a novel form of regulated necrotic cell death, called ferroptosis. To study the relevance of Gpx4 and of another vitally important selenoprotein, cytosolic thioredoxin reductase (Txnrd1, for liver function, mice with conditional deletion of Gpx4 in hepatocytes were studied, along with those lacking Txnrd1 and selenocysteine (Sec tRNA (Trsp in hepatocytes. Unlike Txnrd1- and Trsp-deficient mice, Gpx4−/− mice died shortly after birth and presented extensive hepatocyte degeneration. Similar to Txnrd1-deficient livers, Gpx4−/− livers manifested upregulation of nuclear factor (erythroid-derived-like 2 (Nrf2 response genes. Remarkably, Gpx4−/− pups born from mothers fed a vitamin E-enriched diet survived, yet this protection was reversible as subsequent vitamin E deprivation caused death of Gpx4-deficient mice ~4 weeks thereafter. Abrogation of selenoprotein expression in Gpx4−/− mice did not result in viable mice, indicating that the combined deficiency aggravated the loss of Gpx4 in liver. By contrast, combined Trsp/Txnrd1-deficient mice were born, but had significantly shorter lifespans than either single knockout, suggesting that Txnrd1 plays an important role in supporting liver function of mice lacking Trsp. In sum our study demonstrates that the ferroptosis regulator Gpx4 is critical for hepatocyte survival and proper liver function, and that vitamin E can compensate for its loss by protecting cells against deleterious lipid peroxidation.

  9. The Roles of Glutathione Peroxidases during Embryo Development

    Science.gov (United States)

    Ufer, Christoph; Wang, Chi Chiu

    2011-01-01

    Embryo development relies on the complex interplay of the basic cellular processes including proliferation, differentiation, and apoptotic cell death. Precise regulation of these events is the basis for the establishment of embryonic structures and the organ development. Beginning with fertilization of the oocyte until delivery the developing embryo encounters changing environmental conditions such as varying levels of oxygen, which can give rise to reactive oxygen species (ROS). These challenges are met by the embryo with metabolic adaptations and by an array of anti-oxidative mechanisms. ROS can be deleterious by modifying biological molecules including lipids, proteins, and nucleic acids and may induce abnormal development or even embryonic lethality. On the other hand ROS are vital players of various signaling cascades that affect the balance between cell growth, differentiation, and death. An imbalance or dysregulation of these biological processes may generate cells with abnormal growth and is therefore potentially teratogenic and tumorigenic. Thus, a precise balance between processes generating ROS and those decomposing ROS is critical for normal embryo development. One tier of the cellular protective system against ROS constitutes the family of selenium-dependent glutathione peroxidases (GPx). These enzymes reduce hydroperoxides to the corresponding alcohols at the expense of reduced glutathione. Of special interest within this protein family is the moonlighting enzyme glutathione peroxidase 4 (Gpx4). This enzyme is a scavenger of lipophilic hydroperoxides on one hand, but on the other hand can be transformed into an enzymatically inactive cellular structural component. GPx4 deficiency – in contrast to all other GPx family members – leads to abnormal embryo development and finally produces a lethal phenotype in mice. This review is aimed at summarizing the current knowledge on GPx isoforms during embryo development and tumor development with an emphasis

  10. Quickly-released peroxidase of moss in defense against fungal invaders.

    Science.gov (United States)

    Lehtonen, Mikko T; Akita, Motomu; Kalkkinen, Nisse; Ahola-Iivarinen, Elina; Rönnholm, Gunilla; Somervuo, Panu; Thelander, Mattias; Valkonen, Jari P T

    2009-01-01

    Mosses (Bryophyta) are nonvascular plants that constitute a large part of the photosynthesizing biomass and carbon storage on Earth. Little is known about how this important portion of flora maintains its health status. This study assessed whether the moss, Physcomitrella patens, responds to treatment with chitosan, a fungal cell wall-derived compound inducing defense against fungal pathogens in vascular plants. Application of chitosan to liquid culture of P. patens caused a rapid increase in peroxidase activity in the medium. For identification of the peroxidase(s), matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF)/MS, other methods and the whole-genome sequence of P. patens were utilized. Peroxidase gene knock-out mutants were made and inoculated with fungi. The peroxidase activity resulted from a single secreted class III peroxidase (Prx34) which belonged to a P. patens specific phylogenetic cluster in analysis of the 45 putative class III peroxidases of P. patens and those of Arabidopsis and rice. Saprophytic and pathogenic fungi isolated from another moss killed the Prx34 knockout mutants but did not damage wild-type P. patens. The data point out the first specific host factor that is pivotal for pathogen defense in a nonvascular plant. Furthermore, results provide conclusive evidence that class III peroxidases in plants are needed in defense against hostile invasion by fungi.

  11. Use of an immuno-peroxidase staining method for the detection of ...

    African Journals Online (AJOL)

    Immunopurified antigens of axenic E. histolytica were used to produce rabbit hyper-immune sera. Immunoglobulin G (IgG) was purified from hyper-immune sera and coupled to peroxidase using a two-step procedure. The IgG-peroxidase conjugate was then evaluated by detection of E. histolytica in 128 stool samples and ...

  12. Purification and characterization of an intracellular catalase-peroxidase from Penicillium simplicissimum

    NARCIS (Netherlands)

    Fraaije, Marco W.; Roubroeks, Hanno P.; Hagen, Wilfred R.; Berkel, Willem J.H. van

    1996-01-01

    The first dimeric catalase-peroxidase of eucaryotic origin, an intracellular hydroperoxidase from Penicillium simplicissimum which exhibited both catalase and peroxidase activities, has been isolated. The enzyme has an apparent molecular mass of about 170 kDa and is composed of two identical

  13. The nop gene from Phanerochaete chrysosporium encodes a peroxidase with novel structural features

    Science.gov (United States)

    Luis F. Larrondo; Angel Gonzalez; Tomas Perez-Acle; Dan Cullen; Rafael Vicuna

    2005-01-01

    Inspection of the genome of the ligninolytic basidiomycete Phanerochaete chrysosporium revealed an unusual peroxidase-like sequence. The corresponding full length cDNA was sequenced and an archetypal secretion signal predicted. The deduced mature protein (NoP, novel peroxidase) contains 295 aa residues and is therefore considerably shorter than other Class II (fungal)...

  14. Peroxidase-mediated cross-linking of a tyrosine-containing peptide with ferulic acid

    NARCIS (Netherlands)

    Oudgenoeg, G.; Hilhorst, H.; Piersma, S.R.; Boeriu, C.G.; Gruppen, H.; Voragen, A.G.J.; Laane, C.

    2001-01-01

    The tyrosine-containing peptide Gly-Tyr-Gly (GYG) was oxidatively cross-linked by horseradish peroxidase in the presence of hydrogen peroxide. As products, covalently coupled di- to pentamers of the peptide were identified by LC-MS. Oxidative cross-linking of ferulic acid with horseradish peroxidase

  15. Regional localization of the gene for thyroid peroxidase to human chromosome 2pter----p12

    NARCIS (Netherlands)

    de Vijlder, J. J.; Dinsart, C.; Libert, F.; Geurts van Kessel, A.; Bikker, H.; Bolhuis, P. A.; Vassart, G.

    1988-01-01

    A 2.0-kb thyroid peroxidase cDNA of human origin was used as probe for Southern blot hybridization of genomic DNA from human somatic cells and human-rodent somatic cell hybrids. The results showed that the gene coding for human thyroid peroxidase is located on chromosome. 2. Further analysis of

  16. Inflammatory peroxidases promote breast cancer progression in mice via regulation of the tumour microenvironment.

    Science.gov (United States)

    Panagopoulos, Vasilios; Leach, Damien A; Zinonos, Irene; Ponomarev, Vladimir; Licari, Giovanni; Liapis, Vasilios; Ingman, Wendy V; Anderson, Peter; DeNichilo, Mark O; Evdokiou, Andreas

    2017-04-01

    Myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are heme-containing enzymes, well known for their antimicrobial activity, are released in high quantities by infiltrating immune cells in breast cancer. However, the functional importance of their presence within the tumour microenvironment is unclear. We have recently described a new role for peroxidases as key regulators of fibroblast and endothelial cell functionality. In the present study, we investigate for the first time, the ability of peroxidases to promote breast cancer development and progression. Using the 4T1 syngeneic murine orthotopic breast cancer model, we examined whether increased levels of peroxidases in developing mammary tumours influences primary tumour growth and metastasis. We showed that MPO and EPO stimulation increased mammary tumour growth and enhanced lung metastases, effects that were associated with reduced tumour necrosis, increased collagen deposition and neo-vascularisation within the primary tumour. In vitro, peroxidase treatment, robustly stimulated human mammary fibroblast migration and collagen type I and type VI secretion. Mechanistically, peroxidases induced the transcription of pro-tumorigenic and metastatic MMP1, MMP3 and COX-2 genes. Taken together, these findings identify peroxidases as key contributors to cancer progression by augmenting pro-tumorigenic collagen production and angiogenesis. Importantly, this identifies inflammatory peroxidases as therapeutic targets in breast cancer therapy.

  17. Polyvinylpyrrolidone (PVP)-Capped Pt Nanocubes with Superior Peroxidase-Like Activity

    Energy Technology Data Exchange (ETDEWEB)

    Ye, Haihang [Department of Chemistry, Michigan Technological University, Houghton Michigan 49931 United States; Liu, Yuzi [Center for Nanoscale Materials, Argonne National Laboratory, Argonne Illinois 60439 United States; Chhabra, Ashima [Department of Chemistry, Michigan Technological University, Houghton Michigan 49931 United States; Lilla, Emily [Department of Chemistry, Michigan Technological University, Houghton Michigan 49931 United States; Xia, Xiaohu [Department of Chemistry, Michigan Technological University, Houghton Michigan 49931 United States

    2016-12-21

    Peroxidase mimics of inorganic nanoparticles are expected to circumvent the inherent issues of natural peroxidases, providing enhanced performance in important applications such as diagnosis and imaging. Despite the report of a variety of peroxidase mimics in the past decade, very limited progress has been made on improving their catalytic efficiency. The catalytic efficiencies of most previously reported mimics are only up to one order of magnitude higher than those of natural peroxidases. In this work, we demonstrate a type of highly efficient peroxidase mimic – polyvinylpyrrolidone (PVP)-capped Pt nanocubes of sub-10 nm in size. These PVP-capped Pt cubes are ~200-fold more active than the natural counterparts and exhibit a record-high specific catalytic efficiency. In addition to the superior efficiency, the new mimic shows several other promising features, including excellent stabilities, well-controlled uniformity in both size and shape, controllable sizes, and facile and scalable production.

  18. Bromophenol blue discoloration using peroxidase immobilized on highly activated corncob powder

    Directory of Open Access Journals (Sweden)

    Júlio César Vinueza Galárraga

    2013-08-01

    Full Text Available The aim of the present study was to evaluate the efficacy of peroxidase immobilized on corncob powder for the discoloration of dye. Peroxidase was extracted from soybean seed coat, followed by amination of the surface of the tertiary structure. The aminated peroxidase was immobilized on highly activated corncob powder and employed for the discoloration of bromophenol blue. Amination was performed with 10 or 50 mmol.L-1 carbodiimide and 1 mol.L-1 ethylenediamine. The amount of protein in the extract was 0.235 ± 0.011 mg.mL-1 and specific peroxidase activity was 86.06 ± 1.52 µmol min-1 . mg-1, using 1 mmol.L-1 ABTS as substrate. Ten mmol.L-1 and 50 mmol.L-1 aminated peroxidase retained 88 and 100% of the initial activity. Following covalent immobilization on a corncob powder-glyoxyl support, 10 and 50 mmol.L-1 aminated peroxidase retained 74 and 86% of activity, respectively. Derivatives were used for the discoloration of 0.02 mmol.L-1 bromophenol blue solution. After 30 min, 93 and 89% discoloration was achieved with the 10 mmol.L-1 and 50 mmol.L-1 derivatives, respectively. Moreover, these derivatives retained 60% of the catalytic properties when used three times. Peroxidase extracted from soybean seed coat immobilized on a low-cost corncob powder support exhibited improved thermal stability.

  19. A Tomato Peroxidase Involved in the Synthesis of Lignin and Suberin1

    Science.gov (United States)

    Quiroga, Mónica; Guerrero, Consuelo; Botella, Miguel A.; Barceló, Araceli; Amaya, Iraida; Medina, María I.; Alonso, Francisco J.; de Forchetti, Silvia Milrad; Tigier, Horacio; Valpuesta, Victoriano

    2000-01-01

    The last step in the synthesis of lignin and suberin has been proposed to be catalyzed by peroxidases, although other proteins may also be involved. To determine which peroxidases are involved in the synthesis of lignin and suberin, five peroxidases from tomato (Lycopersicon esculentum) roots, representing the majority of the peroxidase activity in this organ, have been partially purified and characterized kinetically. The purified peroxidases with isoelectric point (pI) values of 3.6 and 9.6 showed the highest catalytic efficiency when the substrate used was syringaldazine, an analog of lignin monomer. Using a combination of transgenic expression and antibody recognition, we now show that the peroxidase pI 9.6 is probably encoded by TPX1, a tomato peroxidase gene we have previously isolated. In situ RNA hybridization revealed that TPX1 expression is restricted to cells undergoing synthesis of lignin and suberin. Salt stress has been reported to induce the synthesis of lignin and/or suberin. This stress applied to tomato caused changes in the expression pattern of TPX1 and induced the TPX1 protein. We propose that the TPX1 product is involved in the synthesis of lignin and suberin. PMID:10759507

  20. Characterization of Plant Peroxidases and Their Potential for Degradation of Dyes: a Review.

    Science.gov (United States)

    Kalsoom, Umme; Bhatti, Haq Nawaz; Asgher, Muhammad

    2015-07-01

    Peroxidases are ubiquitously found in all vascular plants and are promising biocatalysts for oxidization of wide range of aromatic substrates including various industrial dyes. Peroxidases can catalyze degradation of chemical structure of aromatic dyes either by precipitation or by opening the aromatic ring structure. Both soluble and immobilized peroxidases have been successfully used in batches as well as in continuous processes for the treatment of aromatic dyes present in industrial effluents. Plant peroxidases are stable catalysts that retain their activities over a broad range of pH and temperatures. The performance of an enzyme for degradation process depends upon the structure of dyes and the operational parameters like concentration of enzyme, H2O2 and dye, incubation time, pH, and temperature. Recalcitrant dyes can also be mineralized by plant peroxidases in the presence of redox mediators. Thus, plant peroxidases are easily available, inexpensive, and ecofriendly biocatalysts for the treatment of wastewaters containing a wide spectrum of textile and non-textile synthetic dyes. This article reviews the recent developments in isolation and characterization of plant peroxidases and their applications for bioremediation of synthetic dyes.

  1. Direct Electrochemistry of Horseradish Peroxidase-Gold Nanoparticles Conjugate

    Directory of Open Access Journals (Sweden)

    Chanchal K. Mitra

    2009-02-01

    Full Text Available We have studied the direct electrochemistry of horseradish peroxidase (HRP coupled to gold nanoparticles (AuNP using electrochemical techniques, which provide some insight in the application of biosensors as tools for diagnostics because HRP is widely used in clinical diagnostics kits. AuNP capped with (i glutathione and (ii lipoic acid was covalently linked to HRP. The immobilized HRP/AuNP conjugate showed characteristic redox peaks at a gold electrode. It displayed good electrocatalytic response to the reduction of H2O2, with good sensitivity and without any electron mediator. The covalent linking of HRP and AuNP did not affect the activity of the enzyme significantly. The response of the electrode towards the different concentrations of H2O2 showed the characteristics of Michaelis Menten enzyme kinetics with an optimum pH between 7.0 to 8.0. The preparation of the sensor involves single layer of enzyme, which can be carried out efficiently and is also highly reproducible when compared to other systems involving the layer-by-layer assembly, adsorption or encapsulation of the enzyme. The immobilized AuNP-HRP can be used for immunosensor applications

  2. The multihued palette of dye-decolorizing peroxidases.

    Science.gov (United States)

    Singh, Rahul; Eltis, Lindsay D

    2015-05-15

    Dye-decolorizing peroxidases (DyPs; EC 1.11.1.19) are heme enzymes that comprise a family of the dimeric α+β barrel structural superfamily of proteins. The first DyP, identified relatively recently in the fungus Bjerkandera adusta, was characterized for its ability to catalyze the decolorization of anthraquinone-based industrial dyes. These enzymes are now known to be present in all three domains of life, but do not appear to occur in plants or animals. They are involved in a range of physiological processes, although in many cases their roles remain unknown. This has not prevented the development of their biocatalytic potential, which includes the transformation of lignin. This review highlights the functional diversity of DyPs in the light of phylogenetic, structural and biochemical data. The phylogenetic analysis reveals the existence of at least five classes of DyPs. Their potential physiological roles are discussed based in part on synteny analyses. Finally, the considerable biotechnological potential of DyPs is summarized. Crown Copyright © 2015. Published by Elsevier Inc. All rights reserved.

  3. Thermal and high pressure inactivation kinetics of blueberry peroxidase.

    Science.gov (United States)

    Terefe, Netsanet Shiferaw; Delon, Antoine; Versteeg, Cornelis

    2017-10-01

    This study for the first time investigated the stability and inactivation kinetics of blueberry peroxidase in model systems (McIlvaine buffer, pH=3.6, the typical pH of blueberry juice) during thermal (40-80°C) and combined high pressure-thermal processing (0.1-690MPa, 30-90°C). At 70-80°C, the thermal inactivation kinetics was best described by a biphasic model with ∼61% labile and ∼39% stable fractions at temperature between 70 and 75°C. High pressure inhibited the inactivation of the enzyme with no inactivation at pressures as high as 690MPa and temperatures less than 50°C. The inactivation kinetics of the enzyme at 60-70°C, and pressures higher than 500MPa was best described by a first order biphasic model with ∼25% labile fraction and 75% stable fraction. The activation energy values at atmospheric pressure were 548.6kJ/mol and 324.5kJ/mol respectively for the stable and the labile fractions. Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.

  4. Thyroid Peroxidase Antibody and Screening for Postpartum Thyroid Dysfunction

    Directory of Open Access Journals (Sweden)

    Mohamed A. Adlan

    2011-01-01

    Full Text Available Postpartum thyroid dysfunction (PPTD is a common disorder which causes considerable morbidity in affected women. The availability of effective treatment for hypothyroid PPTD, the occurrence of the disease in subsequent pregnancies and the need to identify subjects who develop long term hypothyroidism, has prompted discussion about screening for this disorder. There is currently no consensus about screening as investigations hitherto have been variable in their design, definitions and assay frequency and methodology. There is also a lack of consensus about a suitable screening tool although thyroid peroxidase antibody (TPOAb is a leading contender. We present data about the use of TPOAb in early pregnancy and its value as a screening tool. Although its positive predictive value is moderate, its sensitivity and specificity when used in early pregnancy are comparable or better compared to other times during pregnancy and the postpartum period. Recent studies have also confirmed this strategy to be cost effective and to compare favourably with other screening strategies. We also explore the advantages of universal screening.

  5. Colorimetric peroxidase mimetic assay for uranyl detection in sea water

    KAUST Repository

    Zhang, Dingyuan

    2015-03-04

    Uranyl (UO2 2+) is a form of uranium in aqueous solution that represents the greatest risk to human health because of its bioavailability. Different sensing techniques have been used with very sensitive detection limits especially the recently reported uranyl-specific DNAzymes systems. However, to the best of our knowledge, few efficient detection methods have been reported for uranyl sensing in seawater. Herein, gold nanoclusters (AuNCs) are employed in an efficient spectroscopic method to detect uranyl ion (UO2 2+) with a detection limit of 1.86 ÎM. In the absence of UO2 2+, the BSA-stabilized AuNCs (BSA-AuNCs) showed an intrinsic peroxidase-like activity. In the presence of UO2 2+, this activity can be efficiently restrained. The preliminary quenching mechanism and selectivity of UO2 2+ was also investigated and compared with other ions. This design strategy could be useful in understanding the binding affinity of protein-stabilized AuNCs to UO2 2+ and consequently prompt the recycling of UO2 2+ from seawater.

  6. Increasing the scale of peroxidase production by Streptomyces sp. strain BSII#1.

    Science.gov (United States)

    Musengi, A; Khan, N; Le Roes-Hill, M; Pletschke, B I; Burton, S G

    2014-03-01

    To optimize peroxidase production by Streptomyces sp. strain BSII#1, up to 3 l culture volumes. Peroxidase production by Streptomyces sp. strain BSII#1 was optimized in terms of production temperature and pH and the use of lignin-based model chemical inducers. The highest peroxidase activity (1·30 ± 0·04 U ml(-1) ) in 10 ml culture volume was achieved in a complex production medium (pH 8·0) at 37°C in the presence of 0·1 mmol l(-1) veratryl alcohol, which was greater than those reported previously. Scale-up to 100 and 400 ml culture volumes resulted in decreased peroxidase production (0·53 ± 0·10 and 0·26 ± 0·08 U ml(-1) , respectively). However, increased aeration improved peroxidase production with the highest production achieved using an airlift bioreactor (4·76 ± 0·46 U ml(-1) in 3 l culture volume). Veratryl alcohol (0·1 mmol l(-1) ) is an effective inducer of peroxidase production by Streptomyces sp. strain BSII#1. However, improved aeration increased peroxidase production in larger volumes without the use of an inducer, surpassing induced yields in an optimized small-scale process. Only a limited number of reports in literature have focused on the up-scaling of bacterial peroxidase production. There remains opportunity for feasible large-scale production of bacterial peroxidases with potentially novel biocatalytic properties. © 2013 The Society for Applied Microbiology.

  7. Molecular characterization of the lignin-forming peroxidase: Role in growth, development and response to stress. Progress summary report, April 1, 1992--March 31, 1993

    Energy Technology Data Exchange (ETDEWEB)

    Lagrimini, L.M.

    1993-03-01

    This laboratory has continued its comprehensive study of the structure and function of plant peroxidases and their genes. Specifically, we are characterizing the anionic peroxidase of tobacco. During the past year we have completed the nucleotide sequence of the tobacco anionic peroxidase gene, joined the anionic peroxidase promoter to {Beta}-glucuronidase and demonstrated expression in transformed plants, measured lignin, auxin, and ethylene levels in transgenic tobacco plants over-expressing the anionic peroxidase, developed chimeric peroxidase genes to over-or under-express the anionic peroxidase in tissue specific manner in transgenic plants, and over-expressed the tobacco anionic peroxidase in transgenic tomato and sweetgum plants.

  8. Inhibition of Heme Peroxidase During Phenol Derivatives Oxidation. Possible Molecular Cloaking of the Active Center

    Directory of Open Access Journals (Sweden)

    Juozas Kulys

    2005-10-01

    Full Text Available Abstract: Ab initio quantum chemical calculations have been applied to the study of the molecular structure of phenol derivatives and oligomers produced during peroxidasecatalyzed oxidation. The interaction of substrates and oligomers with Arthromyces ramosus peroxidase was analyzed by docking methods. The most possible interaction site of oligomers is an active center of the peroxidase. The complexation energy increases with increasing oligomer length. However, the complexed oligomers do not form a precise (for the reaction hydrogen bonding network in the active center of the enzyme. It seems likely that strong but non productive docking of the oligomers determines peroxidase inhibition during the reaction.

  9. Polyphenol oxidase and peroxidase in different sugarcane cultivars, in Presidente Prudente region; Polifenoloxidases e peroxidase em diferentes variedades de cana-de-acucar na regiao de Presidente Prudente

    Energy Technology Data Exchange (ETDEWEB)

    Marques, Tadeu A.; Gomes, Danilo B.; Marques, Patricia A.A.; Alves, Vagner C. [Universidade do Oeste Paulista (UNOESTE), Presidente Prudente, SP (Brazil). Curso de Agronomia], Emails: tmarques@unoeste.br, pmarques@unoeste.br, vagner@unoeste.br

    2009-07-01

    The objective in present work was compare three sugarcane cultivars (RB 72-454, RB 86-7515, IAC 86-2480), evaluating the content of polyphenoloxidase and peroxidase. These determinations had aimed at to detect possible differences between varieties thus and being to differentiate them with regard to the products most interesting to be elaborated, ethanol production or sugar production. The varieties had presented differences of behavior for studied enzymes. The activity of polyphenoloxidase was superior the activity of peroxidase. The enzyme peroxidase was presented in bigger indices in the dry and cold periods. The enzyme polyphenoloxidase was presented well changeable, but with strong trend of bigger values in the rainy periods. It can be said that distinct periods for the best use of the varieties in the sugar production or alcohol exist. (author)

  10. Purification of a membrane-bound trypsin-like enzyme from the gut of the velvetbean caterpillar (Anticarsia gemmatalis Hübner =Purificação de uma enzima “tipo tripsina” não-solúvel do intestino da lagarta da soja (Anticarsia gemmatalis Hübner

    Directory of Open Access Journals (Sweden)

    Marcelo Matos Santoro

    2012-07-01

    Full Text Available Disruption of protein digestion in insects by specific endoprotease inhibitors is being regarded as an alternative to conventional insecticides for pest control. To optimize the effectiveness of this strategy, the understanding of the endoprotease diversity of the target insect is crucial. In this sense, a membrane-bound trypsin-like enzyme from the gut of Anticarsia gemmatalis fifth-instar larvae was purified. Non-soluble fraction of the gut extract was solubilized with 3-[(3-cholamidopropyldimethylammonio]-1-propanesulfonate (CHAPS and subjected to a p-aminobenzamidine affinity chromatography followed by anion-exchange chromatography. The yield of the purified enzyme was 11% with a purification factor of 143 and a final specific activity of 18.6 µM min.-1 mg-1 protein using N-α-benzoyl-L- Arg-p-nitroanilide (L-BApNA as substrate. The purified sample showed a single band with proteolytic activity active and apparent molecular mass of 25 kDa on SDS-PAGE. Molecular mass determined by MALDI-TOF mass spectrometry was 28,632 ± 26 Da. Although the low recovery and the difficulties in purifying large enzyme amounts limited its further characterization, the results contribute for the understanding of the proteases present on A. gemmatalis gut, which are potential targets for natural or specifically designed protease inhibitors.Comprometer a digestão de proteínas dos insetos pelo uso de inibidores específicos de endoproteases tem sido amplamente estudado como um método de controle de pragas alternativo ao uso dos inseticidas convencionais. No processo de otimização desta estratégia, o conhecimento da diversidade das endoproteases do inseto alvo torna-se crucial. Neste sentido, uma enzima “tipo-tripsina” ligada à membrana obtida do intestino de larvas do 5° instar de A. gemmatalis foi purificada. A fração insolúvel do extrato do intestino foi solubilizada com 3-[(3-cholamidopropyldimethylammonio]-1-propanesulfonate (CHAPS e submetida

  11. Prostaglandin endoperoxide H synthases: peroxidase hydroperoxide specificity and cyclooxygenase activation.

    Science.gov (United States)

    Liu, Jiayan; Seibold, Steve A; Rieke, Caroline J; Song, Inseok; Cukier, Robert I; Smith, William L

    2007-06-22

    The cyclooxygenase (COX) activity of prostaglandin endoperoxide H synthases (PGHSs) converts arachidonic acid and O2 to prostaglandin G2 (PGG2). PGHS peroxidase (POX) activity reduces PGG2 to PGH2. The first step in POX catalysis is formation of an oxyferryl heme radical cation (Compound I), which undergoes intramolecular electron transfer forming Intermediate II having an oxyferryl heme and a Tyr-385 radical required for COX catalysis. PGHS POX catalyzes heterolytic cleavage of primary and secondary hydroperoxides much more readily than H2O2, but the basis for this specificity has been unresolved. Several large amino acids form a hydrophobic "dome" over part of the heme, but when these residues were mutated to alanines there was little effect on Compound I formation from H2O2 or 15-hydroperoxyeicosatetraenoic acid, a surrogate substrate for PGG2. Ab initio calculations of heterolytic bond dissociation energies of the peroxyl groups of small peroxides indicated that they are almost the same. Molecular Dynamics simulations suggest that PGG2 binds the POX site through a peroxyl-iron bond, a hydrogen bond with His-207 and van der Waals interactions involving methylene groups adjoining the carbon bearing the peroxyl group and the protoporphyrin IX. We speculate that these latter interactions, which are not possible with H2O2, are major contributors to PGHS POX specificity. The distal Gln-203 four residues removed from His-207 have been thought to be essential for Compound I formation. However, Q203V PGHS-1 and PGHS-2 mutants catalyzed heterolytic cleavage of peroxides and exhibited native COX activity. PGHSs are homodimers with each monomer having a POX site and COX site. Cross-talk occurs between the COX sites of adjoining monomers. However, no cross-talk between the POX and COX sites of monomers was detected in a PGHS-2 heterodimer comprised of a Q203R monomer having an inactive POX site and a G533A monomer with an inactive COX site.

  12. In vitro free radical metabolism of phenolphthalein by peroxidases.

    Science.gov (United States)

    Sipe, H J; Corbett, J T; Mason, R P

    1997-04-01

    Phenolphthalein, a widely used laxative, is the active ingredient in more than a dozen commercial nonprescription formulations. Fast-flow EPR studies of the reaction of phenolphthalein with horseradish peroxidase (HRP) and hydrogen peroxide permit the direct detection of two free radicals. One has EPR parameters characteristic of phenoxyl radicals. The other has a broad unresolved spectrum, possibly arising from free radical polymeric products of the initial phenoxyl radical. EPR spin-trapping studies of incubations of phenolphthalein with lactoperoxidase, reduced glutathione (GSH), and hydrogen peroxide with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) demonstrate stimulated production of DMPO/.SG compared with an identical incubation lacking phenolphthalein. In the absence of DMPO, measurements with a Clark-type oxygen electrode show that molecular oxygen is consumed by a sequence of reactions initiated by the glutathione thiyl radical. Enhanced production of DMPO superoxide radical adduct is also found in a system of phenolphthalein, NADH, and lactoperoxidase. In this system the phenolphthalein phenoxyl radical abstracts hydrogen from NADH to generate NAD., which is not spin trapped by DMPO, but reacts with molecular oxygen to produce the superoxide radical detected by EPR. In the absence of DMPO, the oxygen consumption is measured using the Clark-type electrode. Production of ascorbate radical anion is also enhanced in a system of phenolphthalein, ascorbic acid, hydrogen peroxide, and lactoperoxidase. Ascorbate inhibits oxygen consumption when phenolphthalein is metabolized in the presence of either glutathione or NADH by reducing radical intermediates to their parent molecules and forming the relatively stable ascorbate anion radical. The detection of enhanced free radical production in these three systems, a consequence of futile metabolism (or redox cycling), suggests that phenolphthalein may be a significant source of oxidative stress in physiological systems

  13. Degradation of disperse dye from textile effluent by free and immobilized Cucurbita pepo peroxidase

    Directory of Open Access Journals (Sweden)

    Abouseoud M.

    2012-06-01

    Full Text Available Disperse dyes constitute the largest group of dyes used in local textile industry. This work evaluates the potential of the Cucurbita peroxidase(C-peroxidase extracted from courgette in the decolourization of disperse dye in free and immobilized form. The optimal conditions for immobilization of C-peroxidase in Ca-alginate were identified. The immobilization was optimized at 2%(w/v of sodium alginate and 0.2 M of calcium chloride. After optimization of treatment parameters, the results indicate that at pH 2, dye concentration: 80 mg/L(for FCP and 180 mg/L(for ICP, H2O2 dose: 0,02M (for FCP and 0,12M(for ICP, the decolourization by free and immobilized C-peroxidase were 72.02% and 69.71 % respectively. The degradation pathway and the metabolic products formed after the degradation were also predicted using UV–vis spectroscopy analysis.

  14. Degradation of disperse dye from textile effluent by free and immobilized Cucurbita pepo peroxidase

    Science.gov (United States)

    Boucherit, N.; Abouseoud, M.; Adour, L.

    2012-06-01

    Disperse dyes constitute the largest group of dyes used in local textile industry. This work evaluates the potential of the Cucurbita peroxidase(C-peroxidase) extracted from courgette in the decolourization of disperse dye in free and immobilized form. The optimal conditions for immobilization of C-peroxidase in Ca-alginate were identified. The immobilization was optimized at 2%(w/v) of sodium alginate and 0.2 M of calcium chloride. After optimization of treatment parameters, the results indicate that at pH 2, dye concentration: 80 mg/L(for FCP) and 180 mg/L(for ICP), H2O2 dose: 0,02M (for FCP) and 0,12M(for ICP), the decolourization by free and immobilized C-peroxidase were 72.02% and 69.71 % respectively. The degradation pathway and the metabolic products formed after the degradation were also predicted using UV-vis spectroscopy analysis.

  15. The effect of acid rain stress on chlorophyll, peroxidase of the conservation of rare earth elements

    International Nuclear Information System (INIS)

    Chongling, Y.; Yetang, H.; Xianke, Y.; Shunzhen, F.; Shanql, W.

    1998-01-01

    Full text: Based on pot experiment, the effect of acid rain stress on chlorophyll, peroxidase of wheat, the relationship of them and the conservation of rare earth elements has been studied. The result showed: stress of acid rain resulted in decrease of chlorophyll content and a/b values, chlorophyll a/b value and chlorophyll content is positive correlation with pH value of acid rain: peroxidase activity was gradually rise with pH value decrease, which indirectly increased decomposition intensity of chlorophyll. Decreased content and a/b value of chlorophyll further speeded blade decay affected the transport and transformation of light energy and metabolism of carbohydrates. After being treated by rare earth elements content and pH value of chlorophyll and peroxidase activity could be relatively stable. Therefore, under lower acidity condition, rare earth elements can influence the effect of acid rain on chlorophyll and peroxidase activity of wheat

  16. Removal of Phenol from Synthetic and Industrial Wastewater by Potato Pulp Peroxidases.

    Science.gov (United States)

    Kurnik, Katarzyna; Treder, Krzysztof; Skorupa-Kłaput, Monika; Tretyn, Andrzej; Tyburski, Jarosław

    Plant peroxidases have strong potential utility for decontamination of phenol-polluted wastewater. However, large-scale use of these enzymes for phenol depollution requires a source of cheap, abundant, and easily accessible peroxidase-containing material. In this study, we show that potato pulp, a waste product of the starch industry, contains large amounts of active peroxidases. We demonstrate that potato pulp may serve as a tool for peroxidase-based remediation of phenol pollution. The phenol removal efficiency of potato pulp was over 95 % for optimized phenol concentrations. The potato pulp enzymes maintained their activity at pH 4 to 8 and were stable over a wide temperature range. Phenol solutions treated with potato pulp showed a significant reduction in toxicity compared with untreated phenol solutions. Finally we determined that this method may be employed to remove phenol from industrial effluent with over 90 % removal efficiency under optimal conditions.

  17. Molecular-level insights into intrinsic peroxidase-like activity of nanocarbon oxides.

    Science.gov (United States)

    Zhao, Ruisheng; Zhao, Xiang; Gao, Xingfa

    2015-01-12

    Nanocarbon oxides have been proved to possess great peroxidase-like activity, catalyzing the oxidation of many peroxidase substrates, such as 3,3',5,5'-tetramethylbenzidine (TMB) and o-phenylenediamine dihydrochloride (OPD), accompanied by a significant color change. This chromogenic reaction is widely used to detect glucose and occult blood. The chromogenic reaction was intensively investigated with density functional theory and molecular-level insights into the nature of peroxidase-like activity were gained. A radical mechanism was unraveled and the carboxyl groups of nanocarbon oxides were identified as the reactive sites. Aromatic domains connected with the carboxyl groups were critical to the peroxidase-like activity. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Epitope recognition patterns of thyroid peroxidase autoantibodies in healthy individuals and patients with Hashimoto's thyroiditis*

    DEFF Research Database (Denmark)

    Nielsen, Claus H; Brix, Thomas H; Gardas, Andrzej

    2008-01-01

    Thyroid peroxidase antibodies (TPOAb) are markers of autoimmune thyroid disease (AITD), including Hashimoto's thyroiditis (HT), but naturally occurring TPOAb are also detectable in healthy, euthyroid individuals. In AITD, circulating TPOAb react mainly with two immunodominant regions (IDR), IDR...

  19. A catalytic approach to estimate the redox potential of heme-peroxidases

    International Nuclear Information System (INIS)

    Ayala, Marcela; Roman, Rosa; Vazquez-Duhalt, Rafael

    2007-01-01

    The redox potential of heme-peroxidases varies according to a combination of structural components within the active site and its vicinities. For each peroxidase, this redox potential imposes a thermodynamic threshold to the range of oxidizable substrates. However, the instability of enzymatic intermediates during the catalytic cycle precludes the use of direct voltammetry to measure the redox potential of most peroxidases. Here we describe a novel approach to estimate the redox potential of peroxidases, which directly depends on the catalytic performance of the activated enzyme. Selected p-substituted phenols are used as substrates for the estimations. The results obtained with this catalytic approach correlate well with the oxidative capacity predicted by the redox potential of the Fe(III)/Fe(II) couple

  20. CDNA cloning, characterization and expression of an endosperm-specific barley peroxidase

    DEFF Research Database (Denmark)

    Rasmussen, Søren Kjærsgård; Welinder, K.G.; Hejgaard, J.

    1991-01-01

    A barley peroxidase (BP 1) of pI ca. 8.5 and M(r) 37000 has been purified from mature barley grains. Using antibodies towards peroxidase BP 1, a cDNA clone (pcR7) was isolated from cDNA expression library. The nucleotide sequence of pcR7 gave a derived amino acid sequence identical to the 158 C...

  1. The Ustilago maydis effector Pep1 suppresses plant immunity by inhibition of host peroxidase activity.

    Directory of Open Access Journals (Sweden)

    Christoph Hemetsberger

    Full Text Available The corn smut Ustilago maydis establishes a biotrophic interaction with its host plant maize. This interaction requires efficient suppression of plant immune responses, which is attributed to secreted effector proteins. Previously we identified Pep1 (Protein essential during penetration-1 as a secreted effector with an essential role for U. maydis virulence. pep1 deletion mutants induce strong defense responses leading to an early block in pathogenic development of the fungus. Using cytological and functional assays we show that Pep1 functions as an inhibitor of plant peroxidases. At sites of Δpep1 mutant penetrations, H₂O₂ strongly accumulated in the cell walls, coinciding with a transcriptional induction of the secreted maize peroxidase POX12. Pep1 protein effectively inhibited the peroxidase driven oxidative burst and thereby suppresses the early immune responses of maize. Moreover, Pep1 directly inhibits peroxidases in vitro in a concentration-dependent manner. Using fluorescence complementation assays, we observed a direct interaction of Pep1 and the maize peroxidase POX12 in vivo. Functional relevance of this interaction was demonstrated by partial complementation of the Δpep1 mutant defect by virus induced gene silencing of maize POX12. We conclude that Pep1 acts as a potent suppressor of early plant defenses by inhibition of peroxidase activity. Thus, it represents a novel strategy for establishing a biotrophic interaction.

  2. Structure-activity relationships and molecular docking of thirteen synthesized flavonoids as horseradish peroxidase inhibitors.

    Science.gov (United States)

    Mahfoudi, Reguia; Djeridane, Amar; Benarous, Khedidja; Gaydou, Emile M; Yousfi, Mohamed

    2017-10-01

    For the first time, the structure-activity relationships of thirteen synthesized flavonoids have been investigated by evaluating their ability to modulate horseradish peroxidase (HRP) catalytic activity. Indeed, a modified spectrophotometrically method was carried out and optimized using 4-methylcatechol (4-MC) as peroxidase co-substrate. The results show that these flavonoids exhibit a great capacity to inhibit peroxidase with Ki values ranged from 0.14±0.01 to 65±0.04mM. Molecular docking has been achieved using Auto Dock Vina program to discuss the nature of interactions and the mechanism of inhibition. According to the docking results, all the flavonoids have shown great binding affinity to peroxidase. These molecular modeling studies suggested that pyran-4-one cycle acts as an inhibition key for peroxidase. Therefore, potent peroxidase inhibitors are flavonoids with these structural requirements: the presence of the hydroxyl (OH) group in 7, 5 and 4' positions and the absence of the methoxy (O-CH 3 ) group. Apigenin contributed better in HRP inhibitory activity. The present study has shown that the studied flavonoids could be promising HRP inhibitors, which can help in developing new molecules to control thyroid diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Eosinophil peroxidase signals via epidermal growth factor-2 to induce cell proliferation.

    LENUS (Irish Health Repository)

    Walsh, Marie-Therese

    2011-11-01

    Eosinophils exert many of their inflammatory effects in allergic disorders through the degranulation and release of intracellular mediators, including a set of cationic granule proteins that include eosinophil peroxidase. Studies suggest that eosinophils are involved in remodeling. In previous studies, we showed that eosinophil granule proteins activate mitogen-activated protein kinase signaling. In this study, we investigated the receptor mediating eosinophil peroxidase-induced signaling and downstream effects. Human cholinergic neuroblastoma IMR32 and murine melanoma B16.F10 cultures, real-time polymerase chain reaction, immunoprecipitations, and Western blotting were used in the study. We showed that eosinophil peroxidase caused a sustained increase in both the expression of epidermal growth factor-2 (HER2) and its phosphorylation at tyrosine 1248, with the consequent activation of extracellular-regulated kinase 1\\/2. This, in turn, promoted a focal adhesion kinase-dependent egress of the cyclin-dependent kinase inhibitor p27(kip) from the nucleus to the cytoplasm. Eosinophil peroxidase induced a HER2-dependent up-regulation of cell proliferation, indicated by an up-regulation of the nuclear proliferation marker Ki67. This study identifies HER2 as a novel mediator of eosinophil peroxidase signaling. The results show that eosinophil peroxidase, at noncytotoxic levels, can drive cell-cycle progression and proliferation, and contribute to tissue remodeling and cell turnover in airway disease. Because eosinophils are a feature of many cancers, these findings also suggest a role for eosinophils in tumorigenesis.

  4. Characterization of Peroxidase Changes in Tolerant and Susceptible Soybeans Challenged by Soybean Aphid (Hemiptera: Aphididae).

    Science.gov (United States)

    Marchi-Werle, L; Heng-Moss, T M; Hunt, T E; Baldin, E L L; Baird, L M

    2014-10-01

    Changes in protein content, peroxidase activity, and isozyme profiles in response to soybean aphid feeding were documented at V1 (fully developed leaves at unifoliate node, first trifoliate leaf unrolled) and V3 (fully developed leaf at second trifoliate node, third trifoliate leaf unrolled) stages of soybean aphid-tolerant (KS4202) and -susceptible (SD76R) soybeans. Protein content was similar between infested and control V1 and V3 stage plants for both KS4202 and SD76R at 6, 16, and 22 d after aphid introduction. Enzyme kinetics studies documented that control and aphid-infested KS4202 V1 stage and SD76R V1 and V3 stages had similar levels of peroxidase activity at the three time points evaluated. In contrast, KS4202 aphid-infested plants at the V3 stage had significantly higher peroxidase activity levels than control plants at 6 and 22 d after aphid introduction. The differences in peroxidase activity observed between infested and control V3 stage KS4202 plants at these two time points suggest that peroxidases may be playing multiple roles in the tolerant plant. Native gels stained for peroxidase were able to detect differences in the isozyme profiles of aphid-infested and control plants for both KS4202 and SD76R. © 2014 Entomological Society of America.

  5. Peroxidases from root exudates of Medicago sativa and Sorghum bicolor: Catalytic properties and involvement in PAH degradation.

    Science.gov (United States)

    Dubrovskaya, Ekaterina; Pozdnyakova, Natalia; Golubev, Sergey; Muratova, Anna; Grinev, Vyacheslav; Bondarenkova, Anastasiya; Turkovskaya, Olga

    2017-02-01

    Peroxidases from root exudates of sorghum (Sorghum bicolor L. Moench) and alfalfa (Medicago sativa L.) were purified and characterized, and their ability to oxidize native PAHs and PAH-derivatives was evaluated. The obtained data confirm that peroxidases are involved in the rhizosphere degradation of PAHs. Nondenaturing PAGE showed that the peroxidases of both plants were represented by a range of isoforms/isoenzymes (five to eight). Minor forms were lost during further purification, and as a result, the major anionic form from alfalfa root exudates and the major cationic form from those of sorghum were obtained. Both electrophoretically homogeneous peroxidases were monomeric proteins with a molecular weight of about 46-48 kDa. The pH optima and the main catalytic constants for the test substrates were determined. On the basis of their molecular and catalytic properties, the obtained enzymes were found to be typical plant peroxidases. Derivatives of PAHs and potential products of their microbial degradation (9-phenanthrol and 9,10-phenanthrenequinone), unlike the parent PAH (phenanthrene), inhibited the catalytic activity of the peroxidases, possibly indicating greater availability of the enzymes' active centers to these substances. Peroxidase-catalyzed decreases in the concentrations of a number of PAHs and their derivatives were observed. Sorghum peroxidase oxidized anthracene and phenanthrene, while alfalfa peroxidase oxidized only phenanthrene. 1-Hydroxy-2-naphthoic acid was best oxidized by peroxidase of alfalfa. However, quinone derivatives of PAHs were unavailable to sorghum peroxidase, but were oxidized by alfalfa peroxidase. These results indicate that the major peroxidases from root exudates of alfalfa and sorghum can have a role in the rhizosphere degradation of PAHs. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Lignin Peroxidase Activity Is Not Important in Biological Bleaching and Delignification of Unbleached Kraft Pulp by Trametes versicolor

    Science.gov (United States)

    Archibald, Frederick S.

    1992-01-01

    The discovery in 1983 of fungal lignin peroxidases able to catalyze the oxidation of nonphenolic aromatic lignin model compounds and release some CO2 from lignin has been seen as a major advance in understanding how fungi degrade lignin. Recently, the fungus Trametes versicolor was shown to be capable of substantial decolorization and delignification of unbleached industrial kraft pulps over 2 to 5 days. The role, if any, of lignin peroxidase in this biobleaching was therefore examined. Several different assays indicated that T. versicolor can produce and secrete peroxidase proteins, but only under certain culture conditions. However, work employing a new lignin peroxidase inhibitor (metavanadate ions) and a new lignin peroxidase assay using the dye azure B indicated that secreted lignin peroxidases do not play a role in the T. versicolor pulp-bleaching system. Oxidative activity capable of degrading 2-keto-4-methiolbutyric acid (KMB) appeared unique to ligninolytic fungi and always accompanied pulp biobleaching. PMID:16348775

  7. Peroxidase activity in Raphanus sativus and its relationship with soil heavy metals

    International Nuclear Information System (INIS)

    Alipour, H.; Zare Myvan, H.; Sharifi, M.

    2009-01-01

    Today heavy metals are important environmental pollutants which generated from human activities and are one of the most important environmental stresses that cause molecular damages to plants through reactive oxygen species formation such as H2O2. Heavy metals are absorbed and accumulated by plants thus are absorbed by human bodies through the food chain. Raphanus sativus is a herbaceous plant within the Brassicaceae family that has different varieties and is used as a food plant in different parts of Iran. Peroxidase is one of the most important enzyme in oxidoreductase super family that can metabolize H2O2. In this research we studied some growth parameters, peroxidase activity and their relationships with heavy metal content and other soil factors in three different populations of radish collected from Sari, Semnan and south of Tehran. After harvesting the plants shoots and roots Peroxidase activity was assayed spectrophotometrically at 470 nm. Our results showed total heavy metal content of shomal 3 station soil and radish plants was higher than other stations, so plants collected from this station had lowest root and shoot lengths, fresh weights, dry weights, protein content and leaf collrophyll content. The peroxidase activity in both leaves and roots of these plants was higher than plants of other stations Therefore our results showed that with increasing heavy metal concentrations in soils peroxidase activity increased.

  8. COMPARISON OF METHODS FOR ALKALINE PHOSPHATASE AND PEROXIDASE DETECTION IN MILK

    Directory of Open Access Journals (Sweden)

    felipe Nael Seixas

    2014-02-01

    Full Text Available This study evaluated the performance of strips for colorimetric detection of alkaline phosphatase and peroxidase in milk, comparing them with a kit of reagents for alkaline phosphatase and the official methodology for peroxidase. The samples were analyzed at the Laboratory Inspection of Products of Animal Origin, State University of Londrina. For the comparison tests for the detection of alkaline phosphatase four treatments were made by adding different percentages of raw milk (1%, 2%, 5% and 10% in the pasteurized milk, plus two control treatments. Thirty-eight samples triplicate for each treatment were analyzed. To compare the performance of tests for peroxidase 80 pasteurized milk samples were evaluated simultaneously by official methodology and by colorimetric strips. The performance of the alkaline phosphatase were different for the treatments with 1% and 2% of raw milk which had all the strips change color as the reagent kit showed the presence of phosphatase in just 2.63% and 5.26% the cases, respectively for each treatment. The colorimetric strips for alkaline phosphatase are more sensitive for the identification of small quantities compared to the reagent kit. The performance of tests for peroxidase showed no difference. The strips for the detection of peroxidase or alkaline phosphatase were effective and can replace traditional methods.

  9. Activity and Isoenzyme Profile of Peroxidase as Affected by Microgravity Stress

    Science.gov (United States)

    Sarnatska, V. V.; Gladun, H. O.; Padalko, S. F.

    2008-06-01

    To investigate microgravity (clinorotation) effect on activity and isoenzyme pattern of peroxidase the culture of primary explants of potato tubers with normal activation of proliferation in vitro, explants inoculated with Agrobacterium tumefaciens(A.t.), where crown-gall tumors were formed and dormant potato tubers were used. Substantial decrease of total peroxidase activity after one day-clinorotation of potato explants, normal and inoculated with A.t., was revealed. Seven day- clinorotation resulted in the decreased peroxidase activity in normal clinorotated explants, while peroxidase activity in clinorotated explants, inoculated with A.t., returned to the level of its stationary control. When peroxidase of potato explants was analyzed by PAGE, the result obtained show the decrease in activity of one electrophoretic fractions with low migrating mobility and two fractions with moderate mobility in clinorotated explants, normal and with crown gall, as compared with the ones in stationary conditions. The decrease in activity of these fractions under microgravity was less pronounced in explants with crown-galls.

  10. Oxidative Burst, Peroxidase Activity, and Lignin Content of Sclerotium rolfsii Infected Peanut Tissue

    Directory of Open Access Journals (Sweden)

    ENDANG PUDJIHARTATI

    2006-12-01

    Full Text Available The objectives of this experiment were to analyse physiological responses, such as oxidative burst reaction, peroxidase activity, and lignin content of healthy and S. rolfsii-infected peanut tissues. Differences in physiological responses among 24 peanut genotypes were determined, the disease severity was calculated and used to group resistance of tested genotypes. The regressions among observed peroxidase activity, lignin content and disease severity were used to determine the possible mechanisms of S. rolfsii resistance in peanut. Peanut seeds were grown in polybag and the growing plants were inoculated at the crown, stem, and leaf tissues. Results of the experiment indicated that infection of S. rolfsii in peanut did not induce oxidative burst. However, infection of the pathogen resulted in increased peroxidase activity and lignin content in the infected tissues. Regression analysis between peroxidase activity and disease severity showed negative slopes, indicating the more resistance the genotype, the more peroxidase activity in the tissue. Regression analysis between lignin content and disease severity was not significant.

  11. PATHOGEN IMPACT ON THE ACTIVITY DYNAMICS OF POTATO SUSPENSION CELLS EXTRA-CELLULAR PEROXIDASE

    Directory of Open Access Journals (Sweden)

    Graskova I.A.

    2005-08-01

    Full Text Available Changes in the activity of extracellular peroxidases were measured in cell suspension cultures of potato infected by Clavibacter michiganensis subsp. sepedonicus (Spieck. et Kotth. Skapt et Burkh. The total extracellular peroxidases activity of the resistant potato variety was higher than that of the sensitive variety both before and after infection. The enzyme of the resistant variety had a рН optimum of 6.2, while that of the sensitive variety was 5.4. Extracellular peroxidases of the sensitive potato variety were activated 10 minutes after infection, and displayed highest activity 1.5-2 hours later. In the resistant variety, peroxidase activity rose sharply in the first minutes of infection, and second peak of activity occurred 1.5-2 hours later. The increase of extracellular peroxidases activity of the sensitive potato variety under pathogenesis is connected with the change of genome expression and synthesis of proteins. The increase of enzyme activity of resistant potato variety in the first moments of infection is not related to proteins synthesis and is apparently conditioned by the change of kinetic parameters.

  12. Peptide Inhibitor of Complement C1 Inhibits the Peroxidase Activity of Hemoglobin and Myoglobin

    Directory of Open Access Journals (Sweden)

    Pamela S. Hair

    2017-01-01

    Full Text Available Hemoglobin is the natural carrier of oxygen in red blood cells (RBCs. While intracellular hemoglobin provides life-sustaining oxygen transport, extracellular free hemoglobin displays toxicity due to inherent peroxidase activity generating reactive oxygen species that subsequently react with the hemoglobin molecule to produce toxic heme degradation products resulting in free radicals, oxidative stress damage, and lipid peroxidation. We have recently demonstrated that Peptide Inhibitor of Complement C1 (PIC1 inhibits peroxidase activity of the heme-based enzyme myeloperoxidase. To elucidate whether PIC1 could inhibit peroxidase activity of hemoglobin, we evaluated the consequence of PIC1 on RBC lysates, methemoglobin, and myoglobin using tetramethylbenzidine (TMB as an oxidation target. PIC1 reversibly and dose-dependently prevented TMB oxidation to tetramethylbenzidine diimine by RBC lysates, methemoglobin, and myoglobin, having comparable activity to the inhibitor 4-aminobenzoic acid hydrazide. PIC1 inhibited TMB oxidation of RBC lysates similar to L-cysteine suggesting that the two cysteine residues contained in PIC1 may mediate peroxidase activity. PIC1 also inhibited heme destruction by NaOCl for RBC lysates, hemoglobin, and myoglobin as assayed by preservation of the Soret absorbance peak in the presence of NaOCl and reduction in free iron release. In conclusion, PIC1 inhibits peroxidase activity of hemoglobin and myoglobin likely via an antioxidant mechanism.

  13. Use of anti-horseradish peroxidase antibody-gold complex in the ABC technique.

    Science.gov (United States)

    Gee, B; Warhol, M J; Roth, J

    1991-06-01

    We report a modification of the avidin-biotin-peroxidase complex (ABC) technique for the light and electron microscopic detection of antigens in tissue sections. An immunological approach was used instead of the DAB reaction to reveal ABC bound to antigen-antibody complexes. Affinity-purified polyclonal antibodies against horseradish peroxidase were complexed to particles of colloidal gold and applied for reaction with the horseradish peroxidase molecules of the ABC. For light microscopic immunolabeling, the signal produced by the anti-horseradish peroxidase antibody-gold complex required silver intensification. The ABC immunogold reaction as compared with the standard ABC technique, in particular with silver intensification of the DAB reaction product, provided superior resolution in paraffin sections. Furthermore, section pre-treatment to block endogenous peroxidase activity could be omitted and no potentially hazardous substrate was used. The ABC immunogold reaction was successfully applied for electron microscopic immunolabeling on Lowicryl K4M thin sections. We propose that the ABC immunogold reaction is a useful alternative to the standard ABC technique and can be equally well applied to light and electron microscopy.

  14. Overcoming Reductant Interference in Peroxidase-Based Assays for Hydrogen Peroxide Quantification.

    Science.gov (United States)

    Jiang, Shu; Penner, Michael H

    2017-09-20

    A problem commonly encountered when using peroxidase-based methods for hydrogen peroxide quantification in biobased matrixes is interference due to the presence of endogenous reductants. Such assays are typically based on the generation of an oxidized reporter molecule in direct proportion to the amount of hydrogen peroxide reduced in the peroxidase-catalyzed reaction. Endogenous reductants confound such assays by reducing the oxidized reporter molecule, thus resulting in underestimates of hydrogen peroxide content. In the present work, we demonstrate how this problem can be circumvented by selectively oxidizing offending compounds by treatment with the oxidized reporter molecule prior to initiating the peroxidase reaction for hydrogen peroxide quantification. The approach is demonstrated using horseradish peroxidase, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), as the reporter molecule and a representative garlic paste as the hydrogen peroxide-containing biobased matrix. The approach is expected to be generally applicable to a wide range of peroxidase-based assays when applied to complex biobased systems.

  15. PEROXIDASE FROM SOYBEAN MEAL: OBTENTION, PURIFICATION AND APPLICATION IN REDUCTION OF DEOXYNIVALENOL LEVELS

    Directory of Open Access Journals (Sweden)

    Ana Carla Penteado Feltrin

    Full Text Available This study established the conditions for the extraction of the enzyme peroxidase (PO from soybean meal (SBM. An experimental design methodology was carried out in order to evaluate the effects of stirring rate time, pH and extracting solvent volume on the enzyme extraction. By using 5 g SBM and 50 mL phosphate buffer 10 mmol L-1 pH 4.7, 60 min stirring rate at 100 rpm, an enzyme with specific activity of 100 U mg-1 for SBM was obtained. Two techniques of purification were tested and compared for purification of peroxidase from soybean meal: triphasic partition (TPP and molecular exclusion chromatography. TPP showcased a greater efficiency with 50% recuperation and a purification factor of 13.6. Peroxidase in crude and pure forms was characterized for kinetics, thermodynamics and biochemistry. The parameter of thermal inactivation indicates high stability to exposure time and temperature increase, showing that enzyme activity is not altered by the presence of constituents of the reaction medium. Peroxidase in crude form represented a greater upkeep in activity, keeping 50% activity for 114 days at 0 °C. Peroxidase in pure form had greater affinity for substrate and reduced Deoxynivalenol levels by 80%, 20% more than the crude form.

  16. Investigation of hydrazide derivatives inhibitory effect on peroxidase enzyme purified from turnip roots

    Science.gov (United States)

    Almaz, Züleyha; Öztekin, Aykut; Özdemir, Hasan

    2017-04-01

    Peroxidases (EC: 1.11.1.7) are haem proteins and contain iron (III) protoporphyrin IX (ferriprotoporphyrin IX) as the prosthetic group [1]. They are found in all cells and play a critical role in many biological processes, such as the host-defense mechanism [2]. Peroxidases (PODs) are widely used in clinical biochemistry, enzyme immunoassays, synthesis of various aromatic chemicals, treatment of waste water containing phenolic compounds [3, 4]. In this study, peroxidase enzyme was purified with Para amino benzohydrazide (PABH)-L-Tyrosine Sepharose 4B affinity chromatography to investigate the inhibitory effect of hydrazide derivatives on Turnip (Brassica rapa L.). IC50 values and Ki constants were calculated for the molecules of 6-Amino nicotinic hydrazide, 6-Amino-5-bromo nicotinic hydrazide, 2-Amino-5-hydroxy benzohydrazide, 4-Amino-3-hydroxy benzohydrazide on purified enzyme and inhibition type of these molecules were determined.

  17. Differential leaf resistance to insects of transgenic sweetgum (Liquidambar styraciflua) expressing tobacco anionic peroxidase.

    Science.gov (United States)

    Dowd, P F; Lagrimini, L M; Herms, D A

    1998-07-01

    Leaves of transgenic sweetgum (Liquidambar styraciflua) trees that expressed tobacco anionic peroxidase were compared with leaves of L. styraciflua trees that did not express the tobacco enzyme. Leaves of the transgenic trees were generally more resistant to feeding by caterpillars and beetles than wild-type leaves. However, as for past studies with transgenic tobacco and tomato expressing the tobacco anionic peroxidase, the degree of relative resistance depended on the size of insect used and the maturity of the leaf. Decreased growth of gypsy moth larvae appeared mainly due to decreased consumption, and not changes in the nutritional quality of the foliage. Transgenic leaves were more susceptible to feeding by the corn earworm, Helicoverpa zea. Thus, it appears the tobacco anionic peroxidase can contribute to insect resistance, but its effects are more predictable when it is expressed in plant species more closely related to the original gene source.

  18. Expression of a defence-related intercellular barley peroxidase in transgenic tobacco

    DEFF Research Database (Denmark)

    Kristensen, B.K.; Brandt, J.; Bojsen, K.

    1997-01-01

    Tobacco plants (Nicotiana benthamiana L.) have been transformed with a T-DNA vector construct carrying the cDNA pBH6-301, encoding the major pathogen induced leaf peroxidase (Prx8) of barley, under control of an enhanced CaMV 35S promoter. Progeny from three independent transformants were analyzed...... genetically, phenotypically and biochemically. The T-DNA was steadily inherited through three generations. The barley peroxidase is expressed and sorted to the intercellular space in the transgenic tobacco plants. The peroxidase can be extracted from the intercellular space in two molecular forms from both...... barley and transgenic tobacco. The tobacco expressed forms are indistinguishable from the barley expressed forms as determined by analytical isoelectric focusing (pI 8.5) and Western-blotting. Staining for N-glycosylation showed that one form only was glycosylated. The N-terminus of purified Prx8 from...

  19. [Isolation and purification of Mn-peroxidase from Azospirillum brasilense Sp245].

    Science.gov (United States)

    Kupriashina, M A; Selivanov, N Iu; Nikitina, V E

    2012-01-01

    Homogenous Mn-peroxidase of a 26-fold purity grade was isolated from a culture of Azospirillum brasilense Sp245 cultivated on a medium containing 0.1 mM pyrocatechol. The molecular weight of the enzyme is 43 kD as revealed by electrophoresis in SDS-PAAG. It was shown that the use of pyrocatechol and 2,2'-azino-bis(3-ethylbenzotiazoline-6-sulfonate) at concentrations of 0.1 and I mM as inductors increased the Mn-peroxidase activity by a factor of 3.

  20. Role of the Chalcogen (S, Se, Te) in the Oxidation Mechanism of the Glutathione Peroxidase Active Site

    NARCIS (Netherlands)

    Bortoli, Marco; Torsello, Mauro; Bickelhaupt, F. Matthias; Orian, Laura

    2017-01-01

    The oxidation by H2O2 of the human phospholipid hydroperoxide glutathione peroxidase (GPx4), used as a model peroxidase selenoenzyme, as well as that of its cysteine (Cys) and tellurocysteine (Tec) mutants, was investigated in silico through a combined classic and quantum mechanics approach to

  1. 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

  2. Thyroid peroxidase antibodies in pregnant women with type 1 diabetes: impact on thyroid function, metabolic control and pregnancy outcome

    DEFF Research Database (Denmark)

    Vestgaard, Marianne; Nielsen, Lene Ringholm; Rasmussen, Åse Krogh

    2008-01-01

    In pregnant women with type 1 diabetes, we evaluated whether the presence of thyroid peroxidase autoantibodies (anti-TPO) was associated with changes in thyroid function, metabolic control and pregnancy outcome.......In pregnant women with type 1 diabetes, we evaluated whether the presence of thyroid peroxidase autoantibodies (anti-TPO) was associated with changes in thyroid function, metabolic control and pregnancy outcome....

  3. Effects of gamma-irradiation and granozan on the proliferative activity of plant tissue and its modification by peroxidase

    Energy Technology Data Exchange (ETDEWEB)

    Agabeili, R.A.; Melikova, N.K. (AN Azerbajdzhanskoj SSR, Baku. Biologicheskij Tsentr)

    1984-01-01

    Mitotic activity of meristem cells of seedlings of welsh onion Allium fistulesum L. under the effect of gamma-rays, granosane fungicide is studied as well as its modification by peroxidase. Antimitotic granosane activity is established, as well as peroxidase ability to stimulate proliferative ability of cells in the case of its inhibition by gamma-rays and granosane.

  4. Calculated ionisation potentials to determine the oxidation of vanillin precursors by lignin peroxidase.

    NARCIS (Netherlands)

    Have, ten R.; Rietjens, I.M.C.M.; Hartmans, S.; Swarts, H.J.; Field, J.A.

    1998-01-01

    In view of the biocatalytic production of vanillin, this research focused on the lignin peroxidase (LiP) catalysed oxidation of naturally occurring phenolic derivatives: O-methyl ethers, O-acetyl esters, and O-glucosyl ethers. The ionisation potential (IP) of a series of model compounds was

  5. Lignin peroxidase mediated biotransformations useful in the biocatalytic production of vanillin

    NARCIS (Netherlands)

    Have, ten R.

    2000-01-01

    This research concentrates on lignin peroxidase (LiP) mediated biotrans-formations that are useful in producing vanillin.

    In order to obtain this extracellular enzyme, the white-rot fungus Bjerkandera sp. strain BOS55 was cultivated on nitrogen rich

  6. Efferent neurons to the labyrinth of Salamandra salamandra as revealed by retrograde transport of horseradish peroxidase.

    Science.gov (United States)

    Fritzsch, B

    1981-11-04

    Application of horseradish peroxidase to the severed VIIIth nerve of Salamandra salamandra resulted in heavy bilateral labeling of neurons of the medullary reticular formation. These neurons closely resemble the Mauthner neuron in their widespread dendritic ramification. In most preparations axon collaterals are seen to leave the medulla via the contralateral VIIIth nerve. It is suggested that these neurons are labyrinthine efferents.

  7. 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

  8. A peroxidase gene expressed during early developmental stages of the parasitic plant Orobanche ramosa.

    Science.gov (United States)

    González-Verdejo, Clara Isabel; Barandiaran, Xabier; Moreno, Maria Teresa; Cubero, José Ignacio; Di Pietro, Antonio

    2006-01-01

    Broomrapes (Orobanche spp.) are holoparasitic weeds that cause devastating losses in many economically important crops. The molecular mechanisms that control the early stages of host infection in Orobanche are poorly understood. In the present study, the role of peroxidase has been examined during pre-infection growth and development of O. ramosa, using an in vitro model system. Peroxidase activity was histochemically localized at the tips of actively growing radicles and nascent attachment organs. Addition of exogenous catalase resulted in a significant reduction in the apical growth rate of the radicle. The prx1 gene encoding a putative class III peroxidase was cloned from a cDNA library of O. ramosa and was found to be expressed specifically during the early stages of the parasitic life cycle. The exogenous addition of sucrose resulted in significantly reduced prx1 transcript levels and in a dramatic change in radicle development from polarized apical growth to isotropic growth and the formation of tubercle-like structures. The results indicate an important role of peroxidases during the early parasitic stages of Orobanche.

  9. Peroxidase-mediated cross-linking of bovine a-lactalbumin

    NARCIS (Netherlands)

    Heijnis, W.H.

    2010-01-01

    The research presented in this thesis aimed at controlling the horseradish peroxidase-catalyzed cross-linking of bovine α lactalbumin and the implications of this cross-linking for the foam stabilizing properties. Attention is also given to microreactors and their potential to control the enzymatic

  10. Structure and organ specificity of an anionic peroxidase from Arabidopsis thaliana cell suspension culture

    DEFF Research Database (Denmark)

    Ostergaard, L; Abelskov, A K; Mattsson, O

    1996-01-01

    The predominant peroxidase (pI 3.5) (E.C. 1.11.1.7) of an Arabidopsis thaliana cell suspension culture was purified and partially sequenced. Oligonucleotides were designed and a specific probe was obtained. A cDNA clone was isolated from an Arabidopsis cell suspension cDNA library and completely...

  11. Organization and differential regulation of a cluster of lignin peroxidase genes of Phanerochaete chrysosporium

    Science.gov (United States)

    Philip. Stewart; Daniel. Cullen

    1999-06-01

    The lignin peroxidases of Phanerochaete chrysosporium are encoded by a minimum of 10 closely related genes. Physical and genetic mapping of a cluster of eight lip genes revealed six genes occurring in pairs and transcriptionally convergent, suggesting that portions of the lip family arose by gene duplication events. The completed sequence of 1ipG and lipJ, together...

  12. Oxidative cross-linking of casein by horseradish peroxidase and its ...

    African Journals Online (AJOL)

    The central composite design using response surface methodology was used to optimize cross-linking conditions of casein. The optimal cross-linking conditions of casein ... Cross-linking of food proteins induced by horseradish peroxidase might serve as an alternative approach to modify functional property of the proteins.

  13. Peroxidase-Generated Apoplastic ROS Impair Cuticle Integrity and Contribute to DAMP-Elicited Defenses

    Science.gov (United States)

    Survila, Mantas; Davidsson, Pär R.; Pennanen, Ville; Kariola, Tarja; Broberg, Martin; Sipari, Nina; Heino, Pekka; Palva, Erkki T.

    2016-01-01

    Cuticular defects trigger a battery of reactions including enhanced reactive oxygen species (ROS) production and resistance to necrotrophic pathogens. However, the source of ROS generated by such impaired cuticles has remained elusive. Here, we report the characterization of Arabidopsis thaliana ohy1 mutant, a Peroxidase 57 (PER57) – overexpressing line that demonstrates enhanced defense responses that result both from increased accumulation of ROS and permeability of the leaf cuticle. The ohy1 mutant was identified in a screen of A. thaliana seedlings for oligogalacturonides (OGs) insensitive/hypersensitive mutants that exhibit altered growth retardation in response to exogenous OGs. Mutants impaired in OG sensitivity were analyzed for disease resistance/susceptibility to the necrotrophic phytopathogens Botrytis cinerea and Pectobacterium carotovorum. In the ohy1 line, the hypersensitivity to OGs was associated with resistance to the tested pathogens. This PER57 overexpressing line exhibited a significantly more permeable leaf cuticle than wild-type plants and this phenotype could be recapitulated by overexpressing other class III peroxidases. Such peroxidase overexpression was accompanied by the suppressed expression of cutin biosynthesis genes and the enhanced expression of genes associated with OG-signaling. Application of ABA completely removed ROS, restored the expression of genes associated with cuticle biosynthesis and led to decreased permeability of the leaf cuticle, and finally, abolished immunity to B. cinerea. Our work demonstrates that increased peroxidase activity increases permeability of the leaf cuticle. The loss of cuticle integrity primes plant defenses to necrotrophic pathogens via the activation of DAMP-responses. PMID:28066496

  14. Decolorization of direct dyes using peroxidase from raphanus sativus (F04 SL)

    International Nuclear Information System (INIS)

    Bhatti, H.N.; Kalsoom, U.; Habib, A.

    2012-01-01

    An acidic peroxidase was isolated and partially purified from Raphanus sativus. The purified enzyme was characterized in terms of kinetics and thermodynamic aspects. Finally the enzyme was assessed to see its potential for decolorization of direct dyes. The specific activity of Raphanus sativus peroxidase increased from 44.77 to 65.20 U/mg of protein using 80 % ammonium sulphate precipitation. The optimum pH and temperature of the enzyme was 4 and 55 deg. C respectively. The activation energy of Raphanus sativus peroxidase was 25.44 kJ/mol and average value of Km was 0.25 mM. The activation energy of thermal denaturation of Raphanus sativus peroxidase was 17.79 kJ/mol. It was observed that with an increase in temperature, there was decrease in a half life and enthalpy, which showed that the enzyme was unstable at higher temperature. A maximum decolorization of 97 and 77 % was observed for Solar Blue A and Solar Flavine 5G at pH 4 and temperature 50 deg. C respectively. It was observed that % decolorization of both the dyes increased with an increase in enzyme units and incubation time. H/sub 2/O/sub 2/ dose of 0.8 mM for Solar Blue A and 0.7 mM for Solar Flavine 5G was sufficient for the maximum dye degradation. (author)

  15. Electrochemical determination of hydrogen peroxide using Rhodobacter capsulatus cytochrome c peroxidase at a gold electrode

    NARCIS (Netherlands)

    De Wael, K.; Buschop, H.; Heering, H.A.; De Smet, L.; Van Beeumen, J.; Devreese, B.; Adriaens, A.

    2007-01-01

    We describe the redox behaviour of horse heart cytochrome c (HHC) and Rhodobacter capsulatus cytochrome c peroxidase (RcCCP) at a gold electrode modified with 4,4?-bipyridyl. RcCCP shows no additional oxidation or reduction peaks compared to the electrochemistry of only HHC, which indicates that it

  16. Improved manganese-oxidizing activity of DypB, a peroxidase from a lignolytic bacterium.

    Science.gov (United States)

    Singh, Rahul; Grigg, Jason C; Qin, Wei; Kadla, John F; Murphy, Michael E P; Eltis, Lindsay D

    2013-04-19

    DypB, a dye-decolorizing peroxidase from the lignolytic soil bacterium Rhodococcus jostii RHA1, catalyzes the peroxide-dependent oxidation of divalent manganese (Mn(2+)), albeit less efficiently than fungal manganese peroxidases. Substitution of Asn246, a distal heme residue, with alanine increased the enzyme's apparent k(cat) and k(cat)/K(m) values for Mn(2+) by 80- and 15-fold, respectively. A 2.2 Å resolution X-ray crystal structure of the N246A variant revealed the Mn(2+) to be bound within a pocket of acidic residues at the heme edge, reminiscent of the binding site in fungal manganese peroxidase and very different from that of another bacterial Mn(2+)-oxidizing peroxidase. The first coordination sphere was entirely composed of solvent, consistent with the variant's high K(m) for Mn(2+) (17 ± 2 mM). N246A catalyzed the manganese-dependent transformation of hard wood kraft lignin and its solvent-extracted fractions. Two of the major degradation products were identified as 2,6-dimethoxybenzoquinone and 4-hydroxy-3,5-dimethoxybenzaldehyde, respectively. These results highlight the potential of bacterial enzymes as biocatalysts to transform lignin.

  17. Peroxidase catalyzed conjugation of peptides, proteins and polysaccharides via endogenous and exogenous phenols.

    NARCIS (Netherlands)

    Oudgenoeg, G.

    2004-01-01

    The research was directed towards peroxidase mediated cross-linking of proteins and polysaccharides. Two approaches were explored, cross-linking by use of ferulic acid (FA)oand cross-linking by use

  18. Lignin peroxidase isoenzyme: a novel approach to biodegrade the toxic synthetic polymer waste.

    Science.gov (United States)

    Khatoon, Nazia; Jamal, Asif; Ali, Muhammad Ishtiaq

    2018-01-05

    Fungal metabolites are playing an immense role in developing various sustainable waste treatment processes. The present study aimed at production and characterization of fungal lignin peroxidase (EC 1.11.1.14) with a potential to degrade Polyvinyl Chloride. Optimization studies revealed that the maximum enzyme production occurred at a temperature 25°C, pH 5 in the 4th week of the incubation period with fungal strain. Enzyme assay was performed to find out the dominating enzyme in the culture broth. The molecular weight of the enzyme was found to be 46 kDa. Partially purified lignin peroxidase from Phanerocheate chrysosporium was used for the degradation of PVC films. A significant reduction in the weight of PVC film was observed (31%) in shake flask experiment. FTIR spectra of the enzyme-treated plastic film revealed structural changes in the chemical composition, indicating a specific peak at 2943 cm -1 that corresponded to alkenyl C-H stretch. Moreover, deterioration on the surface of PVC films was confirmed by Scanning Electron Microscopy tracked through activity assay for the lignin peroxidase. Extracellular lignin peroxidases from P. chrysosporium play a significant role in the degradation of complex polymeric compounds like PVC.

  19. 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…

  20. Degradation of textile dyes using immobilized lignin peroxidase-like metalloporphines under mild experimental conditions

    Directory of Open Access Journals (Sweden)

    Zucca Paolo

    2012-12-01

    Full Text Available Abstract Background Synthetic dyes represent a broad and heterogeneous class of durable pollutants, that are released in large amounts by the textile industry. The ability of two immobilized metalloporphines (structurally emulating the ligninolytic peroxidases to bleach six chosen dyes (alizarin red S, phenosafranine, xylenol orange, methylene blue, methyl green, and methyl orange was compared to enzymatic catalysts. To achieve a green and sustainable process, very mild conditions were chosen. Results IPS/MnTSPP was the most promising biomimetic catalyst as it was able to effectively and quickly bleach all tested dyes. Biomimetic catalysis was fully characterized: maximum activity was centered at neutral pH, in the absence of any organic solvent, using hydrogen peroxide as the oxidant. The immobilized metalloporphine kept a large part of its activity during multi-cycle use; however, well-known redox mediators were not able to increase its catalytic activity. IPS/MnTSPP was also more promising for use in industrial applications than its enzymatic counterparts (lignin peroxidase, laccase, manganese peroxidase, and horseradish peroxidase. Conclusions On the whole, the conditions were very mild (standard pressure, room temperature and neutral pH, using no organic solvents, and the most environmental-friendly oxidant and a significant bleaching and partial mineralization of the dyes was achieved in approximately 1 h. Therefore, the process was consistent with large-scale applications. The biomimetic catalyst also had more promising features than the enzymatic catalysts.

  1. Antioxidant Vitamin Levels and Glutathione Peroxidase Activity During Ischemia/Reperfusion in Myocardial Infarction

    Czech Academy of Sciences Publication Activity Database

    Mužáková, V.; Kanďár, R.; Vojtíšek, P.; Skalický, J.; Vaňková, Radomíra; Čegan, A.; Červinková, Z.

    2001-01-01

    Roč. 50, č. 4 (2001), s. 389-396 ISSN 0862-8408 R&D Projects: GA ČR GA522/99/1130 Institutional research plan: CEZ:AV0Z5038910 Keywords : Myocardial infarction * Oxidative stress * Glutathione peroxidase Subject RIV: CE - Biochemistry Impact factor: 1.027, year: 2001

  2. In silico molecular modeling and docking studies on the leishmanial tryparedoxin peroxidase

    Directory of Open Access Journals (Sweden)

    Ozal Mutlu

    2014-04-01

    Full Text Available Leishmaniasis is one of the most common form of neglected parasitic disease that affects about 350 million people worldwide. Leishmanias have a trypanothione mediated hydroperoxide metabolism to eliminate endogenous or exogenous oxidative agents. Both of 2-Cys peroxiredoxin (Prx and glutathione peroxidase type tryparedoxin peroxidase (Px are the terminal enzymes in the trypanothione dependent detoxification system. Therefore absence of trypanothione redox system in mammals and the sensitivity of trypanosomatids against oxidative stress, enzymes of this pathway are drug targets candidates. In this study, 3D structure of tryparedoxin peroxidase (2-Cys peroxiredoxin type from Leishmania donovani (LdTXNPx was described by homology modeling method based on the template of tryparedoxin peroxidase from Crithidia fasciculata and selected compounds were docked to the active site pocket. The quality of the 3D structure of the model was confirmed by various web based validation programs. When compared secondary and tertiary structure of the model, it showed a typical thioredoxin fold containing a central beta-sheet and three alpha-helices. Docking study showed that the selected compound 2 (CID 16073813 interacted with the active site amino acids and binding energy was -118.675 kcal/mol.

  3. Glutathione peroxidase activity in the selenium-treated alga Scenedesmus quadricauda

    Czech Academy of Sciences Publication Activity Database

    Vítová, Milada; Bišová, Kateřina; Hlavová, Monika; Zachleder, Vilém; Rucki, M.; Čížková, Mária

    2011-01-01

    Roč. 102, 1-2 (2011), s. 87-94 ISSN 0166-445X R&D Projects: GA ČR GA525/09/0102 Institutional research plan: CEZ:AV0Z50200510 Keywords : Cell cycle * Enzyme activity * Glutathione peroxidase Subject RIV: EE - Microbiology, Virology Impact factor: 3.761, year: 2011

  4. [Biosynthesis of silver nanoparticles with the participation of extracellular Mn-dependent peroxidase from Azospirillum].

    Science.gov (United States)

    Kupryashina, M A; Vetchinkina, E P; Nikitina, V E

    2016-01-01

    The accumulation of nanoparticles of colloidal silver with spherical shape in culture liquid of Azospirillum brasilense has been shown by transmission electron microscopy. Bacterial extracellular Mn-peroxidases were found to participate in silver reduction from silver nitrate with the formation of nanoparticles. A mechanism of extracellular biosynthesis of silver nanoparticles by A. brasilense bacteria was proposed

  5. Pathophysiological aspects of thyroid hormone disorders/thyroid peroxidase autoantibodies and reproduction

    NARCIS (Netherlands)

    Vissenberg, R.; Manders, V. D.; Mastenbroek, S.; Fliers, E.; Afink, G. B.; Ris-Stalpers, C.; Goddijn, M.; Bisschop, P. H.

    2015-01-01

    Thyroid hormone disorders and thyroid peroxidase autoantibodies (TPO-Ab) in women are associated with subfertility and early pregnancy loss. Here, we aim to provide a comprehensive overview of the literature on the pathophysiology of these associations. A review of the literature in the English

  6. Iron impurities as the active sites for peroxidase-like catalytic reaction on graphene and its derivatives.

    Science.gov (United States)

    Dong, Ying; Li, Jing; Shi, Lei; Guo, Zhiguang

    2015-07-22

    We established four kinds of good dispersing systems of graphene and its derivatives with different structural characteristics to estimate their peroxidase-like activity. Besides graphene oxide (GO), it is demonstrated that defect-free graphene, low-oxygen graphene, and iron(III)-doped graphene oxide (GO-Fe) are all capable of H2O2 activation to oxidize peroxidase substrates. As for defect-free graphene, the dispersibility in reaction medium exerts great impact on its catalytic activity and our further judgements concerning the nature of active sites. Improved stability and further exfoliation of defect-free graphene in reaction medium are beneficial to the access of reactants to active sites on the basal planes and enhance its peroxidase-like activity, which is superior to that of low-oxygen graphene and much higher than that of GO. In addition, their peroxidase-like activity can be greatly inhibited by the addition of iron chelators. Interestingly, the introduction of trace ferric ions into GO does not lead to an apparent change except for remarkable increase of its peroxidase-like activity. Therefore, we propose that the observed iron impurities rather than the doped nonmetallic heteroatoms play an important role in the peroxidase-like activity of graphene and its derivatives. In this light, saturated iron(III) was immobilized onto the oxygen-donor coordination of GO to immensely promote its activity. The peroxidase-like activity of the prepared GO-Fe was systematically evaluated by using 3,3',5,5'-tetramethylbenzidine and pyrogallol as peroxidase substrates and was compared to that of horseradish peroxidase and hemin. As a result, GO-Fe shows excellent peroxidase-like catalytic activity, which is comparable to that of hemin. Furthermore, GO-Fe was used for the quantitative detection of H2O2 and glucose.

  7. Molecular characterization of the lignin-forming peroxidase: Role in growth, development and response to stress. Progress summary report, April 1, 1993--March 31, 1994

    Energy Technology Data Exchange (ETDEWEB)

    Lagrimini, L.M.

    1994-05-01

    Our group continues to focus on the characterization of the tobacco anionic peroxidase and its genes. Throughout this past year we have generated transgenic plants expressing {beta}-glucuronidase under control of the anionic peroxidase promoter, characterized effectors of peroxidase gene expression in transformed protoplasts, generated numerous transgenic plants which over- and under-express the anionic peroxidase in a tissue specific manner, characterized the role of the anionic peroxidase in the metabolism of auxin, introduced a marker (flag) into the anionic peroxidase primary protein sequence which will permit the identification of the recombinant protein in plant tissue, and described the enhancement of insect resistance as a result of over-expression of the anionic peroxidase. Although our research program has continued along the lines of the original proposal, we have redirected a significant effort to the role which this enzyme plays in the metabolism of auxin, and conversely, the role which auxin plays in regulating the expression of the anionic peroxidase gene.

  8. Modulation of glutathione peroxidase expression by selenium: effect on human MCF-7 breast cancer cell transfectants expressing a cellular glutathione peroxidase cDNA and doxorubicin-resistant MCF-7 cells.

    OpenAIRE

    Chu, F F; Esworthy, R S; Akman, S; Doroshow, J H

    1990-01-01

    We have studied the effect of selenium on the expression of a cellular glutathione peroxidase, GSHPx-1, in transfected MCF-7 cells and in doxorubicin-resistant (Adrr) MCF-7 cells. A GSHPx-1 cDNA with a Rous Sarcoma virus promoter was transfected into a human mammary carcinoma cell line, MCF-7, which has very low endogenous cytosolic glutathione (GSH) peroxidase activity and no detectable message. The transfectant with the highest GSH peroxidase activity among the isolates, MCF-7H6, was charac...

  9. Activity and isoenzyme spectrum of peroxidases and dehydrins of some plant species, growing on the shores of lake Baikal, under abiotic stress

    Directory of Open Access Journals (Sweden)

    M.A. Zhivet’ev

    2010-11-01

    Full Text Available Termostability and optimal pH of weak-associated with plant cell wall and soluble peroxidases was shown to change in relation to natural conditions and season of year. Also the activity of peroxidase was variable during vegetation period. Dehydrine expression was followed by spike of peroxidase activity (and, a priori, an increase of hydrogen peroxide concentration.

  10. The molecular characterization of the lignin-forming peroxidase. Progress summary report, April 1, 1989--March 31, 1992

    Energy Technology Data Exchange (ETDEWEB)

    Lagrimini, L.M.

    1992-04-01

    This laboratory is committed to understanding the function of plant peroxidases via a multi-disciplinary approach. We have chosen the lignin-forming peroxidase from tobacco as the first isoenzyme to be subjected to this comprehensive approach. The goals which were set out upon the initiation of this project were as follows: (1) utilize a cDNA clone to the tobacco anionic peroxidase to generate transgenic plants which either over-produced this isoenzyme or specifically under-produced this isoenzyme via antisense RNA, (2) describe any phenotypic changes resulting from altered peroxidase expression, (3) perform morphological, physiological, and biochemical analysis of the above mentioned plants to help in determining the in planta function for this enzyme, and (4) clone and characterize the gene for the tobacco anionic peroxidase. A summary of progress thus far which includes both published and unpublished work will be presented in three sections: generation and characterization of transgenic plants, description of phenotypes, and biochemical and physiological analysis of peroxidase function, and cloning and characterization of the tobacco anionic peroxidase gene.

  11. The molecular characterization of the lignin-forming peroxidase. Progress summary report, April 1, 1992--March 31, 1995

    Energy Technology Data Exchange (ETDEWEB)

    Lagrimini, L.M.

    1995-06-01

    My research program focuses entirely on the study of the lignin-forming peroxidase of tobacco. Ever since our cloning and sequencing of the first plant peroxidase cDNA, we have pioneered in the introduction of the tools of molecular biology to the study of plant peroxidases. A significant part of our effort has been focused on the construction and analysis of transgenic plants which either over- or under-express the tobacco anionic peroxidase. This research has not only supported the role for this enzyme in lignification, but has opened the door to our understanding of additional metabolic functions including auxin metabolism and insect defense. As you will learn, this enzyme`s role in auxin catabolism has lead to numerous phenotypes in transgenic plants. More recently, our attention has been directed towards the analysis of peroxidase gene expression. From this work we have learned that the anionic peroxidase gene is expressed at high levels in the xylem-forming cells, epidermis, and trichomes. This expression pattern supports its role lignification and hose defenses. We have also learned that this gene is down-regulated by auxin which indicates a strong relationship between auxin and the anionic peroxidase. 12 figs.

  12. Removal of reactive blue 21 and reactive red 195 dyes using horseradish peroxidase as catalyst

    Directory of Open Access Journals (Sweden)

    S. Farias

    Full Text Available Abstract-Textile effluent is rich in hydrolyzed dyes that need to be removed. This study presents an evaluation of the potential of the enzyme horseradish peroxidase to remove the hydrolyzed dyes Reactive Blue 21 (RB 21 and Reactive Red 195 (RR 195 from cotton fiber and the effluent of the dyeing process. The parameters pH, dye concentration and temperature were evaluated to determine the optimal conditions to remove the dyes. The studies of removal of the dyeing effluent led to an increase of degradation for all tested colors. The use of the enzyme horseradish peroxidase as a biocatalyst can be a viable technological alternative to remove some hydrolyzed dyes.

  13. Size-dependent tuning of horseradish peroxidase bioreactivity by gold nanoparticles

    Science.gov (United States)

    Wu, Haohao; Liu, Yi; Li, Meng; Chong, Yu; Zeng, Mingyong; Lo, Y. Martin; Yin, Jun-Jie

    2015-02-01

    Molecules with diverse biological functions, such as heme peroxidases, can be useful tools for identifying potential biological effects of gold nanoparticles (AuNPs) at the molecular level. Here, using UV-Vis, circular dichroism, dynamic light scattering, and electron spin resonance spectroscopy, we report tuning of horseradish peroxidase (HRP) bioactivity by reactant-free AuNPs with diameters of 5, 10, 15, 30 and 60 nm (Au-5 nm, Au-10 nm, Au-15 nm, Au-30 nm and Au-60 nm). HRP conjugation to AuNPs was observed with only Au-5 nm and Au-10 nm prominently increasing the α-helicity of the enzyme to extents inversely related to their size. Au-5 nm inhibited both HRP peroxidase activity toward 3,3',5,5'-tetramethylbenzidine and HRP compound I/II reactivity toward 5,5-dimethyl-1-pyrroline N-oxide. Au-5 nm enhanced the HRP peroxidase activity toward ascorbic acid and the HRP compound I/II reactivity toward redox-active residues in the HRP protein moiety. Further, Au-5 nm also decreased the catalase- and oxidase-like activities of HRP. Au-10 nm showed similar, but weaker effects, while Au-15 nm, Au-30 nm and Au-60 nm had no effect. Results suggest that AuNPs can size-dependently enhance or inhibit HRP bioreactivity toward substrates with different redox potentials via a mechanism involving extension of the HRP substrate access channel and decline in the redox potentials of HRP catalytic intermediates.Molecules with diverse biological functions, such as heme peroxidases, can be useful tools for identifying potential biological effects of gold nanoparticles (AuNPs) at the molecular level. Here, using UV-Vis, circular dichroism, dynamic light scattering, and electron spin resonance spectroscopy, we report tuning of horseradish peroxidase (HRP) bioactivity by reactant-free AuNPs with diameters of 5, 10, 15, 30 and 60 nm (Au-5 nm, Au-10 nm, Au-15 nm, Au-30 nm and Au-60 nm). HRP conjugation to AuNPs was observed with only Au-5 nm and Au-10 nm prominently increasing the

  14. Photophysics and photochemistry of horseradish peroxidase A2 upon ultraviolet illumination

    DEFF Research Database (Denmark)

    Petersen, Maria Teresa Neves; Petersen, Steffen B.; Klitgaard, Søren

    2007-01-01

    Detailed analysis of the effects of UV and blue light illumination of horseradish peroxidase A2, a heme-containing enzyme that reduces H2O2 to oxidize organic and inorganic compounds, is presented. The effects of increasing illumination time on the protein's enzymatic activity, Reinheitzahl value......, fluorescence emission, fluorescence lifetime distribution, fluorescence mean lifetime and heme absorption are reported. UV illumination leads to an exponential decay of the enzyme activity followed by changes in heme group absorption. Longer UV illumination time leads to lower Tm values as well as helical...... emission of horseradish peroxidase A2 with streak camera shows that UV illumination induces an exponential change in the pre-exponential factors distribution associated to the protein's fluorescence lifetimes, leading to an exponential increase of the mean fluorescence lifetime. Illumination of aromatic...

  15. Determining inhibition effects of some aromatic compounds on peroxidase enzyme purified from white and red cabbage

    Energy Technology Data Exchange (ETDEWEB)

    Öztekin, Aykut, E-mail: aoztekin@agri.edu.tr [Ataturk University, Science Faculty, Department of Chemistry, 25240-Erzurum (Turkey); Agri Ibrahim Cecen University Faculty of Arts and Sciences, Department of Chemistry, 04100-Agri (Turkey); Almaz, Züleyha, E-mail: zturkoglu-2344@hotmail.com [Ataturk University, Science Faculty, Department of Chemistry, 25240-Erzurum (Turkey); Mus Alparslan University Faculty of Sciences, Department of Moleculer Biology, 49250-Mus (Turkey); Özdemir, Hasan, E-mail: hozdemir@atauni.edu.tr [Ataturk University, Science Faculty, Department of Chemistry, 25240-Erzurum (Turkey)

    2016-04-18

    Peroxidases (E.C.1.11.1.7) catalyze the one electron oxidation of wide range of substrates. They are used in synthesis reaction, removal of peroxide from industrial wastes, clinical biochemistry and immunoassays. In this study, the white cabbage (Brassica Oleracea var. capitata f. alba) and red cabbage (Brassica oleracea L. var. capitata f. rubra) peroxidase enzymes were purified for investigation of inhibitory effect of some aromatic compounds on these enzymes. IC{sub 50} values and Ki constants were calculated for the molecules of 6-Amino nicotinic hydrazide, 6-Amino-5-bromo nicotinic hydrazide, 2-Amino-5-hydroxy benzohydrazide, 4-Amino-3-hydroxy benzohydrazide on purified enzymes and inhibition type of these molecules were determined. (This research was supported by Ataturk University. Project Number: BAP-2015/98).

  16. Enhanced chemiluminescence: a sensitive analytical system for detection of sweet potato peroxidase.

    Science.gov (United States)

    Vdovenko, Marina M; Della Ciana, Leopoldo; Sakharov, Ivan Y

    2010-08-01

    3-(10'-Phenothiazinyl)propane-1-sulfonate (SPTZ) was shown to be a potent enhancer of anionic sweet potato peroxidase (aSPP)-induced chemiluminescence. The optimal conditions for aSPP-catalyzed oxidation of luminol were investigated by varying the concentrations of luminol, hydrogen peroxide, Tris, and SPTZ as well as the pH values of the reaction mixture. Addition of 4-morpholinopyridine (MORP) to the reaction mixture markedly increased the light intensity. Using SPTZ and MORP together enhanced the effect 265 times. The lower detection limit (LDL) of SPP was 0.09 pM, approximately in 10 times lower than that for the cationic isozyme c of horseradish peroxidase/4-iodophenol system. It was shown that aSPP in the presence of SPTZ produced a longer lasting chemiluminescent signal.

  17. Stable expression and characterization of a fungal pectinase and bacterial peroxidase genes in tobacco chloroplast

    Directory of Open Access Journals (Sweden)

    Edward Alexander Espinoza-Sánchez

    2015-05-01

    Conclusion: This study demonstrated that hydrolytic genes PelA and MnP-2 could be integrated and expressed correctly from the chloroplast genome of tobacco plants. A whole plant, having ~470 g of biomass could feasibly yield 66,676.25 units of pectin or 21,715.46 units of manganese peroxidase. Also, this study provides new information about methods and strategies for the expression of enzymes with industrial value.

  18. Epitope recognition patterns of thyroid peroxidase autoantibodies in healthy individuals and patients with Hashimoto's thyroiditis*

    DEFF Research Database (Denmark)

    Nielsen, Claus H; Brix, Thomas H; Gardas, Andrzej

    2008-01-01

    Thyroid peroxidase antibodies (TPOAb) are markers of autoimmune thyroid disease (AITD), including Hashimoto's thyroiditis (HT), but naturally occurring TPOAb are also detectable in healthy, euthyroid individuals. In AITD, circulating TPOAb react mainly with two immunodominant regions (IDR), IDR-A......-A and IDR-B. The present study was undertaken in order to compare the epitope recognition pattern of TPOAb in HT patients and healthy subjects....

  19. A one-electron oxidation of carcinogenic nonaminoazo dye Sudan I by horseradish peroxidase

    Czech Academy of Sciences Publication Activity Database

    Semanská, M.; Dračínský, Martin; Martínek, V.; Hudeček, J.; Hodek, P.; Frei, E.; Stiborová, M.

    2008-01-01

    Roč. 29, č. 5 (2008), s. 712-716 ISSN 0172-780X Grant - others:GA MŠk(CZ) 1M0505; GA ČR(CZ) GA203/06/0329 Program:1M Institutional research plan: CEZ:AV0Z40550506 Keywords : carcinogen * Sudan I * peroxidase * NMR spectroscopy * mechanism of oxidation Subject RIV: CC - Organic Chemistry Impact factor: 1.359, year: 2008 http://node.nel.edu

  20. Fe(III)-TAML activator: a potent peroxidase mimic for chemiluminescent determination of hydrogen peroxide.

    Science.gov (United States)

    Vdovenko, Marina M; Demiyanova, Alexandra S; Kopylov, Kirill E; Sakharov, Ivan Yu

    2014-07-01

    Efforts to replace native peroxidase with its low molecular weight alternatives have stimulated a search for peroxidase mimetics. Herein we describe the oxidation of luminol with hydrogen peroxide catalyzed by commercially available Fe(III)-TAML activator 1a, which was shown to be a more active catalyst than hemin. At Fe(III)-TAML activator 1a use in chemiluminescent assay for H2O2 determination the detection limit value (3σ) of 5×10(-8)M was similar to the detection limit obtained with horseradish peroxidase (1×10(-7)M) and significantly lower than that obtained in the presence of hemin (6×10(-7)M). The linear ranges (R(2)=0.98) of the assay were 6×10(-8)-1×10(-6)M and 6×10(-7)-1×10(-6)M H2O2 for Fe(III)-TAML 1a and hemin, respectively. The CV values for Fe(III)-TAML 1a-based assay measured within the working range varied from 1.0% to 3.7% (n=4), whereas in the case of hemin -5.0% to 9.7% (n=4). Moreover, the sensitivity of Fe(III)-TAML 1a-based method was 56 and 5 times higher than that of hemin- and HRP-based methods, respectively. The obtained results open good perspectives to apply Fe(III)-TAML activator 1a in CL analytical methods instead of hemin, a traditionally used peroxidase mimetic. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Improving the oxidative stability of a high redox potential fungal peroxidase by rational design.

    Directory of Open Access Journals (Sweden)

    Verónica Sáez-Jiménez

    Full Text Available Ligninolytic peroxidases are enzymes of biotechnological interest due to their ability to oxidize high redox potential aromatic compounds, including the recalcitrant lignin polymer. However, different obstacles prevent their use in industrial and environmental applications, including low stability towards their natural oxidizing-substrate H2O2. In this work, versatile peroxidase was taken as a model ligninolytic peroxidase, its oxidative inactivation by H2O2 was studied and different strategies were evaluated with the aim of improving H2O2 stability. Oxidation of the methionine residues was produced during enzyme inactivation by H2O2 excess. Substitution of these residues, located near the heme cofactor and the catalytic tryptophan, rendered a variant with a 7.8-fold decreased oxidative inactivation rate. A second strategy consisted in mutating two residues (Thr45 and Ile103 near the catalytic distal histidine with the aim of modifying the reactivity of the enzyme with H2O2. The T45A/I103T variant showed a 2.9-fold slower reaction rate with H2O2 and 2.8-fold enhanced oxidative stability. Finally, both strategies were combined in the T45A/I103T/M152F/M262F/M265L variant, whose stability in the presence of H2O2 was improved 11.7-fold. This variant showed an increased half-life, over 30 min compared with 3.4 min of the native enzyme, under an excess of 2000 equivalents of H2O2. Interestingly, the stability improvement achieved was related with slower formation, subsequent stabilization and slower bleaching of the enzyme Compound III, a peroxidase intermediate that is not part of the catalytic cycle and leads to the inactivation of the enzyme.

  2. Thyroid peroxidase forms thionamide-sensitive homodimers: relevance for immunomodulation of thyroid autoimmunity

    OpenAIRE

    McDonald, David O.; Pearce, Simon H. S.

    2009-01-01

    Thyroid peroxidase (TPO) is the key enzyme in thyroid hormone production and a universal autoantigen in Graves? and other autoimmune thyroid diseases. We wished to explore the expression of TPO and whether it was affected by thionamide antithyroid drugs. We studied recombinant TPO, stably expressed by a Chinese hamster ovary cell line (CHO-TPO) and transiently expressed TPO-enhanced green fluorescent protein (eGFP) and -FLAG fusion proteins. Immunoblotting of CHO-TPO cell extracts showed high...

  3. Gut Microbiota Conversion of Dietary Ellagic Acid into Bioactive Phytoceutical Urolithin A Inhibits Heme Peroxidases.

    Directory of Open Access Journals (Sweden)

    Piu Saha

    Full Text Available Numerous studies signify that diets rich in phytochemicals offer many beneficial functions specifically during pathologic conditions, yet their effects are often not uniform due to inter-individual variation. The host indigenous gut microbiota and their modifications of dietary phytochemicals have emerged as factors that greatly influence the efficacy of phytoceutical-based intervention. Here, we investigated the biological activities of one such active microbial metabolite, Urolithin A (UA or 3,8-dihydroxybenzo[c]chromen-6-one, which is derived from the ellagic acid (EA. Our study demonstrates that UA potently inhibits heme peroxidases i.e. myeloperoxidase (MPO and lactoperoxidase (LPO when compared to the parent compound EA. In addition, chrome azurol S (CAS assay suggests that EA, but not UA, is capable of binding to Fe3+, due to its catechol-like structure, although its modest heme peroxidase inhibitory activity is abrogated upon Fe3+-binding. Interestingly, UA-mediated MPO and LPO inhibition can be prevented by innate immune protein human NGAL or its murine ortholog lipocalin 2 (Lcn2, implying the complex nature of host innate immunity-microbiota interactions. Spectral analysis indicates that UA inhibits heme peroxidase-catalyzed reaction by reverting the peroxidase back to its inactive native state. In support of these in vitro results, UA significantly reduced phorbol myristate acetate (PMA-induced superoxide generation in neutrophils, however, EA failed to block the superoxide generation. Treatment with UA significantly reduced PMA-induced mouse ear edema and MPO activity compared to EA treated mice. Collectively, our results demonstrate that microbiota-mediated conversion of EA to UA is advantageous to both host and microbiota i.e. UA-mediated inhibition of pro-oxidant enzymes reduce tissue inflammation, mitigate non-specific killing of gut bacteria, and abrogate iron-binding property of EA, thus providing a competitive edge to the

  4. Purification and characterization of novel cationic peroxidases from Asparagus acutifolius L. with biotechnological applications.

    Science.gov (United States)

    Guida, Vincenzo; Cantarella, Maria; Chambery, Angela; Mezzacapo, Maria C; Parente, Augusto; Landi, Nicola; Severino, Valeria; Di Maro, Antimo

    2014-08-01

    Four novel basic peroxidases, named AaP-1, AaP-2, AaP-3, and AaP-4, were purified from Asparagus acutifolius L. seeds by cation-exchange and gel filtration chromatographies. The four proteins showed a similar electrophoretic mobility of 46 kDa while, by MALDI-TOF MS, different Mr values of 42758.3, 41586.9, 42796.3, and 41595.5 were determined for AaP-1, AaP-2, AaP-3, and AaP-4, respectively. N-terminal sequences of AaPs 1-4 up to residue 20 showed a high percentage of identity with the peroxidase from Glycine max. In addition, AaP-1, AaP-2, AaP-3, and AaP-4 were found to be glycoproteins, containing 21.75, 22.27, 25.62, and 18.31 % of carbohydrates, respectively. Peptide mapping and MALDI-TOF MS analysis of AaPs 1-4 showed that the structural differences between AaP-1 and AaP-2 and AaP-3 and AaPs-4 were mainly due to their glycan content. We also demonstrate that AaPs were able to remove phenolic compounds from olive oil mill wastewaters with a higher catalytic efficiency with respect to horseradish peroxidase, thus representing candidate enzymes for potential biotechnological applications in the environmental field.

  5. A Novel Colorimetric Immunoassay Utilizing the Peroxidase Mimicking Activity of Magnetic Nanoparticles

    Directory of Open Access Journals (Sweden)

    Hyun Gyu Park

    2013-05-01

    Full Text Available A simple colorimetric immunoassay system, based on the peroxidase mimicking activity of Fe3O4 magnetic nanoparticles (MNPs, has been developed to detect clinically important antigenic molecules. MNPs with ca. 10 nm in diameter were synthesized and conjugated with specific antibodies against target molecules, such as rotaviruses and breast cancer cells. Conjugation of the MNPs with antibodies (MNP-Abs enabled specific recognition of the corresponding target antigenic molecules through the generation of color signals arising from the colorimetric reaction between the selected peroxidase substrate, 3,3',5,5'-tetramethylbenzidine (TMB and H2O2. Based on the MNP-promoted colorimetric reaction, the target molecules were detected and quantified by measuring absorbance intensities corresponding to the oxidized form of TMB. Owing to the higher stabilities and economic feasibilities of MNPs as compared to horseradish peroxidase (HRP, the new colorimetric system employing MNP-Abs has the potential of serving as a potent immunoassay that should substitute for conventional HRP-based immunoassays. The strategy employed to develop the new methodology has the potential of being extended to the construction of simple diagnostic systems for a variety of biomolecules related to human cancers and infectious diseases, particularly in the realm of point-of-care applications.

  6. Study of Horseradish Peroxidase Fixed on Mesoporous Materials as a Chemical Reaction Catalyst

    Science.gov (United States)

    Gao, Mengdan; Dai, Rongji

    2017-12-01

    Nanostructured mesoporous materials is a new type of porous materials, which has been widely used. It has excellent capability in enzymes immobilization, but modification on the chemical bonds of the enzyme reduce the enzymatic activity and rarely used in chemical reactions. The horseradish peroxidase was immobilized on the mesoporous materials with appropriate aperture and its activity and stability was evaluated when catalyzing the nitration reaction of amines and oxidation reaction of thiourea. The optimum mesoporous material to fix the horseradish peroxidase can be obtained by mixing polyoxyethylene - polyoxypropylene-pol, yoxyethylene(P123), 1,3,5-trimethylbenzene(TMB), and tetramethoxysilane (TMOS) at a ratio of 10:1:1, whose surface area and pore volume and pore diameter calculated by BET and BJH model were 402.903m2/g, 1.084cm2/g, 1.084cm2/g respectively. The horseradish peroxidase, immobilized on the mesoporous materials, was applied for catalyzing the nitration reaction of anilines and oxidation reaction of thiourea, produced a high product yield and can be recycled. Thus, it is a strong candidate as a catalysts for oxidation reactions, to be produced at industral scale, due to its high efficiency and low cost.

  7. Degradation of direct azo dye by Cucurbita pepo free and immobilized peroxidase.

    Science.gov (United States)

    Boucherit, Nabila; Abouseoud, Mahmoud; Adour, Lydia

    2013-06-01

    Enzymatic decolourization of the azo dye, Direct Yellow (DY106) by Cucurbita pepo (courgette) peroxidase (CP) is a complex process, which is greatly affected by pH, temperature, enzyme activity and the concentrations of H2O2 and dye. Courgette peroxidase was extracted and its performance was evaluated by using the free-CP (FCP) and immobilized-CP (ICP) forms in the decolourization of DY106. Immobilization of peroxidase in calcium alginate beads was performed according to a strategy aiming to minimize enzyme leakage and keep its activity at a maximum value by optimizing sodium alginate content, enzyme loading and calcium chloride concentration. The initial conditions at which the highest DY106 decolourization yield was obtained were found at pH 2, temperature 20 degrees C, H2O2 dose 1 mmol/L (FCP) and 100 mmol/L (ICP). The highest decolourization rates were obtained for dye concentrations 50 mg/L (FCP) and 80 mg/L (ICP). Under optimal conditions, the FCP was able to decolorize more than 87% of the dye within 2 min. While with ICP, the decolourization yield was 75% within 15 min. The decolourization and removal of DY106 was proved by UV-Vis analysis. Fourier transform infrared (FT-IR) spectroscopy analysis was also performed on DY106 and enzymatic treatment precipitated byproduct.

  8. Effects of ageing on peroxidase activity and localization in radish (Raphanus sativus L. seeds

    Directory of Open Access Journals (Sweden)

    A. Scialabba

    2010-05-01

    Full Text Available Peroxidase activity was assayed in crude extracts of integument, cotyledons and embryo axis of radish seeds, deteriorated under accelerated ageing conditions. Over five days of ageing, in which germination decreased from 100 to 52%, the enzyme activity in integument was higher than that in other seed parts, increasing in the first days of ageing and then decreasing sharply in extremely aged seeds. Polyacrylamide gel electrophoresis analysis showed four peroxidase isoenzymes with MM of 98, 52.5, 32.8 and 29.5 kDa in the embryo axis of unaged seeds, and only the 32.8 and 29.5 kDa MM isoforms in the integument and cotyledons. In these parts of the seed, only the 29.5 kDa MM isoenzyme increased in activity in early days of ageing and decreased thereafter. In the embryo axis, the 29.5 kDa MM isoenzyme activity increased slowly in the first day of ageing, while the 98 and 52.5 kDa MM isoenzyme activities disappeared. A cytochemical localization of peroxidase activity in the various tissues showed that main differences between unaged and extremely aged seeds occurred in the embryo axis.

  9. Molecular Cloning, Expression and Peroxidase Conjugation of Staphylococcus aureus Protein A

    Science.gov (United States)

    Seyfi Abad Shapouri, Masoud Reza; Mahmoodi, Pezhman; Gharibi, Dariush; Ghorbanpoor, Masoud; Yaghoubi, Sara; Rezaei, Elham; Rashno, Mohammad; Mehravar, Neda

    2016-01-01

    Background Staphylococcal protein A (SPA) is a cell wall component of Staphylococcus aureus that binds to different IgG subclasses of human and several animal species. This bacterial protein can be used as an antibody detector in various immunological assays or as an isolation reagent for the purification of antibody molecules via immuno-chromatography procedures. Objectives Molecular cloning and expression of SPA followed by the purification and conjugation of the recombinant protein to peroxidase enzyme. Material and Methods Encoding DNA fragment of SPA was amplified and inserted into a prokaryotic plasmid vector for the expression of recombinant SPA fused to a maltose binding protein (MBP). The recombinant protein was purified using amylose resin column chromatography and conjugated to horseradish peroxidase (HRP) enzyme. Finally, the reactivity of the recombinant SPA was examined against human IgG molecules in ELISA. Results The results indicated that the recombinant peroxidase-conjugated SPA has a good recognition capacity for human IgG molecules and it was able to produce significant OD values after reacting with human IgG molecules at a concentration up to 0.06 μg.well-1. Conclusions This recombinant protein can be very useful in all research laboratories and may decrease some of the expenses, e.g. those for preparing conjugated anti-antibodies. PMID:28959340

  10. Enhanced Delignification of Lignocellulosic Biomass by Recombinant Fungus Phanerochaete chrysosporium Overexpressing Laccases and Peroxidases.

    Science.gov (United States)

    Coconi Linares, Nancy; Fernández, Francisco; Loske, Achim M; Gómez-Lim, Miguel A

    2018-02-27

    Ligninolytic enzyme production and lignin degradation are typically the rate-limiting steps in the biofuel industry. To improve the efficiency of simultaneous bio-delignification and enzyme production, Phanerochaete chrysosporium was transformed by shock wave-induced acoustic cavitation to co-overexpress 3 peroxidases and 1 laccase and test it on the degradation of sugarcane bagasse and wheat bran. Lignin depolymerization was enhanced by up to 25% in the presence of recombinant fungi in comparison with the wild-type strain. Sugar release on lignocellulose was 2- to 6-fold higher by recombinant fungi as compared with the control. Wheat bran ostensibly stimulated the production of ligninolytic enzymes. The highest peroxidase activity from the recombinant strains was 2.6-fold higher, whereas the increase in laccase activity was 4-fold higher in comparison to the control. The improvement of lignin degradation was directly proportional to the highest peroxidase and laccase activity. Because various phenolic compounds released during lignocellulose degradation have proven to be toxic to cells and to inhibit enzyme activity, a significant reduction (over 40%) of the total phenolic content in the samples treated with recombinant strains was observed. To our knowledge, this is the first report that engineering P. chrysosporium enhances biodegradation of lignocellulosic biomass. © 2018 S. Karger AG, Basel.

  11. Purification, crystallization and preliminary X-ray diffraction analysis of royal palm tree (Roystonea regia) peroxidase

    Energy Technology Data Exchange (ETDEWEB)

    Watanabe, Leandra; Nascimento, Alessandro S. [Instituto de Física de São Carlos, Departamento de Física e Informática, Universidade de São Paulo, Avenida Trabalhador São Carlense 400, CEP 13566-590 São Carlos, SP (Brazil); Zamorano, Laura S. [Departamento de Química Física, Facultad de Química, Universidad de Salamanca, 37008 Salamanca (Spain); Shnyrov, Valery L. [Departamento de Bioquímica y Biología Molecular, Facultad de Biología, Universidad de Salamanca, 37007 Salamanca (Spain); Polikarpov, Igor, E-mail: ipolikarpov@if.sc.usp.br [Instituto de Física de São Carlos, Departamento de Física e Informática, Universidade de São Paulo, Avenida Trabalhador São Carlense 400, CEP 13566-590 São Carlos, SP (Brazil); Laboratório Nacional de Luz Síncrotron, Campinas, SP (Brazil)

    2007-09-01

    The purification, crystallization, X-ray diffraction data acquisition and molecular-replacement results of royal palm tree (R. regia) peroxidase are described. Royal palm tree peroxidase (RPTP), which was isolated from Roystonea regia leaves, has an unusually high stability that makes it a promising candidate for diverse applications in industry and analytical chemistry [Caramyshev et al. (2005 ▶), Biomacromolecules, 6, 1360–1366]. Here, the purification and crystallization of this plant peroxidase and its X-ray diffraction data collection are described. RPTP crystals were obtained by the hanging-drop vapour-diffusion method and diffraction data were collected to a resolution of 2.8 Å. The crystals belong to the trigonal space group P3{sub 1}21, with unit-cell parameters a = b = 116.83, c = 92.24 Å, and contain one protein molecule per asymmetric unit. The V{sub M} value and solvent content are 4.07 Å{sup 3} Da{sup −1} and 69.8%, respectively.

  12. Photosynthetic pigments and peroxidase activity of Lepidium sativum L. during assisted Hg phytoextraction.

    Science.gov (United States)

    Smolinska, Beata; Leszczynska, Joanna

    2017-05-01

    The study was conducted to evaluate metabolic answer of Lepidium sativum L. on Hg, compost, and citric acid during assisted phytoextraction. The chlorophyll a and b contents, total carotenoids, and activity of peroxidase were determined in plants exposed to Hg and soil amendments. Hg accumulation in plant shoots was also investigated. The pot experiments were provided in soil artificially contaminated by Hg and/or supplemented with compost and citric acid. Hg concentration in plant shoots and soil substrates was determined by cold vapor atomic absorption spectroscopy (CV-AAS) method after acid mineralization. The plant photosynthetic pigments and peroxidase activity were measured by standard spectrophotometric methods. The study shows that L. sativum L. accumulated Hg in its aerial tissues. An increase in Hg accumulation was noticed when soil was supplemented with compost and citric acid. Increasing Hg concentration in plant shoots was correlated with enhanced activation of peroxidase activity and changes in total carotenoid concentration. Combined use of compost and citric acid also decreased the chlorophyll a and b contents in plant leaves. Presented study reveals that L. sativum L. is capable of tolerating Hg and its use during phytoextraction assisted by combined use of compost and citric acid lead to decreasing soil contamination by Hg.

  13. Study on a hydrogen peroxide biosensor based on horseradish peroxidase/GNPs-thionine/chitosan

    International Nuclear Information System (INIS)

    Kang Xiaobin; Pang Guangchang; Liang Xinyi; Wang Meng; Liu Jing; Zhu Weiming

    2012-01-01

    Highlights: ► Glutaraldehyde was used as the bridge linking agent to covalently bonded thionine in chitosan, which is more stable and could effectively prevalent leakage of the electronic mediator. ► The effect of GNPs adsorbed HRP was first accurately characterized by bio-layer interferometry using the ForteBio Octer system. ► The application of self-assembly technology increases the biosensor stability. - Abstract: A novel hydrogen peroxide biosensor based on horseradish peroxidase/GNPs-thionine/chitosan has been developed. Gold nanoparticles fixed with horseradish peroxidase were adsorbed on glassy carbon electrode by the chitosan which cross-linked with the electron mediator of horseradish peroxidase as the bridge linking agent. The assembly procedures were monitored by UV–visible spectral scanning, bio-layer interferometry, cyclic voltammetric and alternating current impedance. The chronoamperometry was used to measure hydrogen peroxide. The hydrogen peroxide biosensor linear range of detection is 1 × 10 −7 –1 × 10 −4 mol/L, detection limit up to 5.0 × 10 −8 mol/L. Moreover the stability, reproducibility and selectivity of the biosensor were also studied and the results confirmed that the biosensor exhibit fast response to hydrogen peroxide and possess high sensitivity, good reproducibility and long-term stability.

  14. Cadmium-induced oxidative damage and protective effects of N-acetyl-L-cysteine against cadmium toxicity in Solanum nigrum L

    International Nuclear Information System (INIS)

    Deng Xiaopeng; Xia Yan; Hu Wei; Zhang Hongxiao; Shen Zhenguo

    2010-01-01

    The effects of cadmium (Cd) on the accumulation of hydrogen peroxide (H 2 O 2 ) and antioxidant enzyme activities in roots of Solanum nigrum L. and the role of N-acetyl-L-cysteine (NAC) as a cysteine (Cys) donor against Cd toxicity were investigated. Cd at 50 and 200 μM significantly increased the contents of thiobarbituric acid-reactive substances (TBARS), the production of H 2 O 2 and superoxide anion (O 2 · - ), and the activities of catalase, guaiacol peroxidase, ascorbate peroxidase, glutathione peroxidase (GSH-Px), glutathione reductase, and superoxide dismutase. Experiments with diphenylene iodonium as an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and NaN 3 as an inhibitor of peroxidase showed that the major source of Cd-induced reactive oxygen species in the roots may include plasma membrane-bound NADPH oxidase and peroxidase. In addition, the effects of NAC on plant growth, antioxidant enzyme activity, and non-protein thiol content were analyzed. Under Cd stress, the addition of 500 μM NAC decreased the contents of TBARS and production of H 2 O 2 and O 2 · - , but increased levels of Cys and reduced glutathione (GSH), phytochelatins, and activity of GSH-Px in roots. These results suggest that NAC could protect plants from oxidative stress damage, and this protection seems to be performed via increased GSH biosynthesis. Furthermore, NAC treatment also increased the contents of protein thiols in S. nigrum roots. By using size-exclusion chromatography, we found involvement of NAC in the Cd tolerance mechanism through increased biosynthesis of Cd-binding proteins.

  15. Cadmium-induced oxidative damage and protective effects of N-acetyl-L-cysteine against cadmium toxicity in Solanum nigrum L

    Energy Technology Data Exchange (ETDEWEB)

    Deng Xiaopeng; Xia Yan; Hu Wei [College of Life Sciences, Nanjing Agricultural University, Weigang 1, Nanjing 210095 (China); Zhang Hongxiao, E-mail: hxzhang@njau.edu.cn [College of Life Sciences, Nanjing Agricultural University, Weigang 1, Nanjing 210095 (China); Shen Zhenguo, E-mail: zgshen@njau.edu.cn [College of Life Sciences, Nanjing Agricultural University, Weigang 1, Nanjing 210095 (China)

    2010-08-15

    The effects of cadmium (Cd) on the accumulation of hydrogen peroxide (H{sub 2}O{sub 2}) and antioxidant enzyme activities in roots of Solanum nigrum L. and the role of N-acetyl-L-cysteine (NAC) as a cysteine (Cys) donor against Cd toxicity were investigated. Cd at 50 and 200 {mu}M significantly increased the contents of thiobarbituric acid-reactive substances (TBARS), the production of H{sub 2}O{sub 2} and superoxide anion (O{sub 2}{center_dot}{sup -}), and the activities of catalase, guaiacol peroxidase, ascorbate peroxidase, glutathione peroxidase (GSH-Px), glutathione reductase, and superoxide dismutase. Experiments with diphenylene iodonium as an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and NaN{sub 3} as an inhibitor of peroxidase showed that the major source of Cd-induced reactive oxygen species in the roots may include plasma membrane-bound NADPH oxidase and peroxidase. In addition, the effects of NAC on plant growth, antioxidant enzyme activity, and non-protein thiol content were analyzed. Under Cd stress, the addition of 500 {mu}M NAC decreased the contents of TBARS and production of H{sub 2}O{sub 2} and O{sub 2}{center_dot}{sup -}, but increased levels of Cys and reduced glutathione (GSH), phytochelatins, and activity of GSH-Px in roots. These results suggest that NAC could protect plants from oxidative stress damage, and this protection seems to be performed via increased GSH biosynthesis. Furthermore, NAC treatment also increased the contents of protein thiols in S. nigrum roots. By using size-exclusion chromatography, we found involvement of NAC in the Cd tolerance mechanism through increased biosynthesis of Cd-binding proteins.

  16. Effect of caffeine on peroxidase activity and gamma-ray-induced oxic and anoxic damage in Hordeum vulgare

    International Nuclear Information System (INIS)

    Balachandran, R.; Kesavan, P.C.

    1978-01-01

    The influence of caffeine during and after gamma radiation of barley seeds was studied using seedling injury and peroxidase activity as parameters. The radiation-induced stimulation of peroxidase activity is evident in eight-day only seedlings but not in embryos (i.e. immediately after irradiation). Caffeine present during irradiation of seeds soaked in oxygenated water diminishes seedling injury and also reduces the peroxidase activity to the level observed in eight-day old seedlings of unirradiated seeds. Caffeine, however, produces just the opposite effect (i.e. enhances the seedling injury and peroxidase activity of eight-day old seedlings) when applied during irradiation of seeds soaked in oxygen-free water. There is no evidence that caffeine effects enzyme activity under in vitro conditions. (author)

  17. Thyroid peroxidase antibodies in pregnant women with type 1 diabetes: impact on thyroid function, metabolic control and pregnancy outcome

    DEFF Research Database (Denmark)

    Vestgaard, Marianne; Nielsen, Lene Ringholm; Rasmussen, Åse Krogh

    2008-01-01

    In pregnant women with type 1 diabetes, we evaluated whether the presence of thyroid peroxidase autoantibodies (anti-TPO) was associated with changes in thyroid function, metabolic control and pregnancy outcome....

  18. The involvement of polyphenols and peroxidase activities in heavy-metal accumulation by epidermal glands of the waterlily (Nymphaeaceae).

    Science.gov (United States)

    Lavid, N; Schwartz, A; Yarden, O; Tel-Or, E

    2001-02-01

    Co-localization of polyphenols and peroxidase activity was demonstrated in epidermal glands of the waterlily (Nymphaea) by histochemistry. Total phenols, tannins and peroxidase activity were determined quantitatively in plant extracts. Polyphenols were partially identified and were found to consist mainly of hydrolyzable tannins, gallic and tannic acid derivatives. Nymphaea polyphenols were shown to chelate Cr, Hg, and Pb in vitro, and Cd-binding by polymerized polyphenols was demonstrated in leaves exposed to Cd in vivo. Both polyphenols and peroxidases were found at very high constitutive levels, which were not induced or altered by external conditions, such as light and heavy-metal stress. It is suggested that the polymerization of polyphenols by peroxidases, enhanced after heavy-metal uptake and detoxification, is responsible for the binding of heavy metals in Nymphaea epidermal glands.

  19. Maternal and fetal plasma concentrations of endothelin, lipidhydroperoxides, glutathione peroxidase and fibronectin in relation to abnormal umbilical artery velocimetry.

    NARCIS (Netherlands)

    Karsdorp, V.H.M.; Bast, A.; van Kamp, G.J.; Bouman, A.A.; Dekker, G.A.; van Vugt, J.M.G.; van Geijn, H.

    1998-01-01

    Objective: To study plasma concentrations of endothelin (ET), lipidhydroperoxides (LOOH), glutathione peroxidase (GSHpx) and fibronectin in relation to abnormal umbilical artery velocimetry. Study design: Plasma concentrations of ET, LOOH, GSHpx and fibronectin were measured in fetal and maternal

  20. Aluminum-induced cell wall peroxidase activity and lignin synthesis are differentially regulated by jasmonate and nitric oxide.

    Science.gov (United States)

    Xue, Yao Juan; Tao, Ling; Yang, Zhi Min

    2008-10-22

    Cassia tora is an annual legume and cultivated as a traditional medicinal herb for multiple therapies including regulation of blood pressure and blood lipid. Because of naturally occurring acidic soils in southeastern China, this plant species may possess strategies for tolerance to low pH and aluminum toxicity. In the search for the regulatory basis of biochemical response to Al, cell wall-bound peroxidases, including lignin-generated peroxidases and NADH oxidases, were investigated in the root tips of C. tora. Activities of both types of peroxidases significantly increased with Al concentrations. Analysis with native PAGE also demonstrated the strong induction of cell wall peroxidases by Al. The Al-induced increasing activities of peroxidases were closely correlated with lignin accumulation and H 2O 2 production. The biochemical effect of exogenous nitric oxide (NO) and methyl jasmonic acid (MJ) was examined to investigate signal properties and lignin synthesis under Al stress. Application of MJ at 10 microM promoted root sensitivity to Al by activating apoplastic peroxidase activity and accumulating H 2O 2 and lignin, whereas the opposite action was found for NO. The sensitivity of apoplastic peroxidases under Al stress was associated with the cross-talk of MJ and NO signals. The analysis reveals that the activity of lipoxygenase (an enzyme for MJ biosynthesis), with its transcripts increased in Al-exposed roots, was depressed by NO exposure. The effect of MJ on intracellular NO production was also investigated. It is shown that NO staining with 4,5-diaminofluorescein diacetate fluorescence was intensified by Al but was suppressed by MJ. These results suggest that NO and MJ may interplay in signaling the cell wall peroxidase activity and lignin synthesis in the roots exposed to Al.

  1. [Direction of strategic use: a new classification of non-steroidal anti-inflammatory drugs based on reactivity with peroxidase].

    Science.gov (United States)

    Miura, Toshiaki

    2013-01-01

      The pharmaceutical effects of non-steroidal anti-inflammatory drugs (NSAIDs) occur through the inhibition of prostaglandin H synthase (PGHS). Prostaglandin H2 is produced from arachidonic acid via peroxidase and cyclooxygenase cycles in PGHS. NSAIDs exhibit different levels of reactivity in these reaction cycles. To prevent the development of side effect while maintaining the beneficial effects of drugs, a therapeutic strategy should be used. A new classification of NSAIDs has been proposed based on reactivity to peroxidase. Class 1 includes the majority of NSAIDs, which react with horseradish peroxidase (HRP) compounds I and II. Also, their drugs exhibit spectral changes induced by PGHS peroxidase and diminished ESR signals of the tyrosyl radical of metmyoglobin. They reduce compounds I and II of HRP and scavenge tyrosyl radicals. The branched chain mechanism by which the porphyrin radical is transferred to the tyrosine residue of the protein might be blocked by these NSAIDs. Class 2 includes salicylic acid derivatives that react only with the porphyrin radical and do not react with HRP compound II (oxoferryl species). Class 3 includes aspirin, nimesulide, tolmetin, and arylpropionic acid derivatives, including ibuprofen and the coxibs such as celecoxib and rofecoxib, which are not substrates for HRP or PGHS peroxidase. It seems that the selectivity of NSAIDs to PGHS1 and PGHS2 depends on their reactivity with cyclooxygenase rather than with the peroxidase of PGHS. The best drug for each inflammatory disease should therefore be selected for therapy.

  2. Changes in Peroxidase Activity in the Peel of Unshiub Mandarin (Citrus unshiu Marc. Fruit with Different Storage Treatments

    Directory of Open Access Journals (Sweden)

    Hrvoje Lepeduš

    2005-01-01

    Full Text Available The Unshiu mandarin (Citrus unshiu Marc. is the major Citrus crop in Croatia. Limiting factors for longer consumption of Unshiu mandarin are low storage performance and the appearance of chilling injuries during storage. Previous studies indicated that oxidative stress might be involved in cold-induced peel damage of harvested Citrus fruit. The aim of the present study was to investigate peroxidase distribution, isoenzyme pattern and activity in the peel of Unshiu mandarin fruit. Special goal of our study was to investigate the changes of peroxidase activity in respect to two different hot water dipping (HWD treatments (3 min at 48 and 52 °C and two different storage temperatures (1 and 3 °C combined. Peroxidase activity was detected at the border of oil glands, in the peel surface and in the conducting elements positioned in the inner part of the peel. Electrophoretic analysis revealed the presence of two peroxidase isoenzymes. There were no differences in the electrophoretic pattern after the HWD treatments and cold storage. Lowering of both total and specific peroxidase activity was measured in HWD-treated samples in comparison with the control ones. However, it appeared that significant decrease in total peroxidase activity was influenced by the storage temperatures, while the increase in total soluble protein content was influenced by the HWD pretreatment.

  3. Differential expression on mitochondrial tryparedoxin peroxidase (mTcTXNPx in Trypanosoma cruzi after ferrocenyl diamine hydrochlorides treatments

    Directory of Open Access Journals (Sweden)

    Andréa A.N. Kohatsu

    2017-03-01

    Full Text Available Resistance to benznidazole in certain strains of Trypanosoma cruzi may be caused by the increased production of enzymes that act on the oxidative metabolism, such as mitochondrial tryparedoxin peroxidase which catalyses the reduction of peroxides. This work presents cytotoxicity assays performed with ferrocenyl diamine hydrochlorides in six different strains of T. cruzi epimastigote forms (Y, Bolivia, SI1, SI8, QMII, and SIGR3. The last four strains have been recently isolated from triatominae and mammalian host (domestic cat. The expression of mitochondrial tryparedoxin peroxidase was analyzed by the Western blotting technique using polyclonal antibody anti mitochondrial tryparedoxin peroxidase obtained from a rabbit immunized with the mitochondrial tryparedoxin peroxidase recombinant protein. All the tested ferrocenyl diamine hydrochlorides were more cytotoxic than benznidazole. The expression of the 25.5 kDa polypeptide of mitochondrial tryparedoxin peroxidase did not increase in strains that were more resistant to the ferrocenyl compounds (SI8 and SIGR3. In addition, a 58 kDa polypeptide was also recognized in all strains. Ferrocenyl diamine hydrochlorides showed trypanocidal activity and the expression of 25.5 kDa mitochondrial tryparedoxin peroxidase is not necessarily increased in some T. cruzi strains. Most likely, other mechanisms, in addition to the over expression of this antioxidative enzyme, should be involved in the escape of parasites from cytotoxic oxidant agents.

  4. Differential expression on mitochondrial tryparedoxin peroxidase (mTcTXNPx) in Trypanosoma cruzi after ferrocenyl diamine hydrochlorides treatments.

    Science.gov (United States)

    Kohatsu, Andréa A N; Silva, Flávia A J; Francisco, Acácio I; Rimoldi, Aline; Silva, Marco T A; Vargas, Maria D; Rosa, João A da; Cicarelli, Regina M B

    Resistance to benznidazole in certain strains of Trypanosoma cruzi may be caused by the increased production of enzymes that act on the oxidative metabolism, such as mitochondrial tryparedoxin peroxidase which catalyses the reduction of peroxides. This work presents cytotoxicity assays performed with ferrocenyl diamine hydrochlorides in six different strains of T. cruzi epimastigote forms (Y, Bolivia, SI1, SI8, QMII, and SIGR3). The last four strains have been recently isolated from triatominae and mammalian host (domestic cat). The expression of mitochondrial tryparedoxin peroxidase was analyzed by the Western blotting technique using polyclonal antibody anti mitochondrial tryparedoxin peroxidase obtained from a rabbit immunized with the mitochondrial tryparedoxin peroxidase recombinant protein. All the tested ferrocenyl diamine hydrochlorides were more cytotoxic than benznidazole. The expression of the 25.5kDa polypeptide of mitochondrial tryparedoxin peroxidase did not increase in strains that were more resistant to the ferrocenyl compounds (SI8 and SIGR3). In addition, a 58kDa polypeptide was also recognized in all strains. Ferrocenyl diamine hydrochlorides showed trypanocidal activity and the expression of 25.5kDa mitochondrial tryparedoxin peroxidase is not necessarily increased in some T. cruzi strains. Most likely, other mechanisms, in addition to the over expression of this antioxidative enzyme, should be involved in the escape of parasites from cytotoxic oxidant agents. Copyright © 2016 Sociedade Brasileira de Infectologia. Published by Elsevier Editora Ltda. All rights reserved.

  5. High frequency of positive anti-thyroid peroxidase antibodies (ATPO) in adult subjects without known thyroid disease, Santiago de Chile

    International Nuclear Information System (INIS)

    Lanas, Alejandra; Letelier, Carolina; Caamano, Edgardo; Massardo, Teresa; Gonzalez, Patricio; Araya, Veronica

    2010-01-01

    Background: Anti-thyroid peroxidase antibodies have a pathogenic role in Hashimoto thyroiditis. Between 10 and 19% of individuals without thyroid disease, have positive titers of these antibodies. Aim: To study the frequency of positive titers of anti-thyroid peroxidase antibodies in healthy individuals. Material and Methods: A blood sample, to measure anti-thyroid peroxidase antibodies and thyroid stimulating hormone (TSH) by chemiluminescence assay, was obtained from 67 women and 62 men aged 45 ± 14 years, without a personal or familiar history of thyroid diseases and normal thyroid palpation. The cutoff point of the manufacturer to consider positive a titer of anti-thyroid peroxidase antibodies was set at 35 IU/ml. Results: Twenty-eight women and 28 men had positive antibody titers (43% of the sample). Subjects in the upper tercile of anti-thyroid peroxidase antibody titers had a higher TSH than those in the second tercile, although within normal limits (1.73 ± 0.74 and 1.37 ± 0.59 mlU/L, respectively p = 0.02) Conclusions: Forty three percent of the studied subjects without personal or familial history of thyroid diseases had positive titers of anti-thyroid peroxidase antibodies. Further prospective studies should evaluate whether this observation discloses an increase in thyroid autoimmune disease in a population with increased iodine intake

  6. Obtenção de nova fonte de peroxidase de folha de Copaifera langsdorffii Desf. com alta atividade Obtention of a new source of peroxidase from Copaifera langsdorffii leaf, Desf. with high activity

    Directory of Open Access Journals (Sweden)

    Hermelinda Penha Freire Maciel

    2006-12-01

    Full Text Available Objetivou-se neste trabalho extrair peroxidase de folha de Copaifera langsdorffii (COP, medir sua atividade, compará-la com a peroxidase de raiz forte (Horseradish peroxidase - HRP e determinar o pH ótimo, a melhor solução extratora e o efeito de aditivos sobre a atividade da COP. Os resultados mostraram que a COP atingiu 81,6% da atividade de HRP e a faixa de pH ótimo foi de 5,5 a 6,0. A melhor solução extratora da enzima foi o tampão fosfato de sódio 50 mM, pH 6,0 e o melhor aditivo foi o PVPP. Concluindo, a COP apresenta atividade mais alta que outras peroxidases de diferentes fontes citadas na literatura.The purpose of this work was to extract peroxidase from Copaifera langsdorffii leaves (COP, measure its activity, compare it to that of Horseradish peroxidase and determine the optimum pH, the best extraction solution and the effect of additives on the COP activity. The results showed that COP has 81.6% of the activity of HRP and an optimum pH range between 5.5-6.0. The best extraction solution was a sodium phosphate buffer 50 mM, pH 6.0 and the best additive was PVPP. In conclusion, COP presents higher activity than peroxidases from different sources reported in the literature.

  7. Changes in the activity of ascorbate peroxidase under anaerobiosis in cocoyam (Colocasia esculenta).

    Science.gov (United States)

    Chibueze, Nwose

    2014-01-01

    This study was conducted to determine the activity of ascorbate peroxidase in the cormels of cocoyam (Colocasia esculenta var. antiquorum) immediately after harvest and in storage under anaerobiosis for one and three weeks, respectively. During stress condition in plants, hydrogen peroxide is released and mechanisms to detoxify it must be maintained. The cocoyam tubers that were neither damaged nor affected by disease were harvested from a local farm in Ugbogui, Ovia North Local Government Area in Edo State, Nigeria. The selected cocoyam tubers were peeled manually, washed with ice cold water and cut into pieces. The root tissues (50 g) were homogenised with 100 mL of ice cold 0.05 M phosphate buffer. The extract obtained was clarified by centrifugation for 15 min at 8000 g at 4 degrees C. Ascorbate-peroxidising activity was assayed using the initial rate of decrease in ascorbate concentration as measured by its absorbance at 290 nm using Milton Roy Spectron 21D. Results showed the weight of the cormels decreased all through during storage. Immediately after harvest the activity of ascorbate peroxidase was 15.49 unit mL(-1) with a significant increase (p < 0.05) after one week to 73.05 U mL(-1). Thereafter there was a significant decrease in activity of the enzyme after three weeks of storage to 33.33 U mL(-1). This increase in activity of ascorbate peroxidase after three weeks of storage may be related to increase in response to various biotic stresses. Therefore, manipulation of the capacity of cocoyam to tolerate anaerobiosis is a function of its ability to modulate the antioxidant enzymes' armory in case of need.

  8. Peroxidase-like activity of nanocrystalline cobalt selenide and its application for uric acid detection

    Directory of Open Access Journals (Sweden)

    Zhuang QQ

    2017-04-01

    Full Text Available Quan-Quan Zhuang,1 Zhi-Hang Lin,1 Yan-Cheng Jiang,1 Hao-Hua Deng,2 Shao-Bin He,1,3 Li-Ting Su,4 Xiao-Qiong Shi,2 Wei Chen2 1Department of Pharmacy, Affiliated Quanzhou First Hospital of Fujian Medical University, Quanzhou, 2Department of Pharmaceutical Analysis, School of Pharmacy, Fujian Medical University, Fuzhou, 3Department of Pharmacy, Quanzhou Infectious Disease Hospital, 4Department of Pharmaceutical Analysis, Quanzhou Medical College, Quanzhou, People’s Republic of China Abstract: Dendrite-like cobalt selenide nanostructures were synthesized from cobalt and selenium powder precursors by a solvothermal method in anhydrous ethylenediamine. The as-prepared nanocrystalline cobalt selenide was found to possess peroxidase-like activity that could catalyze the reaction of peroxidase substrates in the presence of H2O2. A spectrophotometric method for uric acid (UA determination was developed based on the nanocrystalline cobalt selenide-catalyzed coupling reaction between N-ethyl-N-(3-sulfopropyl-3-methylaniline sodium salt and 4-aminoantipyrine (4-AAP in the presence of H2O2. Under optimum conditions, the absorbance was proportional to the concentration of UA over the range of 2.0–40 µM with a detection limit of 0.5 µM. The applicability of the proposed method has been validated by determination of UA in human serum samples with satisfactory results. Keywords: enzyme mimics, cobalt selenide, peroxidase-like activity, uric acid, human serum

  9. Effect of distal His mutation on the peroxynitrite reactivity of Leishmania major peroxidase.

    Science.gov (United States)

    Saha, Rina; Bose, Moumita; Santara, Sumit Sen; Roy, Jayasree; Yadav, Rajesh K; Adak, Subrata

    2013-10-01

    The conserved distal histidine in peroxidases has been considered to play a major role as a general acid-base catalyst for heterolytic cleavage of an OO bond in H2O2. However, heme peroxidases react with peroxynitrite to form transient intermediates but the role of the distal histidine in this reaction is still unknown. In order to investigate catalytic roles of the histidine at the distal cavity, two Leishmania major peroxidase (LmP) mutants (H68E, H68V) were prepared. The rate of transition from ferric H68V to Compound ES by H2O2 is decreased by approximately five orders of magnitude relative to wild type, which is consistent with electron donor oxidation data where the H68V is ~1000 fold less active than wild type. In the reaction with peroxynitrite, the formation rate of intermediates in the mutants is not significantly lower than that for the wild type, indicating that the His68 has no major role in homolytic cleavage of an OO bond in peroxynitrite. EPR spectroscopic data suggest that the transient intermediates formed by the reaction of LmP with H2O2 exhibits an intense and stable signal similar to CCP Compound ES whereas in case of the reaction with peroxynitrite, this signal disappears, indicating that the transient intermediate is Compound II. Rapid kinetics data suggest that the distal His68 mutants display higher decay rates of Compound II than wild type. Thus, His68 mutations minimize Compound II formation (inactive species in peroxynitrite scavenging cycles) by increasing decay rates during the steady state and results in higher peroxynitrite degrading activity. © 2013.

  10. Antioxidant activity and peroxidase inhibition of Amazonian plants extracts traditionally used as anti-inflammatory.

    Science.gov (United States)

    de Vargas, Fabiano S; Almeida, Patricia D O; de Boleti, Ana Paula A; Pereira, Maria M; de Souza, Tatiane P; de Vasconcellos, Marne C; Nunez, Cecilia Veronica; Pohlit, Adrian M; Lima, Emerson S

    2016-02-27

    The Amazon is the largest rainforest in the world and is home to a rich biodiversity of medicinal plants. Several of these plants are used by the local population for the treatment of diseases, many of those with probable anti-inflammatory effect. The aim of the present investigation was to evaluate the in vitro antioxidant and anti-peroxidases potential of the ethanol extracts of five plants from the Brazilian Amazon (Byrsonima japurensis, Calycophyllum spruceanum, Maytenus guyanensis, Passiflora nitida and Ptychopetalum olacoides). DPPH, ABTS, superoxide anion radical, singlet oxygen and the β-carotene bleaching methods were employed for characterization of free radical scavenging activity. Also, total polyphenols were determined. Antioxidant activities were evaluated using murine fibroblast NIH3T3 cell. Inhibition of HRP and MPO were evaluated using amplex red® as susbtract. The stem bark extracts of C. spruceanum and M. guyanensis provided the highest free radical scavenging activities. C. spruceanum exhibited IC50 = 7.5 ± 0.9, 5.0 ± 0.1, 18.2 ± 3.0 and 92.4 ± 24.8 μg/mL for DPPH(•), ABTS(+•), O2 (-•) and (1)O2 assays, respectively. P. olacoides and C. spruceanum extracts also inhibited free radicals formation in the cell-based assay. At a concentration of 100 μg/mL, the extracts of C. spruceanum, B. japurensis inhibited horseradish peroxidase by 62 and 50 %, respectively. C. spruceanum, M. guyanensis, B. japurensis also inhibited myeloperoxidase in 72, 67 and 56 %, respectively. This work supports the folk use these species that inhibited peroxidases and exhibited significant free radical scavenging and antioxidant activities what can be related to treatment of inflammation.

  11. Cytosolic peroxidases protect the lysosome of bloodstream African trypanosomes from iron-mediated membrane damage.

    Directory of Open Access Journals (Sweden)

    Corinna Hiller

    2014-04-01

    Full Text Available African trypanosomes express three virtually identical non-selenium glutathione peroxidase (Px-type enzymes which preferably detoxify lipid-derived hydroperoxides. As shown previously, bloodstream Trypanosoma brucei lacking the mitochondrial Px III display only a weak and transient proliferation defect whereas parasites that lack the cytosolic Px I and Px II undergo extremely fast lipid peroxidation and cell lysis. The phenotype can completely be rescued by supplementing the medium with the α-tocopherol derivative Trolox. The mechanism underlying the rapid cell death remained however elusive. Here we show that the lysosome is the origin of the cellular injury. Feeding the px I-II knockout parasites with Alexa Fluor-conjugated dextran or LysoTracker in the presence of Trolox yielded a discrete lysosomal staining. Yet upon withdrawal of the antioxidant, the signal became progressively spread over the whole cell body and was completely lost, respectively. T. brucei acquire iron by endocytosis of host transferrin. Supplementing the medium with iron or transferrin induced, whereas the iron chelator deferoxamine and apo-transferrin attenuated lysis of the px I-II knockout cells. Immunofluorescence microscopy with MitoTracker and antibodies against the lysosomal marker protein p67 revealed that disintegration of the lysosome precedes mitochondrial damage. In vivo experiments confirmed the negligible role of the mitochondrial peroxidase: Mice infected with px III knockout cells displayed only a slightly delayed disease development compared to wild-type parasites. Our data demonstrate that in bloodstream African trypanosomes, the lysosome, not the mitochondrion, is the primary site of oxidative damage and cytosolic trypanothione/tryparedoxin-dependent peroxidases protect the lysosome from iron-induced membrane peroxidation. This process appears to be closely linked to the high endocytic rate and distinct iron acquisition mechanisms of the infective

  12. Calculated ionisation potentials to determine the oxidation of vanillin precursors by lignin peroxidase.

    OpenAIRE

    Have, ten, R.; Rietjens, I.M.C.M.; Hartmans, S.; Swarts, H.J.; Field, J.A.

    1998-01-01

    In view of the biocatalytic production of vanillin, this research focused on the lignin peroxidase (LiP) catalysed oxidation of naturally occurring phenolic derivatives: O-methyl ethers, O-acetyl esters, and O-glucosyl ethers. The ionisation potential (IP) of a series of model compounds was calculated and compared to their experimental conversion by LiP, defining a relative IP threshold of approximately 9.0 eV. Based on this threshold value only the O-acetyl esters and glucosides of isoeugeno...

  13. Lignin peroxidase mediated biotransformations useful in the biocatalytic production of vanillin

    OpenAIRE

    Have, ten, R.

    2000-01-01

    This research concentrates on lignin peroxidase (LiP) mediated biotrans-formations that are useful in producing vanillin.

    In order to obtain this extracellular enzyme, the white-rot fungus Bjerkandera sp. strain BOS55 was cultivated on nitrogen rich medium. This procedure resulted in a successful LiP production of 600 U/L. Peptone in the culture medium was shown to interfere with the standard LiP assay in which the formation of veratraldehyde (V...

  14. Decolorization of Azo, Triphenyl Methane, Heterocyclic, and Polymeric Dyes by Lignin Peroxidase Isoenzymes from Phanerochaete chrysosporium

    OpenAIRE

    Ollikka, Pauli; Alhonmäki, Kirsi; Leppänen, Veli-Matti; Glumoff, Tuomo; Raijola, Timo; Suominen, Ilari

    1993-01-01

    The ligninolytic enzyme system of Phanerochaete chrysosporium decolorizes several recalcitrant dyes. Three isolated lignin peroxidase isoenzymes (LiP 4.65, LiP 4.15, and LiP 3.85) were compared as decolorizers with the crude enzyme system from the culture medium. LiP 4.65 (H2), LiP 4.15 (H7), and LiP 3.85 (H8) were purified by chromatofocusing, and their kinetic parameters were found to be similar. Ten different types of dyes, including azo, triphenyl methane, heterocyclic, and polymeric dyes...

  15. Methylene blue as a lignin surrogate in manganese peroxidase reaction systems.

    Science.gov (United States)

    Goby, Jeffrey D; Penner, Michael H; Lajoie, Curtis A; Kelly, Christine J

    2017-11-15

    Manganese peroxidase (MnP) is associated with lignin degradation and is thus relevant to lignocellulosic-utilization technologies. Technological applications require reaction mixture optimization. A surrogate substrate can facilitate this if its susceptibility to degradation is easily monitored and mirrors that of lignin. The dye methylene blue (MB) was evaluated in these respects as a surrogate substrate by testing its reactivity in reaction mixtures containing relevant redox mediators (dicarboxylic acids, fatty acids). Relative rates of MB degradation were compared to available literature reports of lignin degradation under similar conditions, and suggest that MB can be a useful lignin surrogate in MnP systems. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Effect of biological and chemical preparations on peroxidase activity in leaves of tomato plants

    Directory of Open Access Journals (Sweden)

    Yulia Kolomiets

    2016-10-01

    Full Text Available In terms of treating tomato variety Chaika with chemical preparations with active substances if aluminum phosphate, 570 g/l + phosphorous acid 80 g/,l and mankotseb in concentration of 640 g/kg, the maximum increase in peroxidase activity in leaves of plants was observed in12 hours. In terms of use of biological preparations based on living cells Bacillus subtilis and Azotobacter chroococcum its activity was maximum in 24 hours and ranged from 77.7 to 112.7 un.mg-1•s-1

  17. Catalytic application of selenium and tellurium compounds as glutathione peroxidase enzyme mimetics

    Energy Technology Data Exchange (ETDEWEB)

    Alberto, Eduardo E.; Braga, Antonio L., E-mail: albraga@qmc.ufsc.b [Federal University of Santa Maria (UFSM), RS (Brazil). Dept. of Chemistry; Nascimento, Vanessa do [Federal University of Santa Catarina (UFSC), Florianopolis, SC (Brazil). Dept. of Chemistry

    2010-07-01

    This review covers the past decade of intensive research on the design, synthesis and screening of organoselenides and tellurides as catalyst able to mimic the activity of the selenoenzyme glutathione peroxidase (GPx). This important enzyme forms part of the detoxification system in humans which deals with harmful peroxides and their byproducts formed during oxygen metabolism. Several strategies to enhance the GPx-like activity of compounds such as diselenides, selenides and tellurides have been proposed in recent years. Different mechanisms of action of these compounds are also presented in this review highlighting new advances in this exciting research field. (author)

  18. Effect of Nonsurgical Periodontal Therapy on Crevicular Fluid and Serum Glutathione Peroxidase Levels

    Directory of Open Access Journals (Sweden)

    Swati Pradeep Patel

    2012-01-01

    Full Text Available Background: Plasma glutathione peroxidase (eGPx is an important selenium containing antioxidant in human defense against oxidative stress. While crevicular fluid (GCF eGPx levels and its association with periodontal disease is well documented, there is no data on correlation of GCF and serum eGPx levels in chronic periodontitis. Hence this study was undertaken to further probe into the role of oxidative stress in periodontal diseases and effect of nonsurgical periodontal therapy (NSPT by correlating GCF and serum levels of eGPx.

  19. Removal of triclosan via peroxidases-mediated reactions in water: Reaction kinetics, products and detoxification

    Energy Technology Data Exchange (ETDEWEB)

    Li, Jianhua; Peng, Jianbiao [State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210023 (China); Zhang, Ya [Nanjing Institute of Environmental Sciences, Ministry of Environmental Protection of the People’s Republic of China, Nanjing 210042 (China); Ji, Yuefei [College of Resources and Environmental Science, Nanjing Agricultural University, Nanjing 210095 (China); Shi, Huanhuan; Mao, Liang [State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210023 (China); Gao, Shixiang, E-mail: ecsxg@nju.edu.cn [State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210023 (China)

    2016-06-05

    Highlights: • Enzymatic treatment of triclosan in water by soybean and horseradish peroxidases. • pH, H{sub 2}O{sub 2} concentration and enzyme dosage affected the removal efficiency of TCS. • The removal of TCS by SBP was more efficient than that of HRP. • K{sub CAT} and K{sub CAT}/K{sub M} values for SBP toward TCS were much higher than those for HRP. • Polymers formed via radical coupling mechanism were nontoxic to the growth of alga. - Abstract: This study investigated and compared reaction kinetics, product characterization, and toxicity variation of triclosan (TCS) removal mediated by soybean peroxidase (SBP), a recognized potential peroxidase for removing phenolic pollutants, and the commonly used horseradish peroxidase (HRP) with the goal of assessing the technical feasibility of SBP-catalyzed removal of TCS. Reaction conditions such as pH, H{sub 2}O{sub 2} concentration and enzyme dosage were found to have a strong influence on the removal efficiency of TCS. SBP can retain its catalytic ability to remove TCS over broad ranges of pH and H{sub 2}O{sub 2} concentration, while the optimal pH and H{sub 2}O{sub 2} concentration were 7.0 and 8 μM, respectively. 98% TCS was removed with only 0.1 U mL{sup −1} SBP in 30 min reaction time, while an HRP dose of 0.3 U mL{sup −1} was required to achieve the similar conversion. The catalytic performance of SBP towards TCS was more efficient than that of HRP, which can be explained by catalytic rate constant (K{sub CAT}) and catalytic efficiency (K{sub CAT}/K{sub M}) for the two enzymes. MS analysis in combination with quantum chemistry computation showed that the polymerization products were generated via C−C and C−O coupling pathways. The polymers were proved to be nontoxic through growth inhibition of green alga (Scenedesmus obliquus). Taking into consideration of the enzymatic treatment cost, SBP may be a better alternative to HRP upon the removal and detoxification of TCS in water

  20. Removal of triclosan via peroxidases-mediated reactions in water: Reaction kinetics, products and detoxification

    International Nuclear Information System (INIS)

    Li, Jianhua; Peng, Jianbiao; Zhang, Ya; Ji, Yuefei; Shi, Huanhuan; Mao, Liang; Gao, Shixiang

    2016-01-01

    Highlights: • Enzymatic treatment of triclosan in water by soybean and horseradish peroxidases. • pH, H 2 O 2 concentration and enzyme dosage affected the removal efficiency of TCS. • The removal of TCS by SBP was more efficient than that of HRP. • K CAT and K CAT /K M values for SBP toward TCS were much higher than those for HRP. • Polymers formed via radical coupling mechanism were nontoxic to the growth of alga. - Abstract: This study investigated and compared reaction kinetics, product characterization, and toxicity variation of triclosan (TCS) removal mediated by soybean peroxidase (SBP), a recognized potential peroxidase for removing phenolic pollutants, and the commonly used horseradish peroxidase (HRP) with the goal of assessing the technical feasibility of SBP-catalyzed removal of TCS. Reaction conditions such as pH, H 2 O 2 concentration and enzyme dosage were found to have a strong influence on the removal efficiency of TCS. SBP can retain its catalytic ability to remove TCS over broad ranges of pH and H 2 O 2 concentration, while the optimal pH and H 2 O 2 concentration were 7.0 and 8 μM, respectively. 98% TCS was removed with only 0.1 U mL −1 SBP in 30 min reaction time, while an HRP dose of 0.3 U mL −1 was required to achieve the similar conversion. The catalytic performance of SBP towards TCS was more efficient than that of HRP, which can be explained by catalytic rate constant (K CAT ) and catalytic efficiency (K CAT /K M ) for the two enzymes. MS analysis in combination with quantum chemistry computation showed that the polymerization products were generated via C−C and C−O coupling pathways. The polymers were proved to be nontoxic through growth inhibition of green alga (Scenedesmus obliquus). Taking into consideration of the enzymatic treatment cost, SBP may be a better alternative to HRP upon the removal and detoxification of TCS in water/wastewater treatment.

  1. Apoplastic peroxidases are required for salicylic acid-mediated defense against Pseudomonas syringae.

    Science.gov (United States)

    Mammarella, Nicole D; Cheng, Zhenyu; Fu, Zheng Qing; Daudi, Arsalan; Bolwell, G Paul; Dong, Xinnian; Ausubel, Frederick M

    2015-04-01

    Reactive oxygen species (ROS) generated by NADPH oxidases or apoplastic peroxidases play an important role in the plant defense response. Diminished expression of at least two Arabidopsis thaliana peroxidase encoding genes, PRX33 (At3g49110) and PRX34 (At3g49120), as a consequence of anti-sense expression of a heterologous French bean peroxidase gene (asFBP1.1), were previously shown to result in reduced levels of ROS following pathogen attack, enhanced susceptibility to a variety of bacterial and fungal pathogens, and reduced levels of callose production and defense-related gene expression in response to the microbe associated molecular pattern (MAMP) molecules flg22 and elf26. These data demonstrated that the peroxidase-dependent oxidative burst plays an important role in the elicitation of pattern-triggered immunity (PTI). Further work reported in this paper, however, shows that asFBP1.1 antisense plants are not impaired in all PTI-associated responses. For example, some but not all flg22-elicited genes are induced to lower levels by flg22 in asFPB1.1, and callose deposition in asFPB1.1 is similar to wild-type following infiltration with a Pseudomonas syringae hrcC mutant or with non-host P. syringae pathovars. Moreover, asFPB1.1 plants did not exhibit any apparent defect in their ability to mount a hypersensitive response (HR). On the other hand, salicylic acid (SA)-mediated activation of PR1 was dramatically impaired in asFPB1.1 plants. In addition, P. syringae-elicited expression of many genes known to be SA-dependent was significantly reduced in asFBP1.1 plants. Consistent with this latter result, in asFBP1.1 plants the key regulator of SA-mediated responses, NPR1, showed both dramatically decreased total protein abundance and a failure to monomerize, which is required for its translocation into the nucleus. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Effects of pH and Temperature on Recombinant Manganese Peroxidase Production and Stability

    Science.gov (United States)

    Jiang, Fei; Kongsaeree, Puapong; Schilke, Karl; Lajoie, Curtis; Kelly, Christine

    The enzyme manganese peroxidase (MnP) is produced by numerous white-rot fungi to overcome biomass recalcitrance caused by lignin. MnP acts directly on lignin and increases access of the woody structure to synergistic wood-degrading enzymes such as cellulases and xylanases. Recombinant MnP (rMnP) can be produced in the yeast Pichia pastoris αMnP1-1 in fed-batch fermentations. The effects of pH and temperature on recombinant manganese peroxidase (rMnP) production by P. pastoris αMnP1-1 were investigated in shake flask and fed-batch fermentations. The optimum pH and temperature for a standardized fed-batch fermentation process for rMnP production in P. pastoris ctMnP1-1 were determined to be pH 6 and 30 °C, respectively. P. pastoris αMnP1-1 constitutively expresses the manganese peroxidase (mnp1) complementary DNA from Phanerochaete chrysosporium, and the rMnP has similar kinetic characteristics and pH activity and stability ranges as the wild-type MnP (wtMnP). Cultivation of P. chrysosporium mycelia in stationary flasks for production of heme peroxidases is commonly conducted at low pH (pH 4.2). However, shake flask and fed-batch fermentation experiments with P. pastoris αMnP1-1 demonstrated that rMnP production is highest at pH 6, with rMnP concentrations in the medium declining rapidly at pH less than 5.5, although cell growth rates were similar from pH 4-7. Investigations of the cause of low rMnP production at low pH were consistent with the hypothesis that intracellular proteases are released from dead and lysed yeast cells during the fermentation that are active against rMnP at pH less than 5.5.

  3. Screening of Coprinus species for the production of extracellular peroxidase and evaluation of its applicability to the treatment of aqueous phenol

    International Nuclear Information System (INIS)

    Ikehata, K.; Buchanan, I.D.

    2002-01-01

    Twenty-nine strains of Coprinus species comprising 16 strains from 12 identified species and 13 unidentified strains as well as one Arthromyces ramosus strain were screened for the production of extracellular peroxidase. Among the fungi examined, three strains of C. cinereus, UAMH 4103, UAMH 7907 and IFO 30116, as well as one Coprinus sp., UAMH 10067, which was isolated from urea treated soil, were shown to produce large amounts of extracellular peroxidase. The performance of crude peroxidase, obtained from liquid culture of C. cinereus, (CIP) on phenol removal from synthetic wastewater was evaluated and compared with that of purified horseradish peroxidase and A. ramosus peroxidase. Although crude CIP performed better than both purified enzymes, its superiority vanished in the presence of poly(ethylene glycol), a known protective agent of peroxidase. This suggests that the residual soluble substances present in crude CIP have protective effects similar to those of poly(ethylene glycol). (author)

  4. Peroxidase activity in scutella of maize in association with anatomical changes during germination and grain storage.

    Science.gov (United States)

    Corona-Carrillo, José Isaac; Flores-Ponce, Mitzi; Chávez-Nájera, Gerardo; Díaz-Pontones, David Manuel

    2014-01-01

    The embryo of the maize grain (Zea mays L.) is separated from the starchy endosperm by a fibrous structure, which is called the fibrous layer (FL). Using histochemical staining, it was determined that the FL is composed of collapsed cellular layers that contain phenols, neutral lipids, and 1,3-β-glucan. Due to its composition, the FL prevents free diffusion and separates the embryo from the endosperm during germination. Twenty-four hours after imbibition, the scutellum epidermis initiated a series of asynchronous spatial modifications, including cell growth, the perforation of cell walls, increased peroxidase activity in the apoplastic space, and elevated levels of superoxide, phenols, and other components that interact with the fibrous layer, enabling its transformation in addition to the free flow between compartments. During storage at high relative humidity levels, which leads to fast or slow deterioration depending on the temperature, the activity of phenol peroxidase in the scutellum was associated with a loss of vigor and reduced germination capacity when compared with low temperature and low relative humidity conditions. Such deterioration is associated with alterations in autofluorescent emissions from endogenous compounds in the scutellum, indicating changes in the microenvironment or in the differential proportions of epidermal and FL components.

  5. Expression and Function of Cell Wall-Bound Cationic Peroxidase in Asparagus Somatic Embryogenesis

    Science.gov (United States)

    Takeda, Hiroyuki; Kotake, Toshihisa; Nakagawa, Naoki; Sakurai, Naoki; Nevins, Donald J.

    2003-01-01

    Cultured asparagus (Asparagus officinalis L. cv Y6) cells induced to regenerate into whole plants through somatic embryogenesis secreted a 38-kD protein into cell walls. The full-length cDNA sequence of this protein (Asparagus officinalis peroxidase 1 [AoPOX1]) determined by reverse transcriptase-polymerase chain reaction showed similarity with plant peroxidases. AoPOX1 transcripts were particularly abundant during early somatic embryogenesis. To evaluate the in vivo function of AoPOX1 protein, purified recombinant AoPOX1 protein was reacted with a series of phenolic substrates. The AoPOX1 protein was effective in the metabolism of feruloyl (o-methoxyphenol)-substituted substrates, including coniferyl alcohol. The reaction product of coniferyl alcohol was fractionated and subjected to gas chromatography-mass spectrometry analysis and 1H-nuclear magnetic resonance analysis, indicating that the oxidation product of coniferyl alcohol in the presence of AoPOX1 was dehydrodiconiferyl alcohol. The concentration of dehydrodiconiferyl alcohol in the cultured medium of the somatic embryos was in the range of 10−8 m. Functions of the AoPOX1 protein in the cell differentiation are discussed. PMID:12692335

  6. Electrospun Chitosan-Gelatin Biopolymer Composite Nanofibers for Horseradish Peroxidase Immobilization in a Hydrogen Peroxide Biosensor

    Directory of Open Access Journals (Sweden)

    Siriwan Teepoo

    2017-10-01

    Full Text Available A biosensor based on chitosan-gelatin composite biopolymers nanofibers is found to be effective for the immobilization of horseradish peroxidase to detect hydrogen peroxide. The biopolymer nanofibers were fabricated by an electrospining technique. Upon optimization of synthesis parameters, biopolymers nanofibers, an average of 80 nm in diameter, were obtained and were then modified on the working electrode surface. The effects of the concentration of enzyme, pH, and concentration of the buffer and the working potential on the current response of the nanofibers-modified electrode toward hydrogen peroxide were optimized to obtain the maximal current response. The results found that horseradish peroxidase immobilization on chitosan-gelatin composite biopolymer nanofibers had advantages of fast response, excellent reproducibility, high stability, and showed a linear response to hydrogen peroxide in the concentration range from 0.1 to 1.7 mM with a detection limit of 0.05 mM and exhibited high sensitivity of 44 µA∙mM−1∙cm−2. The developed system was evaluated for analysis of disinfectant samples and showed good agreement between the results obtained by the titration method without significant differences at the 0.05 significance level. The proposed strategy based on chitosan-gelatin composite biopolymer nanofibers for the immobilization of enzymes can be extended for the development of other enzyme-based biosensors.

  7. Enzymatic decolourisation of Methyl Orange and Bismarck Brown using crude peroxidase from Armoracia rusticana

    Science.gov (United States)

    Ambatkar, Mugdha; Mukundan, Usha

    2015-12-01

    The decolourisation of Methyl Orange (MO) and Bismarck Brown (BB) by crude peroxidase from Armoracia rusticana (Horseradish) was studied by varying different reaction parameters. The pH of the reaction mixture, initial dye concentration, amount of enzyme and hydrogen peroxide concentration were optimised for ambient temperatures (30 ± 2 °C). The optimum pH for decolourisation was 4.0 (72.95 %) and 3.0 (79.24 %) for MO and BB, respectively. Also it was found that the Chemical Oxygen Demand of the enzyme-treated sample was significantly lower than that of the untreated controls for both dyes. The addition of a complex iron salt like Ferric EDTA was found to enhance the decolourisation of both dyes at pH 6.0, showing an increase of 8.69 % and 14.17 % in the decolourisation of MO and of BB, respectively. The present study explores the potential of crude peroxidase from horseradish to decolourise representative monoazo and diazo dyes, MO and BB, respectively. An attempt has been made to utilise a crude enzyme with appreciable activity obtained after minimal processing for the decolourisation of the aforesaid dyes. The findings of this study would find application in the enzymatic treatment of wastewater containing azo dyes.

  8. Online Detection of Peroxidase Using 3D Printing, Active Magnetic Mixing, and Spectra Analysis

    Science.gov (United States)

    2017-01-01

    A new method for online detection of peroxidase (POD) using 3D printing, active magnetic mixing, fluidic control, and optical detection was developed and demonstrated in this study. The proposed POD detection system consisted of a 3D printing and active magnetic mixing based fluidic chip for online catalytic reaction, an optical detector with a fluidic flow cell for quantitative determination of the final catalysate, and a single-chip microcontroller based controller for automatic control of two rotating magnetic fields and four precise peristaltic pumps. Horseradish peroxidase (HRP) was used as research model and a linear relationship between the absorbance at the characteristic wavelength of 450 nm and the concentration of HRP of 1/4–1/128 μg mL−1 was obtained as A  =  0.257ln⁡(C) + 1.425 (R2  = 0.976). For the HRP spiked pork tests, the recoveries of HRP ranged from 93.5% to 110.4%, indicating that this proposed system was capable of detecting HRP in real samples. It has the potential to be extended for online detection of the activity of other enzymes and integration with ELISA method for biological and chemical analysis. PMID:28523275

  9. Soybean peroxidase-mediated degradation of an azo dye- a detailed mechanistic study.

    Science.gov (United States)

    Ali, Liaquat; Algaithi, Rowdha; Habib, Hosam M; Souka, Usama; Rauf, Muhammad A; Ashraf, S Salman

    2013-12-05

    Peroxidases are emerging as an important class of enzymes that can be used for the efficient degradation of organic pollutants. However, detailed studies identifying the various intermediates produced and the mechanisms involved in the enzyme-mediated pollutant degradation are not widely published. In the present study, the enzymatic degradation of an azo dye (Crystal Ponceau 6R, CP6R) was studied using commercially available soybean peroxidase (SBP) enzyme. Several operational parameters affecting the enzymatic degradation of dye were evaluated and optimized, such as initial dye concentration, H2O2 dosage, mediator amount and pH of the solution. Under optimized conditions, 40 ppm dye solution could be completely degraded in under one minute by SBP in the presence of H2O2 and a redox mediator. Dye degradation was also confirmed using HPLC and TOC analyses, which showed that most of the dye was being mineralized to CO2 in the process. Detailed analysis of metabolites, based on LC/MS results, showed that the enzyme-based degradation of the CP6R dye proceeded in two different reaction pathways- via symmetric azo bond cleavage as well as asymmetric azo bond breakage in the dye molecule. In addition, various critical transformative and oxidative steps such as deamination, desulfonation, keto-oxidation are explained on an electronic level. Furthermore, LC/MS/MS analyses confirmed that the end products in both pathways were small chain aliphatic carboxylic acids.

  10. Soybean peroxidase-mediated degradation of an azo dye– a detailed mechanistic study

    Science.gov (United States)

    2013-01-01

    Background Peroxidases are emerging as an important class of enzymes that can be used for the efficient degradation of organic pollutants. However, detailed studies identifying the various intermediates produced and the mechanisms involved in the enzyme-mediated pollutant degradation are not widely published. Results In the present study, the enzymatic degradation of an azo dye (Crystal Ponceau 6R, CP6R) was studied using commercially available soybean peroxidase (SBP) enzyme. Several operational parameters affecting the enzymatic degradation of dye were evaluated and optimized, such as initial dye concentration, H2O2 dosage, mediator amount and pH of the solution. Under optimized conditions, 40 ppm dye solution could be completely degraded in under one minute by SBP in the presence of H2O2 and a redox mediator. Dye degradation was also confirmed using HPLC and TOC analyses, which showed that most of the dye was being mineralized to CO2 in the process. Conclusions Detailed analysis of metabolites, based on LC/MS results, showed that the enzyme-based degradation of the CP6R dye proceeded in two different reaction pathways- via symmetric azo bond cleavage as well as asymmetric azo bond breakage in the dye molecule. In addition, various critical transformative and oxidative steps such as deamination, desulfonation, keto-oxidation are explained on an electronic level. Furthermore, LC/MS/MS analyses confirmed that the end products in both pathways were small chain aliphatic carboxylic acids. PMID:24308857

  11. Manganese peroxidases from Ganoderma applanatum degrade β-carotene under alkaline conditions.

    Science.gov (United States)

    Lanfermann, Isabel; Linke, Diana; Nimtz, Manfred; Berger, Ralf G

    2015-04-01

    A β-carotene-degrading enzyme activity was observed in liquid cultures of the basidiomycete Ganoderma applanatum. Supplementing the cultures with β-carotene induced the bleaching activity. Purification via hydrophobic interaction, ion exchange and size exclusion chromatography followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) resulted in a single protein band. LC-ion-trap-MS analyses and gene amplification identified two manganese peroxidase isoenzymes with 97.8 % identity on the amino acid level. These showed an estimated molecular mass of 48 kDa and an isoelectric point of 2.6. Properties not yet described for other manganese peroxidases were hydrogen-peroxide-independent catalysis and two maxima of the bleaching activity, a distinct one at pH 5 and a lower one at pH 8. During simulated washing studies, the applicability of the isoenzymes for the brightening of carotenoids under alkaline conditions was proven. The new enzymes may replace common bleaching agents to produce environmentally more compatible detergent formulations.

  12. Colorimetric detection of glucose based on ficin with peroxidase-like activity

    Science.gov (United States)

    Pang, Yanjiao; Huang, Zili; Yang, Yufang; Long, Yijuan; Zheng, Huzhi

    2018-01-01

    In this work, we developed a colorimetric biosensing system for glucose detection by coupling the peroxidase-like of ficin and the glucose oxidase (GOx). GOx can catalyze the oxidation of glucose to produce H2O2, then, ficin catalyzes the oxidation of peroxidase substrate 3,3‧,5,5‧-tetramethylbenzidine (TMB) by H2O2 to produce a blue color reaction. The present sensing system showed a linear response toward glucose detection over range of 2.0-100 μM with a detection limit of 0.5 μM. This system is simple, low cost, highly sensitive and selective for glucose detection, and was also applied to measuring glucose in human serum. Furthermore, in order to expand the application of ficin in biological sensing, we immobilized ficin onto the SiO2@Fe3O4 NPs, which exhibited the merits of recycling as well as allowing the repeated detection of glucose. Thus it may provide great potential applications in biomedicine, biotechnology and environmental chemistry.

  13. Tumor suppressor function of the plasma glutathione peroxidase Gpx3 in colitis-associated carcinoma

    Science.gov (United States)

    Barrett, Caitlyn W.; Ning, Wei; Chen, Xi; Smith, J. Joshua; Washington, Mary K; Hill, Kristina E.; Coburn, Lori A.; Peek, Richard M.; Chaturvedi, Rupesh; Wilson, Keith T.; Burk, Raymond F.; Williams, Christopher S.

    2012-01-01

    The glutathione peroxidases, a family of selenocysteine-containing redox enzymes, play pivotal roles in balancing the signaling, immunomodulatory and deleterious effects of reactive oxygen species (ROS). The glutathione peroxidase GPX3 is the only extracellular member of this family, suggesting it may defend cells against ROS in the extracellular environment. Notably, GPX3 hypermethylation and underexpression occurs commonly in prostate, gastric, cervical, thyroid and colon cancers. We took a reverse genetics approach to investigate whether GPX3 would augment inflammatory colonic tumorigenesis, a process characterized by oxidative stress and inflammation, comparing Gpx3−/− mice established two-stage model of inflammatory colon carcinogenesis. Gpx3-deficient mice exhibited an increased tumor number, though not size, along with a higher degree of dysplasia. Additionally, they exhibited increased inflammation with redistribution towards pro-tumorigenic M2 macrophage subsets, increased proliferation, hyperactive WNT signaling, and increased DNA damage. To determine the impact of acute gene loss in an established colon cancer line, we silenced GPX3 in human Caco2 cells, resulting in increased ROS production, DNA damage and apoptosis in response to oxidative stress, combined with decreased contact-independent growth. Taken together, our results suggested an immunomodulatory role for GPX3 that limits the development of colitis-associated carcinoma. PMID:23221387

  14. A manganese catalase from Thermomicrobium roseum with peroxidase and catecholase activity.

    Science.gov (United States)

    Baginski, Robin; Sommerhalter, Monika

    2017-01-01

    An enzyme with catechol oxidase activity was identified in Thermomicrobium roseum extracts via solution assays and activity-stained SDS-PAGE. Yet, the genome of T. roseum does not harbor a catecholase gene. The enzyme was purified with two anion exchange chromatography steps and ultimately identified to be a manganese catalase with additional peroxidase and catecholase activity. Catalase activity (6280 ± 430 IU/mg) clearly dominated over pyrogallol peroxidase (231 ± 53 IU/mg) and catecholase (3.07 ± 0.56 IU/mg) activity as determined at 70 °C. Most enzyme kinetic properties were comparable to previously characterized manganese catalase enzymes. Catalase activity was highest at alkaline pH values and showed inhibition by excess substrate and chloride. The apparent K m and k cat values were 20 mM and 2.02 × 10 4  s -1 subunit -1 at 25 °C and pH 7.0.

  15. Photophysics and photochemistry of horseradish peroxidase A2 upon ultraviolet illumination.

    Science.gov (United States)

    Neves-Petersen, Maria Teresa; Klitgaard, Søren; Carvalho, Ana Sofia Leitão; Petersen, Steffen B; Aires de Barros, Maria Raquel; Pinho e Melo, Eduardo

    2007-03-15

    Detailed analysis of the effects of ultraviolet (UV) and blue light illumination of horseradish peroxidase A2, a heme-containing enzyme that reduces H(2)O(2) to oxidize organic and inorganic compounds, is presented. The effects of increasing illumination time on the protein's enzymatic activity, Reinheitzahl value, fluorescence emission, fluorescence lifetime distribution, fluorescence mean lifetime, and heme absorption are reported. UV illumination leads to an exponential decay of the enzyme activity followed by changes in heme group absorption. Longer UV illumination time leads to lower T(m) values as well as helical content loss. Prolonged UV illumination and heme irradiation at 403 nm has a pronounced effect on the fluorescence quantum yield correlated with changes in the prosthetic group pocket, leading to a pronounced decrease in the heme's Soret absorbance band. Analysis of the picosecond-resolved fluorescence emission of horseradish peroxidase A2 with streak camera shows that UV illumination induces an exponential change in the preexponential factors distribution associated to the protein's fluorescence lifetimes, leading to an exponential increase of the mean fluorescence lifetime. Illumination of aromatic residues and of the heme group leads to changes indicative of heme leaving the molecule and/or that photoinduced chemical changes occur in the heme moiety. Our studies bring new insight into light-induced reactions in proteins. We show how streak camera technology can be of outstanding value to follow such ultrafast processes and how streak camera data can be correlated with protein structural changes.

  16. Ultra-small particles of iron oxide as peroxidase for immunohistochemical detection

    International Nuclear Information System (INIS)

    Wu Yihang; Song Mengjie; Zhang Xiaoqing; Zhang Yu; Wang Chunyu; Gu Ning; Xin Zhuang; Li Suyi

    2011-01-01

    Dimercaptosuccinic acid (DMSA) modified ultra-small particles of iron oxide (USPIO) were synthesized through a two-step process. The first step: oleic acid (OA) capped Fe 3 O 4 (OA-USPIO) were synthesized by a novel oxidation coprecipitation method in H 2 O/DMSO mixing system, where DMSO acts as an oxidant simultaneously. The second step: OA was replaced by DMSA to obtain water-soluble nanoparticles. The as-synthesized nanoparticles were characterized by TEM, FTIR, TGA, VSM, DLS, EDS and UV-vis. Hydrodynamic sizes and Peroxidase-like catalytic activity of the nanoparticles were investigated. The hydrodynamic sizes of the nanoparticles (around 24.4 nm) were well suited to developing stable nanoprobes for bio-detection. The kinetic studies were performed to quantitatively evaluate the catalytic ability of the peroxidase-like nanoparticles. The calculated kinetic parameters indicated that the DMSA-USPIO possesses high catalytic activity. Based on the high activity, immunohistochemical experiments were established: using low-cost nanoparticles as the enzyme instead of expensive HRP, Nimotuzumab was conjugated onto the surface of the nanoparticles to construct a kind of ultra-small nanoprobe which was employed to detect epidermal growth factor receptor (EGFR) over-expressed on the membrane of esophageal cancer cell. The proper sizes of the probes and the result of membranous immunohistochemical staining suggest that the probes can be served as a useful diagnostic reagent for bio-detection.

  17. Enhanced ultrastructural visualization of the horseradish peroxidase-tetramethylbenzidine reaction product.

    Science.gov (United States)

    Henry, M A; Westrum, L E; Johnson, L R

    1985-12-01

    Ultrastructural visualization of the horseradish peroxidase-tetramethylbenzidine (HRP-TMB) reaction product within trigeminal ganglion cells and brain stem axons and terminals following HRP injections into the pulpal chambers of cat teeth is enhanced by utilization of a modified osmication procedure that converts the reaction product to a markedly stable and electron-dense form. The results following the use of the modified osmication procedure (pH 5.0 phosphate buffer at 20 degrees C for 12 hours) are compared to results obtained by following Carson's osmication protocol (Carson KA, Mesulam M-M: J Histochem Cytochem 30:425, 1982; Carson KA, Mesulam M-M: In Tracing Neural Connections with Horseradish Peroxidase. Edited by M-M Mesulam. J Wiley, Chichester, England, 1982, p 153-184) (pH 6.0 phosphate buffer at 45 degrees C for 45 min). The results suggest that the conversion of the HRP-TMB reaction product to an electron-dense form during osmication is intimately associated with the pH of the phosphate buffer and the total time of osmication.

  18. Detection of oligoclonal immunoglobulins in cerebrospinal fluid by an immunofixation-peroxidase method.

    Science.gov (United States)

    Richard, Sophie; Miossec, Véronique; Moreau, Jean-François; Taupin, Jean-Luc

    2002-01-01

    The detection of intrathecal synthesis of immunoglobulins is used in the diagnosis of multiple sclerosis (MS). We tested the semiautomated immunofixation-peroxidase (IFPOD) technique, which uses high-resolution agarose gel electrophoresis (HRAGE) directly followed by immunofixation with a peroxidase-labeled anti-IgG antiserum to detect oligoclonal immunoglobulins in cerebrospinal fluid (CSF). We analyzed 230 consecutive matched serum/CSF pairs that arrived in the laboratory over a 6-month period with both IFPOD and our routine techniques, immunofixation electrophoresis (IFE) and HRAGE. For IFPOD, CSF samples were not concentrated before testing. Among the 230 samples were 12 clinically definite MS, 33 clinically probable, and 20 clinically possible MS samples. IFPOD and HRAGE + IFE each detected oligoclonal IgG in CSF in 10, 16, and 7 cases of these respective groups. For clinically definite MS, sensitivity and specificity (95% confidence intervals) were, respectively, 83% (51-97%) and 79% (73-84%). The IFPOD technique performs comparably to other analytical methods, without the requirement for sample concentration, and may represent an attractive alternative in testing for intrathecal immunoglobulin synthesis.

  19. Thyroid peroxidase (TPO) expressed in thyroid and breast tissues shows similar antigenic properties.

    Science.gov (United States)

    Godlewska, Marlena; Arczewska, Katarzyna D; Rudzińska, Magdalena; Łyczkowska, Anna; Krasuska, Wanda; Hanusek, Karolina; Ruf, Jean; Kiedrowski, Mirosław; Czarnocka, Barbara

    2017-01-01

    Thyroid peroxidase (TPO) is essential for physiological function of the thyroid gland. The high prevalence of thyroid peroxidase antibodies (TPOAbs) in patients with breast cancer and their protective role had previously been demonstrated, indicating a link between breast cancer and thyroid autoimmunity. Recently, TPO was shown to be present in breast cancer tissue samples but its antigenicity has not been analyzed. In this study, we investigated TPO expression levels in a series of fifty-six breast cancer samples paired with normal (peri-tumoral) tissue and its antigenic activity using a panel of well-characterized murine anti-human TPOAbs. We have shown that TPO transcripts were present in both normal and cancer tissue samples, although the amounts in the latter were reduced. Additionally, we observed that TPO levels are lower in more advanced cancers. TPO protein expression was confirmed in all tissue samples, both normal and cancerous. We also found that the antigenicity of the immunodominant regions (IDRs) in breast TPO resembles that of thyroid TPO, which is crucial for effective interactions with human TPOAbs. Expression of TPO in breast cancer together with its antigenic activity may have beneficial effects in TPOAb-positive breast cancer patients. However, further studies are needed to confirm the beneficial role of TPOAbs and to better understand the underlying mechanism.

  20. Lignin Peroxidase from Streptomyces viridosporus T7A: Enzyme Concentration Using Ultrafiltration

    Science.gov (United States)

    Gottschalk, Leda M. F.; Bon, Elba P. S.; Nobrega, Ronaldo

    It is well known that lignin degradation is a key step in the natural process of biomass decay whereby oxidative enzymes such as laccases and high redox potential ligninolytic peroxidases and oxidases play a central role. More recently, the importance of these enzymes has increased because of their prospective industrial use for the degradation of the biomass lignin to increase the accessibility of the cellulose and hemicellulose moieties to be used as renewable material for the production of fuels and chemicals. These biocatalysts also present potential application on environmental biocatalysis for the degradation of xenobiotics and recalcitrant pollutants. However, the cost for these enzymes production, separation, and concentration must be low to permit its industrial use. This work studied the concentration of lignin peroxidase (LiP), produced by Streptomyces viridosporus T7A, by ultrafiltration, in a laboratory-stirred cell, loaded with polysulfone (PS) or cellulose acetate (CA) membranes with molecular weight cutoffs (MWCO) of 10, 20, and 50 KDa. Experiments were carried out at 25 °C and pH 7.0 in accordance to the enzyme stability profile. The best process conditions and enzyme yield were obtained using a PS membrane with 10 KDa MWCO, whereby it was observed a tenfold LiP activity increase, reaching 1,000 U/L and 90% enzyme activity upholding.

  1. Production of phenol-oxidases and peroxidases by fungi isolated from irrigated rice Produção de fenol-oxidases e peroxidases por fungos isolados da cultura de arroz irrigado

    Directory of Open Access Journals (Sweden)

    Célia Maria Maganhotto de Souza Silva

    2003-11-01

    Full Text Available The aim of this work was to study the potential of fungus strains considered as prospective degraders for the herbicides quinclorac and propanil, for ligninolytic enzyme production. Eight fungal strains were grown in King's B liquid culture medium supplemented with 0.05% Remazol Brilliant Blue R (RBBR and in liquid culture medium containing wheat bran as substrate. The enzymatic system assessment were: lignin peroxidase, manganese peroxidase and laccases. Results indicated differential patterns of ligninolytic enzyme production; the highest enzymatic activities were related to the production of lignin peroxidase. Among the strains, two (P3SA1F and P11SA2F showed RBBR discoloration, suggesting the possibility of their application in bioremediation studies.A proposta deste trabalho foi estudar o potencial das linhagens fúngicas, consideradas potenciais degradadoras dos herbicidas quinclorac e propanil, para produção de enzimas ligninolíticas. Oito linhagens fúngicas foram cultivadas em meio de cultura líquido King's B suplementado com 0,05% de Remazol Brilliant Blue R (RBBR e em meio de cultura líquido contendo farelo de trigo como substrato. Os sistemas enzimáticos avaliados foram: lignina peroxidase, manganês peroxidase e lacases. Os resultados demonstraram padrões diferenciados quanto à produção de enzimas ligninolíticas entre as linhagens, sendo que as maiores atividades enzimáticas estiveram relacionadas à produção de lignina peroxidase. Das oito linhagens, duas (P3SA1F e P11SA2F apresentaram descoloração do RBBR, sugerindo a possibilidade de sua aplicabilicação em estudos de biorremediação

  2. Differential Regulation of mnp2, a New Manganese Peroxidase-Encoding Gene from the Ligninolytic Fungus Trametes versicolor PRL 572

    Science.gov (United States)

    Johansson, Tomas; Nyman, Per Olof; Cullen, Daniel

    2002-01-01

    A peroxidase-encoding gene, mnp2, and its corresponding cDNA were characterized from the white-rot basidiomycete Trametes versicolor PRL 572. We used quantitative reverse transcriptase-mediated PCR to identify mnp2 transcripts in nutrient-limited stationary cultures. Although mnp2 lacks upstream metal response elements (MREs), addition of MnSO4 to cultures increased mnp2 transcript levels 250-fold. In contrast, transcript levels of an MRE-containing gene of T. versicolor, mnp1, increased only eightfold under the same conditions. Thus, the manganese peroxidase genes in T. versicolor are differentially regulated, and upstream MREs are not necessarily involved. Our results support the hypothesis that fungal and plant peroxidases arose through an ancient duplication and folding of two structural domains, since we found the mnp1 and mnp2 polypeptides to have internal homology. PMID:11916737

  3. Comparison of plasma malondialdehyde, glutathione, glutathione peroxidase, hydroxyproline and selenium levels in patients with vitiligo and healthy controls

    Directory of Open Access Journals (Sweden)

    Ozturk I

    2008-01-01

    Full Text Available Background: The etiology and pathophysiologic mechanism of vitiligo are still unclear. The relationship between increased oxidative stress due to the accumulation of radicals and reactive oxygen species and the associated changes in blood and epidermal component of vitiliginous skin have been reported many times. We investigated the possible changes of plasma malondialdehyde, glutathione, selenium, hydroxyproline and glutathione peroxidase activity levels in patients with vitiligo in order to evaluate the relationship between oxidative stress and etiopathogenesis of vitiligo. Materials and Methods: Plasma malondialdehyde, glutathione, hydroxyproline and glutathione peroxidase activity levels were measured by spectrophotometric methods, and HPLC was used for measurement of selenium concentrations. Results: Our results showed increased malondialdehyde, hydroxyproline and glutathione peroxidase activity levels in plasma of vitiligo group ( P < 0.05. Conclusion: Support of antioxidant system via nonenzymatic antioxidant compounds and antioxidant enzymes may be useful to prevent of melanocyte degeneration which occur due to oxidative damage in vitiligo.

  4. Modulation in radiation-induced changes in peroxidase activity with gibberellic acid in seedling's growth in chickpea (Cicer arietinum L.)

    International Nuclear Information System (INIS)

    Khan, M.R.; Qureshi, A.S.

    2002-01-01

    Changes in the effects of gamma irradiation (10 to 110 Kr) with gibberellic acid (GA/sub 3/) for peroxidase activity, in relation to early days of seedling's growth in Kabulic chickpea cultivar, Noor-91, were evaluated. Stimulation in peroxidase activity over control was recorded at all the irradiation treatments from 3rd to 8th day of seedling's development. Increase in peroxidase activity at 10 and 20 Kr was due to the increase in metabolic activity, while higher doses of gamma radiation account for the damaging action and production of peroxy radicals. However, stimulation in fresh weight was observed only at 10 Kr of gamma irradiation. Postmutagenic application of Ga/sub 3/ protect the seedlings from radiation injury, by increasing the peroxides activity, and increased the fresh weight of chickpea seedlings. (author)

  5. Activity and multiple forms of peroxidase in Zea mays and Medicago sativa treated and non-treated with lead

    Energy Technology Data Exchange (ETDEWEB)

    Maier, R.

    1978-02-11

    The influence of lead on peroxidase was studied in young plants of Zee mays and Medicago sativa grown in solutions of Pb(NO/sub 3/)/sub 2/. The content of nitrate in the control- and the lead-solutions was equalized by NaNO/sub 3/. In the patterns of the multiple forms in roots and leaves of Zea and Medicago qualitatively no, or respectively negligible, change between control and lead-treated plants takes place. The temperature during the lead-treatment does not effect the numbers and the positions of bands. The bands' activity of peroxidase in lead-treated plants differs clearly from untreated plants, resulting in a different activity of the whole enzyme. In the roots and in the leaves of Zea as well as in the leaves of Medicago the relative activity of the peroxidase in lead-treated plants is raised. In the roots of Medicago the relative activity decreased slightly.

  6. Biosynthesis of N,N-dimethyltryptamine (DMT) in a melanoma cell line and its metabolization by peroxidases.

    Science.gov (United States)

    Gomes, Melissa M; Coimbra, Janine B; Clara, Renan O; Dörr, Felipe A; Moreno, Ana Carolina R; Chagas, Jair R; Tufik, Sérgio; Pinto, Ernani; Catalani, Luiz H; Campa, Ana

    2014-04-01

    Tryptophan (TRP) is essential for many physiological processes, and its metabolism changes in some diseases such as infection and cancer. The most studied aspects of TRP metabolism are the kynurenine and serotonin pathways. A minor metabolic route, tryptamine and N,N-dimethyltryptamine (DMT) biosynthesis, has received far less attention, probably because of the very low amounts of these compounds detected only in some tissues, which has led them to be collectively considered as trace amines. In a previous study, we showed a metabolic interrelationship for TRP in melanoma cell lines. Here, we identified DMT and N,N-dimethyl-N-formyl-kynuramine (DMFK) in the supernatant of cultured SK-Mel-147 cells. Furthermore, when we added DMT to the cell culture, we found hydroxy-DMT (OH-DMT) and indole acetic acid (IAA) in the cell supernatant at 24 h. We found that SK-Mel-147 cells expressed mRNA for myeloperoxidase (MPO) and also had peroxidase activity. We further found that DMT oxidation was catalyzed by peroxidases. DMT oxidation by horseradish peroxidase, H2O2 and MPO from PMA-activated neutrophils produced DMFK, N,N-dimethyl-kynuramine (DMK) and OH-DMT. Oxidation of DMT by peroxidases apparently uses the common peroxidase cycle involving the native enzyme, compound I and compound II. In conclusion, this study describes a possible alternative metabolic pathway for DMT involving peroxidases that has not previously been described in humans and identifies DMT and metabolites in a melanoma cell line. The extension of these findings to other cell types and the biological effects of DMT and its metabolites on cell proliferation and function are key questions for future studies. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Fe-TAML encapsulated inside mesoporous silica nanoparticles as peroxidase mimic: femtomolar protein detection.

    Science.gov (United States)

    Kumari, Sushma; Dhar, Basab B; Panda, Chakadola; Meena, Abhishek; Sen Gupta, Sayam

    2014-08-27

    Peroxidase, such as horseradish peroxidase (HRP), conjugated to antibodies are routinely used for the detection of proteins via an ELISA type assay in which a critical step is the catalytic signal amplification by the enzyme to generate a detectable signal. Synthesis of functional mimics of peroxidase enzyme that display catalytic activity which far exceeds the native enzyme is extremely important for the precise and accurate determination of very low quantities of proteins (fM and lower) that is necessary for early clinical diagnosis. Despite great advancements, analyzing proteins of very low abundance colorimetrically, a method that is most sought after since it requires no equipment for the analysis, still faces great challenges. Most reported HRP mimics that show catalytic activity greater than native enzyme (∼10-fold) are based on metal/metal-oxide nanoparticles such as Fe3O4. In this paper, we describe a second generation hybrid material developed by us in which approximately 25,000 alkyne tagged biuret modified Fe-tetraamido macrocyclic ligand (Fe-TAML), a very powerful small molecule synthetic HRP mimic, was covalently attached inside a 40 nm mesoporous silica nanoparticle (MSN). Biuret-modified Fe-TAMLs represent one of the best small molecule functional mimics of the enzyme HRP with reaction rates in water close to the native enzyme and operational stability (pH, ionic strength) far exceeding the natural enzyme. The catalytic activity of this hybrid material is around 1000-fold higher than that of natural HRP and 100-fold higher than that of most metal/metal oxide nanoparticle based HRP mimics reported to date. We also show that using antibody conjugates of this hybrid material it is possible to detect and, most importantly, quantify femtomolar quantities of proteins colorimetrically in an ELISA type assay. This represents at least 10-fold higher sensitivity than other colorimetric protein assays that have been reported using metal/metal oxide

  8. Inactivation kinetics of peroxidase from coconut waterCinética de inativação da peroxidase em água de coco

    Directory of Open Access Journals (Sweden)

    Gabrielle Cardoso Reis Fontan

    2012-04-01

    Full Text Available The aim of this project was study the thermal inactivation kinetics of the peroxidase enzyme (POD present in the green coconut water, being determined the parameters D values, z value and Q10, besides the Arrhenius’ activation energy (Ea to the process. Nine time/temperature binomials for the thermal treatment at three temperatures (75°C, 85°C and 95°C and maintenance times from 1min to 30 min, besides a control sample without thermal treatment. D values were obtained by linear regression, being equals to 833,3 min, 35,6min and 1,63min at 75°C, 85°C and 95°C, respectively, while the z value was equal to 7,4°C. These values demonstrate that POD of coconut water is thermal resistant to inactivation, being such processes strongly affected by temperature. Such fact is also verified by the high values of Ea (332,3 kJ/mol and Q10 (22,7, which indicate that a high amount of energy is required for the POD inactivation and an increase of temperature elevate the inactivation rate.Este trabalho teve como objetivo estudar a cinética de inativação térmica da enzima peroxidase (POD presente na água de coco verde, determinando-se os valores dos parâmetros D, z e Q10, além da energia de ativação (Ea de Arrhenius para o processo. Foram avaliados nove binômios tempo/temperatura para o tratamento térmico em três temperaturas (75°C, 85°C e 95°C e tempos de manutenção variando de 1min a 30min, além da amostra sem tratamento térmico. Os valores de D foram determinados por meio de regressão linear, obtendo-se os valores de 833,3min., 35,6min. e 1,63min., para as temperaturas de 75°C, 85°C e 95°C, respectivamente, enquanto o valor de z foi igual a 7,4°C. Esses valores demonstraram que a POD da água de coco é resistente à inativação térmica, sendo tal processo fortemente afetado pela temperatura utilizada. Tal fato foi reforçado pelos altos valores da Ea (332,3kJ/mol e Q10 (22,7, que indicaram que uma alta quantidade de energia

  9. Calculated ionisation potentials determine the oxidation of vanillin precursors by lignin peroxidase.

    Science.gov (United States)

    ten Have, R; Rietjens, I M; Hartmans, S; Swarts, H J; Field, J A

    1998-07-03

    In view of the biocatalytic production of vanillin, this research focused on the lignin peroxidase (LiP) catalysed oxidation of naturally occurring phenolic derivatives: O-methyl ethers, O-acetyl esters, and O-glucosyl ethers. The ionisation potential (IP) of a series of model compounds was calculated and compared to their experimental conversion by LiP, defining a relative IP threshold of approximately 9.0 eV. Based on this threshold value only the O-acetyl esters and glucosides of isoeugenol and coniferyl alcohol would be potential LiP substrates. Both O-acetyl esters were tested and were shown to be converted to O-acetyl vanillin in molar yields of 51.8 and 2.3%, respectively.

  10. Plant responses to stresses: role of ascorbate peroxidase in the antioxidant protection

    Directory of Open Access Journals (Sweden)

    Andréia Caverzan

    2012-01-01

    Full Text Available When plants are exposed to stressful environmental conditions, the production of Reactive Oxygen Species (ROS increases and can cause significant damage to the cells. Antioxidant defenses, which can detoxify ROS, are present in plants. A major hydrogen peroxide detoxifying system in plant cells is the ascorbate-glutathione cycle, in which, ascorbate peroxidase (APX enzymes play a key role catalyzing the conversion of H2O2 into H2O, using ascorbate as a specific electron donor. Different APX isoforms are present in distinct subcellular compartments, such as chloroplasts, mitochondria, peroxisome, and cytosol. The expression of APX genes is regulated in response to biotic and abiotic stresses as well as during plant development. The APX responses are directly involved in the protection of plant cells against adverse environmental conditions. Furthermore, mutant plants APX genes showed alterations in growth, physiology and antioxidant metabolism revealing those enzymes involvement in the normal plant development.

  11. Vibroacustic microvibrations enhance kidney blood supply, glomerular filtration and glutathione peroxidase activity in spontaneously hypertensive rats.

    Science.gov (United States)

    Miloradović, Zoran; Mihailović-Stanojević, Nevena; Jovović, Đurđica; Ivanov, Milan; Vajić, Una J; Karanović, Danijela; Grujić Milanović, Jelica

    2015-01-01

    Limited numbers of studies include research of microvibration therapy in experimental models. We examined effects of chronic vibroacustic-microvibration treatment on haemodynamics and anti-oxidative defense in experimental hypertension. Study was performed on chronically treated hypertensive and normotensive Wistar rats. Mean arterial pressure (MAP), cardiac output (CO), renal blood flow (RBF), glomerular filtration and activity of anti-oxidative enzymes were determined after three weeks treatment. Vibroacustic treatment had no influence on MAP and CO, but RBF was increased in both groups of treated rats. Additionally, vibroacustic treatment enhanced diuresis and increased glomerular filtration in hypertensive rats. Glutathione peroxidase (GSH-Px) activity was elevated in both treated rat strains, but activity of superoxide dismutase was unchanged. We conclude that microvibration treatment doesn't ameliorate hypertension but improves renal blood supply (trough diminished renal vascular resistance), glomerular filtration, diuresis, and enhances glutathione dependent anti-oxidant defense with more important beneficials in hypertensive animals.

  12. Validation of spectrophotometric microplate methods for polyphenol oxidase and peroxidase activities analysis in fruits and vegetables

    Directory of Open Access Journals (Sweden)

    Érica Sayuri SIGUEMOTO

    Full Text Available Abstract Enzymes polyphenol oxidase (PPO and peroxidase (POD play important roles in the processing of fruits and vegetables, since they can produce undesirable changes in color, texture and flavor. Classical methods of activity assessment are based on cuvette spectrophotometric readings. This work aims to propose, to validate and to test microplate spectrophotometric methods. Samples of apple juice and lyophilized enzymes from mushroom and horseradish were analyzed by the cuvette and microplate methods and it was possible to validate the microplate assays with satisfactory results regarding linearity, repeatability, accuracy along with quantitation and detection limits. The proposed microplate methods proved to be reliable and reproducible as the classical methods besides having the advantages of allowing simultaneous analysis and requiring a reduced amount of samples and reactants, which can beneficial to the study of enzyme inactivation in the processing of fruits and vegetables.

  13. Southern blot screening for lignin peroxidase and aryl-alcohol oxidase genes in 30 fungal species.

    Science.gov (United States)

    Varela, E; Martínez, A T; Martínez, M J

    2000-10-13

    Screening to detect genes encoding lignin peroxidase (LiP) and aryl-alcohol oxidase (AAO) has been carried out with 30 fungal strain using DNA probes from genes lpo of Phanerochaete chrysosporium (encoding LiP isoenzyme H8) and aao of Pleurotus eryngii. Evidence for the presence of genes closely related to lpo was found in Bjerkandera adusta, Fomes fomentarius, Ganoderma applanatum, Ganoderma australe, Lentinula degener, Peniophora gigantea, P. chrysosporium, Phanerochaete flavido-alba and Trametes tersicolor, whereas the gene aao was detected in Pleurotus species and B. adusta. The presence of both genes was only detected in B. adusta. These results suggest that different enzymatic system, formed by enzymes encoded by different genes, are responsible for lignin degradation by white-rot fungi.

  14. Identification of Novel Genetic Loci Associated with Thyroid Peroxidase Antibodies and Clinical Thyroid Disease

    DEFF Research Database (Denmark)

    Medici, Marco; Porcu, Eleonora; Pistis, Giorgio

    2014-01-01

    Autoimmune thyroid diseases (AITD) are common, affecting 2-5% of the general population. Individuals with positive thyroid peroxidase antibodies (TPOAbs) have an increased risk of autoimmune hypothyroidism (Hashimoto's thyroiditis), as well as autoimmune hyperthyroidism (Graves' disease.......12-1.39, P = 6.2×10(-5)). The MAGI3 variant was also associated with an increased risk of hypothyroidism (OR: 1.57, 95% CI 1.18-2.10, P = 1.9×10(-3)). This first GWAS meta-analysis for TPOAbs identified five newly associated loci, three of which were also associated with clinical thyroid disease....... With these markers we identified a large subgroup in the general population with a substantially increased risk of TPOAbs. The results provide insight into why individuals with thyroid autoimmunity do or do not eventually develop thyroid disease, and these markers may therefore predict which TPOAb...

  15. Differential expression of gastrointestinal glutathione peroxidase (GI-GPx) gene during mouse organogenesis.

    Science.gov (United States)

    Baek, I-J; Yon, J-M; Lee, S-R; Kim, M-R; Hong, J T; Lee, B J; Yun, Y W; Nam, S-Y

    2011-06-01

    Gastrointestinal glutathione peroxidase (GI-GPx) is an antioxidant enzyme that has been known to be restricted to the gastrointestinal tract in rodents. In an effort to determine the expression pattern of GI-GPx mRNA during organogenesis, quantitative real-time PCR and in situ hybridization for GI-GPx mRNA were conducted in whole embryos or each developing organ of mice. GI-GPx mRNA was expressed more abundantly in the extraembryonic tissues, including placenta than in embryos on embryonic days (EDs) 7.5-18.5 (P embryonic organs during organogenesis but also may perform a mutual compensatory role with the cGPx in the protection of embryos and extraembryonic tissues against the reactive oxygen species generated in ontogenetic periods. © 2011 Blackwell Verlag GmbH.

  16. Data on blueberry peroxidase kinetic characterization and stability towards thermal and high pressure processing

    Directory of Open Access Journals (Sweden)

    Netsanet Shiferaw Terefe

    2017-08-01

    Full Text Available The data presented in this article are related to a research article entitled ‘Thermal and high pressure inactivation kinetics of blueberry peroxidase’ (Terefe et al., 2017 [1]. In this article, we report original data on the activity of partially purified blueberry peroxidase at different concentrations of hydrogen peroxide and phenlylenediamine as substrates and the effects of thermal and high pressure processing on the activity of the enzyme. Data on the stability of the enzyme during thermal (at temperatures ranging from 40 to 80 °C and combined thermal-high pressure processing (100–690 MPa, 30–90 °C are included in this report. The data are presented in this format in order to facilitate comparison with data from other researchers and allow statistical analyses and modeling by others in the field.

  17. Detoxification of simulated textile wastewater using a membraneless electrochemical reactor with immobilized peroxidase

    International Nuclear Information System (INIS)

    Cho, Seung-Hee; Shim, Joonmok; Moon, Seung-Hyeon

    2009-01-01

    Simulated textile wastewater was degraded using a membraneless electrochemical reactor with immobilized peroxidase on the porous Celite. The optimal current density was 10 A m -2 , at which the highest amount of hydrogen (H 2 O 2 ) could be generated. The decolorization efficiencies of the simulated wastewater using the electrochemical and electroenzymatic methods were 35% and 92%, respectively. Biodegradability, the ratio of 5-day biochemical oxygen demand to chemical oxygen demand (BOD 5 /COD), was enhanced about 1.88 times when using the electroenzymatic treatment rather than raw wastewater, which could not be achieved by the electrochemical treatment. The toxic unit (TU), calculated using the lethal concentration (LC 50 ) of Daphnia magna (D. Magna), of effluent treated by electroenzymatic method was below 1, whereas those of simulated textile wastewater and effluent treated by electrochemical method were 11.4 and 3.9, respectively

  18. A new assay for lignin-type peroxidases employing the dye azure B.

    Science.gov (United States)

    Archibald, F S

    1992-01-01

    The discovery in 1983 of fungal "ligninases" capable of catalyzing the peroxidation of nonphenolic aromatic lignin components has been seen as a major advance in understanding how certain basidiomycete fungi can completely degrade lignin. The ability of these lignin-type peroxidases to covert millimolar concentrations of veratryl alcohol to veratraldehyde, indicated by a change in the A310 of veratraldehyde, has become the standard assay for routine quantitation of LP activity. A new assay based on the oxidation of micromolar concentrations of the dye Azure B is presented. Although it is as simple and rapid as the veratryl alcohol assay, it appears to overcome some of the shortcomings of that assay. In particular, interference from UV- and short-wavelength visible-light-absorbing materials is greatly reduced and assay specificity is improved. PMID:1444425

  19. cDNA, amino acid carbohydrate sequence of barley seed-specific peroxidase BP 1

    DEFF Research Database (Denmark)

    Johansson, A.; Rasmussen, Søren Kjærsgård; Harthill, J.E.

    1992-01-01

    of 69% in the translated region, a 90% G or C preference in the wobble position of the codons and a typical signal peptide sequence. N-terminal amino acid sequencing and sequence analysis of tryptic peptides verified 98% of the sequence of the mature BP 1 which contains 309 amino acid residues. BP 1...... biological role of this enzyme. The barley peroxidase is processed at the C-terminus and might be targeted to the vacuole. The single site of glycosylation is located near the C-terminus in the N-glycosylation sequon -Asn-Cys-Ser- in which Cys forms part of a disulphide bridge. The major glycan is a typical...

  20. Catalase, Peroxidase and Polyphenoloxidase from Pitaya Amarilla (Acanthocereus pitajaya Fruits: Ripening and Senescense

    Directory of Open Access Journals (Sweden)

    Lucía Estrella Baquero Duarte

    2005-07-01

    Full Text Available We evaluate the relation between some symptoms of deterioration and the activity of enzymes entailed with both the browning and the antioxiding system in fruits of yellow pitaya (Acanthocereus pitajaya, harvested in its physiological maturity and stored for 15 days at 24°C and 85% of relative humidity. In the whole fruits, the respiratory intensity and the external colors were evaluated; further, the activity of catalase (CAT, peroxidase (POD and polyphenoloxidase (PPO was studied in the peel of the fruit. The fruit exhibited a climacteric behavior six days after the date of the harvest. The browning of the peel had a direct relation with the activity of POD and PPO. The maximum observed activity of CAT in the climacterium, responds to the proper balance with the high production of H2O2 expected at that moment.

  1. Automated determination of asulam by enhanced chemiluminescence using luminol/peroxidase system.

    Science.gov (United States)

    Sánchez, Francisco García; Díaz, Aurora Navas; Bracho, Visitación; Aguilar, Alfonso; Algarra, Manuel

    2009-01-01

    A flow injection system with chemiluminescence detection for the determination of asulam, enhancer of the system luminol-H(2)O(2)-horseradish peroxidase, is proposed. The method shows a moderate selectivity against other pesticides usually present in formulations of herbicides and in water. The procedure was applied to the determination of asulam in tap water samples and a recovery study was carried out in order to validate the method. The obtained results show acceptable recovery values (between 88.3 and 93.9%). The detection limit for asulam was 0.12 ng/mL. The precision of the method expressed as relative standard deviation was 1.55% (n = 8), at the 19 ng/mL level.

  2. [Cloning and sequence analysis of thioredoxin peroxidase gene from Taenia multiceps].

    Science.gov (United States)

    Li, Yong-guang; Li, Wen-hui; Gai, Wen-yan; Yao, Ju-xia; Qu, Zi-gang; Jia, Wan-zhong; Radu, Blaga; Fu, Bao-quan

    2011-02-28

    Protoscoleces of Taenia multiceps were collected from the naturally infected sheep and total RNA was extracted. Specific primers were designed according to TaHe2-D11 mRNA sequence and T. multiceps thioredoxin peroxidase gene (TmTPx) was amplified by RT-PCR. PCR products were ligated into pMD18-T vector and transformed to E. coli DH5alpha. The recombinant plasmids were identified by restriction digestion and sequencing. A 614 bp cDNA was amplified. The TmTPx open reading frame (591 bp) encoded a 196-amino acid protein with Mr 21,690, pI 7.61. Bioinformatics analysis indicated that TmTPx had a typical 2-Cys Prx conserved domain. Phylogenetic tree revealed that T. multiceps had the closest relationship to T. asiatica, followed by T. solium and T. crassiceps, E. granulosus and E. multilocularis.

  3. Degradation of orange dyes and carbamazepine by soybean peroxidase immobilized on silica monoliths and titanium dioxide.

    Science.gov (United States)

    Calza, Paola; Zacchigna, Dario; Laurenti, Enzo

    2016-12-01

    In this paper, the removal of three common dyes (orange I, orange II, and methylorange) and of the anticonvulsant drug carbamazepine from aqueous solutions by means of enzymatic and photocatalytic treatment was studied. Soybean peroxidase (SBP) was used as biocatalyst, both free in solution and immobilized on silica monoliths, and titanium dioxide as photocatalyst. The combination of the two catalysts led to a faster (about two to four times) removal of all the orange dyes compared to the single systems. All the dyes were completely removed within 2 h, also in the presence of immobilized SBP. As for carbamazepine, photocatalytic treatment prevails on the enzymatic degradation, but the synergistic effect of two catalysts led to a more efficient degradation; carbamazepine's complete disappearance was achieved within 60 min with combined system, while up to 2 h is required with TiO 2 only.

  4. Electrochemical properties of seamless three-dimensional carbon nanotubes-grown graphene modified with horseradish peroxidase.

    Science.gov (United States)

    Komori, Kikuo; Terse-Thakoor, Trupti; Mulchandani, Ashok

    2016-10-01

    Horseradish peroxidase (HRP) was immobilized through sodium dodecyl sulfate (SDS) on the surface of a seamless three-dimensional hybrid of carbon nanotubes grown at the graphene surface (HRP-SDS/CNTs/G) and its electrochemical properties were investigated. Compared with graphene alone electrode modified with HRP via SDS (HRP-SDS/G electrode), the surface coverage of electroactive HRP at the CNTs/G electrode surface was approximately 2-fold greater because of CNTs grown at the graphene surface. Based on the increase in the surface coverage of electroactive HRP, the sensitivity to H2O2 at the HRP-SDS/CNTs/G electrode was higher than that at the HRP-SDS/G electrode. The kinetics of the direct electron transfer from the CNTs/G electrode to compound I and II of modified HRP was also analyzed. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Congenital goitrous primary hypothyroidism in two German families caused by novel thyroid peroxidase (TPO) gene mutations.

    Science.gov (United States)

    Altmann, K; Hermanns, P; Mühlenberg, R; Fricke-Otto, S; Wentzell, R; Pohlenz, J

    2013-06-01

    Congenital hypothyroidism occurs with a prevalence of approximately 1:3 500. Defects in thyroid hormone synthesis which lead to goitrous hypothyroidism account for 10-15% of these cases. Several genetic defects have been characterized and mutations in the thyroid peroxidase (TPO) gene are the most common cause for dyshormonogenesis.So far, more than 80 mutations in the TPO gene have been described, resulting in a variable decrease in TPO bioactivity. Clinically TPO defects manifest with congenital primary goitrous hypothyroidism.We here present 2 children with congenital primary hypothyroidism, who were identified to have compound heterozygous TPO mutations. They both shared the same novel mutation in the TPO gene (C756R) in exon 13. One case presented with an apparently dominant inheritance of thyroid dyshormonogenesis. © J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart · New York.

  6. Modifying sulfomethylated alkali lignin by horseradish peroxidase to improve the dispersibility and conductivity of polyaniline

    Science.gov (United States)

    Yang, Dongjie; Huang, Wenjing; Qiu, Xueqing; Lou, Hongming; Qian, Yong

    2017-12-01

    Pine and wheat straw alkali lignin (PAL and WAL) were sulfomethylated to improve water solubility, polymerized with horseradish peroxidase (HRP) to improve the molecular weight (Mw) and applied to dope and disperse polyaniline (PANI). The structural effect of lignin from different origins on the reactivities of sulfomethylation and HRP polymerization was investigated. The results show that WAL with less methoxyl groups and lower Mw have higher reactivity in sulfomethylation (SWAL). More phenolic hydroxyl groups and lower Mw benefit the HRP polymerization of sulfomethylated PAL (SPAL). Due to the natural three-dimensional aromatic structure and introduced sulfonic groups, SPAL and SWAL could effectively dope and disperse PANI in water by π-π stacking and electrostatic interaction. HRP modified SPAL (HRP-SPAL) with much higher sulfonation degree and larger Mw significantly increased the conductivity and dispersibility of lignin/PANI composites.

  7. Frequency of thyroid peroxidase antibody and its association with miscarriages among pregnant women

    International Nuclear Information System (INIS)

    Iqbal, S.; Ghani, F.

    2016-01-01

    To measure the frequency of thyroid peroxidase antibody (TPO Ab) among clinically euthyroid pregnant women during first trimester and determine its association with pregnancy outcome as miscarriage or live birth by estimating the relative risk. Study Design: Cohort study. Place and Duration of Study: Section of Chemical Pathology, Department of Pathology and Laboratory Medicine and the Gynaecology and Obstetric outpatient clinics of the Aga Khan University Hospital, Karachi, from July to December 2012. Methodology: The study subjects comprised of apparently euthyroid pregnant women, who were tested for TPO Ab during first trimester of pregnancy and followed till pregnancy outcomes. Pregnancy outcome was noted and relative risk was determined. Results: TPO Ab was found positive in 127 (13.5%) pregnant women from a cohort of 943 subjects. A 2.03% increased risk of miscarriages was observed in TPO Ab positive subjects. Conclusion: There was a positive association of presence of presence of TPO Ab with loss pregnancy. (author)

  8. Irregular-shaped platinum nanoparticles as peroxidase mimics for highly efficient colorimetric immunoassay.

    Science.gov (United States)

    Gao, Zhuangqiang; Xu, Mingdi; Hou, Li; Chen, Guonan; Tang, Dianping

    2013-05-07

    Enzyme-linked immunosorbent assay (ELISA) methods based on natural enzyme-labeled probes have been applied in the immunoassays, but most have some inevitable limitations (e.g. harsh preparation, purification and storage) and are unsuitable for routine use. Herein we synthesized a new class of irregular-shaped platinum nanoparticles (ISPtNP) with a mean length of 7.0 nm and a narrowing width from 2.0 to 5.0 nm along the longitudinal axes, which were utilized as peroxidase-like mimics for the development of colorimetric immunoassays. Compared with bioactive horseradish peroxidase (HRP), the synthesized ISPtNP exhibited a low Km value (~0.12 mM) and a high Kcat value (~2.27×10(4)s(-1)) for 3,3',5,5'-tetramethylbenzidine (TMB) with strong thermal stability and pH tolerance. The catalytic mechanism of the ISPtNP toward TMB/H2O2 was for the first time discussed and deliberated in this work. Based on a sandwich-type assay format, two types of colorimetric immunoassay protocols were designed and developed for the detection of rabbit IgG (RIgG, as a model) by using the synthesized ISPtNP and conventional HRP as the labeling of detection antibodies, respectively. Similar detection limits (LODs) of 2.5 ng mL(-1) vs. 1.0 ng mL(-1) were obtained toward RIgG with the ISPtNP labeling compared to HRP format. Intra- and inter-assay coefficients of variation were less than 13%. Importantly, the ISPtNP-based assay system could be suitable for use in a mass production of miniaturized lab-on-a-chip devices and open new opportunities for protein diagnostics and biosecurity. Copyright © 2013 Elsevier B.V. All rights reserved.

  9. Decolorization of Anthraquinonic Dyes from Textile Effluent Using Horseradish Peroxidase: Optimization and Kinetic Study

    Science.gov (United States)

    Šekuljica, Nataša Ž.; Prlainović, Nevena Ž.; Stefanović, Andrea B.; Žuža, Milena G.; Čičkarić, Dragana Z.; Mijin, Dušan Ž.; Knežević-Jugović, Zorica D.

    2015-01-01

    Two anthraquinonic dyes, C.I. Acid Blue 225 and C.I. Acid Violet 109, were used as models to explore the feasibility of using the horseradish peroxidase enzyme (HRP) in the practical decolorization of anthraquinonic dyes in wastewater. The influence of process parameters such as enzyme concentration, hydrogen peroxide concentration, temperature, dye concentration, and pH was examined. The pH and temperature activity profiles were similar for decolorization of both dyes. Under the optimal conditions, 94.7% of C.I. Acid Violet 109 from aqueous solution was decolorized (treatment time 15 min, enzyme concentration 0.15 IU/mL, hydrogen peroxide concentration 0.4 mM, dye concentration 30 mg/L, pH 4, and temperature 24°C) and 89.36% of C.I. Acid Blue 225 (32 min, enzyme concentration 0.15 IU/mL, hydrogen peroxide concentration 0.04 mM, dye concentration 30 mg/L, pH 5, and temperature 24°C). The mechanism of both reactions has been proven to follow the two substrate ping-pong mechanism with substrate inhibition, revealing the formation of a nonproductive or dead-end complex between dye and HRP or between H2O2 and the oxidized form of the enzyme. Both chemical oxygen demand and total organic carbon values showed that there was a reduction in toxicity after the enzymatic treatment. This study verifies the viability of use of horseradish peroxidase for the wastewaters treatment of similar anthraquinonic dyes. PMID:25685837

  10. Expression of Cryptosporidium parvum thioredoxin peroxidase in COS-7 cells confers radioprotection.

    Science.gov (United States)

    Hong, Semie; Kim, Jae-Hwan; Yoon, Sejoung; Kim, Kyoungjin; Sim, Seobo; Park, Woo-Yoon; Yu, Jae-Ran

    2016-04-01

    Cryptosporidium parvum is one of the most radioresistant organisms identified to date. In a previous study, we found that thioredoxin peroxidase (CpTPx) was significantly upregulated in this species following exposure to high dose (10 kGy) of γ-irradiation. To assess the potential of CpTPx to confer radioprotection in mammalian cells, it was expressed in COS-7 African green monkey kidney cells (CpTPx-COS7). For comparison, the thioredoxin peroxidase of Cryptosporidium muris (CmTPx) was also expressed in these cells (CmTPx-COS7 cells), which has been confirmed to have lesser antioxidant activity than CpTPx in the previous study. Notably, the survival rates of CpTPx-COS7 cells were significantly higher (12-22%) at 72 h after 8 Gy irradiation than CmTPx-COS7 or non-transfected COS-7 (ntCOS-7) counterparts. In addition, CpTPx revealed a 50% of ROS reduction in irradiated CpTPx-COS7 cells, while γ-H2AX DNA damage marker expression was not significantly changed. Furthermore, the amount of apoptosis only increased to about 120% after 2-8 Gy irradiation compared to 200-300% increase observed in ntCOS-7 cells. CmTPx was shown to have antioxidant and DNA damage protection activities; however, these activities were always lower than those of CpTPx. These results suggest that the potent antioxidant and protective activities of CpTPx are well conserved in this cell-based system and that CpTPx contributed to the radioprotection of mammalian cells through its exceptional antioxidant activity. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Decolorization of Anthraquinonic Dyes from Textile Effluent Using Horseradish Peroxidase: Optimization and Kinetic Study

    Directory of Open Access Journals (Sweden)

    Nataša Ž. Šekuljica

    2015-01-01

    Full Text Available Two anthraquinonic dyes, C.I. Acid Blue 225 and C.I. Acid Violet 109, were used as models to explore the feasibility of using the horseradish peroxidase enzyme (HRP in the practical decolorization of anthraquinonic dyes in wastewater. The influence of process parameters such as enzyme concentration, hydrogen peroxide concentration, temperature, dye concentration, and pH was examined. The pH and temperature activity profiles were similar for decolorization of both dyes. Under the optimal conditions, 94.7% of C.I. Acid Violet 109 from aqueous solution was decolorized (treatment time 15 min, enzyme concentration 0.15 IU/mL, hydrogen peroxide concentration 0.4 mM, dye concentration 30 mg/L, pH 4, and temperature 24°C and 89.36% of C.I. Acid Blue 225 (32 min, enzyme concentration 0.15 IU/mL, hydrogen peroxide concentration 0.04 mM, dye concentration 30 mg/L, pH 5, and temperature 24°C. The mechanism of both reactions has been proven to follow the two substrate ping-pong mechanism with substrate inhibition, revealing the formation of a nonproductive or dead-end complex between dye and HRP or between H2O2 and the oxidized form of the enzyme. Both chemical oxygen demand and total organic carbon values showed that there was a reduction in toxicity after the enzymatic treatment. This study verifies the viability of use of horseradish peroxidase for the wastewaters treatment of similar anthraquinonic dyes.

  12. Weak self-association of cytochrome c peroxidase molecules observed by paramagnetic NMR

    Energy Technology Data Exchange (ETDEWEB)

    Schilder, Jesika; Ubbink, Marcellus, E-mail: m.ubbink@chem.leidenuniv.nl [Leiden University, Gorlaeus Laboratories, Leiden Institute of Chemistry (Netherlands)

    2016-05-15

    There is growing experimental evidence that many proteins exhibit a tendency for (ultra)weak homo- or hetero- oligomerization interactions. With the development of paramagnetic relaxation enhancement NMR spectroscopy it has become possible to characterize weak complexes experimentally and even detect complexes with affinities in the 1–25 mM range. We present evidence for a weak complex between cytochrome c peroxidase (CcP) molecules. In a previous study, we attached nitroxide based spin labels at three positions on CcP with the intent of observing intramolecular PRE effects. However, several intermolecular PRE effects were also observed suggesting a weak self-association between CcP molecules. The CcP–CcP complex was characterized using paramagnetic NMR and protein docking. The interaction occurs between the surface that is also part of the stereo-specific binding site for its physiological partner, cytochrome c (Cc), and several small, positively charged patches on the “back” of CcP. The CcP–CcP complex is not a stereo-specific complex. It is a dynamic ensemble of orientations, characteristic of an encounter state. The contact areas resemble those observed for CcP molecules in crystals. The CcP–CcP complex formation competes with that of the CcP-Cc complex. However, the affinity for Cc is much larger and thus it is expected that, under physiological conditions, auto-inhibition will be limited.Graphical AbstractA weak self-association between cytochrome c peroxidase molecules was characterized using paramagnetic NMR.

  13. Mechanistic Insights into Dye-Decolorizing Peroxidase Revealed by Solvent Isotope and Viscosity Effects

    Energy Technology Data Exchange (ETDEWEB)

    Shrestha, Ruben [Department; Huang, Gaochao [Department; Meekins, David A. [Department; Geisbrecht, Brian V. [Department; Li, Ping [Department

    2017-08-18

    Dye-decolorizing peroxidases (DyPs) are a family of H2O2-dependent heme peroxidases that have shown potential applications in lignin degradation and valorization. However, the DyP kinetic mechanism remains underexplored. Using structural biology and solvent isotope (sKIE) and viscosity effects, many mechanistic characteristics have been determined for the B-class ElDyP from Enterobacter lignolyticus. Its structure revealed that a water molecule acts as the sixth axial ligand and two channels at diameters of ~3.0 and 8.0 Å lead to the heme center. A conformational change of ERS* to ERS, which have identical spectral characteristics, was proposed as the final step in DyPs’ bisubstrate Ping-Pong mechanism. This step is also the rate-determining step in ABTS oxidation. The normal KIE of wild-type ElDyP with D2O2 at pD 3.5 suggested that compound 0 deprotonation by the distal aspartate is rate-limiting in the formation of compound I, which is more reactive under acidic pH than under neutral or alkaline pH. The viscosity effects and other biochemical methods implied that the reducing substrate binds with compound I instead of the free enzyme. The significant inverse sKIEs of kcat/KM and kERS* suggested that the aquo release in ElDyP is mechanistically important and may explain the enzyme’s adoption of two-electron reduction for compound I. The distal aspartate is catalytically more important than the distal arginine and plays key roles in determining ElDyP’s optimum acidic pH. The kinetic mechanism of D143H-ElDyP was also briefly studied. The results obtained will pave the way for future protein engineering to improve DyPs’ lignolytic activity.

  14. MacA is a second cytochrome c peroxidase of Geobacter sulfurreducens.

    Science.gov (United States)

    Seidel, Julian; Hoffmann, Maren; Ellis, Katie E; Seidel, Antonia; Spatzal, Thomas; Gerhardt, Stefan; Elliott, Sean J; Einsle, Oliver

    2012-04-03

    The metal-reducing δ-proteobacterium Geobacter sulfurreducens produces a large number of c-type cytochromes, many of which have been implicated in the transfer of electrons to insoluble metal oxides. Among these, the dihemic MacA was assigned a central role. Here we have produced G. sulfurreducens MacA by recombinant expression in Escherichia coli and have solved its three-dimensional structure in three different oxidation states. Sequence comparisons group MacA into the family of diheme cytochrome c peroxidases, and the protein indeed showed hydrogen peroxide reductase activity with ABTS(-2) as an electron donor. The observed K(M) was 38.5 ± 3.7 μM H(2)O(2) and v(max) was 0.78 ± 0.03 μmol of H(2)O(2)·min(-1)·mg(-1), resulting in a turnover number k(cat) = 0.46 · s(-1). In contrast, no Fe(III) reductase activity was observed. MacA was found to display electrochemical properties similar to other bacterial diheme peroxidases, in addition to the ability to electrochemically mediate electron transfer to the soluble cytochrome PpcA. Differences in activity between CcpA and MacA can be rationalized with structural variations in one of the three loop regions, loop 2, that undergoes conformational changes during reductive activation of the enzyme. This loop is adjacent to the active site heme and forms an open loop structure rather than a more rigid helix as in CcpA. For the activation of the protein, the loop has to displace the distal ligand to the active site heme, H93, in loop 1. A H93G variant showed an unexpected formation of a helix in loop 2 and disorder in loop 1, while a M297H variant that altered the properties of the electron transfer heme abolished reductive activation.

  15. A comparison between the oxidation with laccase and horseradish peroxidase for triclosan conversion.

    Science.gov (United States)

    Melo, C F; Dezotti, M; Marques, M R C

    2016-01-01

    Triclosan is a broad-spectrum biocide used in personal-care products that is suspected to be linked to the emergence of antibiotic-resistant bacteria. In the present work, the enzymes horseradish peroxidase and laccase from Trametes versicolor were evaluated for the conversion of triclosan in an aqueous matrix. The removal of antibacterial activity by the enzymatic processes was evaluated by an assay based on the growth inhibition of Escherichia coli K12. The horseradish peroxidase (HRP) process appears more advantageous than the laccase process in removing triclosan from an aqueous matrix, considering the reaction parameters pH, temperature, catalytic efficiency, and enzyme concentration. The highest conversion of triclosan catalysed by laccase was observed at pH 5.0, that is, lower than the typical pH range (6.5-7.5) of sewage treatment plants' effluents. The efficiency of laccase process was much more impacted by variations in the temperature in the range of 10-40°C. Kinetic studies showed that triclosan is a substrate more specific for HRP than for laccase. The protein content for the HRP-catalysed process was 14 times lower than that for the laccase process. Decay kinetics suggest that reaction mechanisms depend on enzyme concentration and its concentration. Both processes were able to reduce the antibacterial activity, and the residual activity of the treated solution is probably due to non-converted triclosan and not due to the reaction products. The laccase-catalysed conversion of triclosan in an environmental relevant concentration required a higher amount of enzyme than that required in the HRP process.

  16. Binding of peroxiredoxin 6 to substrate determines differential phospholipid hydroperoxide peroxidase and phospholipase A2 activities

    Science.gov (United States)

    Manevich, Yefim; Shuvaeva, Tea; Dodia, Chandra; Kazi, Altaf; Feinstein, Sheldon I.; Fisher, Aron B.

    2010-01-01

    Peroxiredoxin 6 (Prdx6) differs from other mammalian peroxiredoxins both in its ability to reduce phospholipid hydroperoxides at neutral pH and in having phospholipase A2 (PLA2) activity that is maximal at acidic pH. We previously showed an active site C47 for peroxidase activity and a catalytic triad S32-H26-D140 necessary for binding of phospholipid and PLA2 activity. This study evaluated binding of reduced and oxidized phospholipid hydroperoxide to Prdx6 at cytosolic pH. Incubation of recombinant Prdx6 with 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine hydroperoxide (PLPCOOH) resulted in peroxidase activity, cys47 oxidation as detected with Prdx6-SO2(3) antibody, and a marked shift in the Prdx6 melting temperature by circular dichroism analysis indicating that PLPCOOH is a specific substrate for Prdx6. Preferential Prdx6 binding to oxidized liposomes was detected by changes in DNS-PE or bis-Pyr fluorescence and by ultrafiltration. Site-specific mutation of S32 or H26 in Prdx6 abolished binding while D140 mutation had no effect. Treatment of A549 cells with peroxides led to lipid peroxidation and translocation of Prdx6 from the cytosol to the cell membrane. Thus, the pH specificity for the two enzymatic activities of Prdx6 can be explained by the differential binding kinetics of the protein; Prdx6 binds to reduced phospholipid at acidic pH but at cytosolic pH binds only phospholipid that is oxidized compatible with a role for Prdx6 in the repair of peroxidized cell membranes. PMID:19236840

  17. Molecular cloning and functional analyses of glutathione peroxidase homologous genes from Chlorella sp. NJ-18.

    Science.gov (United States)

    Wang, Xin; Xu, Xudong

    2012-06-10

    Photosynthetic organisms often encounter oxidative stresses due to changes of environmental conditions. In this study, two glutathione peroxidase (GPX) homologous genes, namely NJ-18Gpx1 and NJ-18Gpx2, were identified in Chlorella sp. NJ-18, a single-celled green alga. The two NJ-18Gpx genes can produce 2 or 3 transcript variants by alternative splicing, predicted to encode 4 non-selenium GPX proteins (NS-GPX). Expression of the two genes was analyzed by quantitative RT-PCR in Chlorella sp. NJ-18 exposed to various treatments known to generate reactive oxygen species. Neutral red, a singlet oxygen-generating photosensitizer, significantly increased the expression of NJ-18Gpx1 within 5 h. Exposure of algal culture to UV-B for 3h caused up-regulation of mRNA levels of NJ-18Gpx1 and NJ-18Gpx2 by 4- and 50-folds, respectively. Similar to CrGPX5 and CrGPX3 in Chlamydomonas reinhardtii, purified recombinant NJ-18GPXs showed activities of thioredoxin-dependent peroxidases that catalyze the reduction of hydrogen peroxide and organic hydroperoxides. The V(max) values for NJ-18GPX1 toward different peroxides were approximately 10-fold higher than those for NJ-18GPX2. In addition, overexpression of NJ-18Gpx1 in Synechocystis sp. PCC 6803, a cyanobacterium, enhanced its tolerance to neutral red and H(2)O(2). These results indicate that NJ-18GPXs can act as efficient peroxide scavengers protecting cells from oxidative damages in Chlorella. Copyright © 2012 Elsevier B.V. All rights reserved.

  18. The importance of anti peroxidase antibodies in patients with nodular thyroid goiter

    Directory of Open Access Journals (Sweden)

    Medenica Sanja

    2012-01-01

    Full Text Available Introduction: Thyroid nodular goiter Nodular goiter is clinically recognizable restricted structure changes of the thyroid gland. Numerous studies show the relationship between thyroid autoimmunity and differentiated thyroid cancer in patients with nodular thyroid goiter. One of the important clinical marker in defining thyroid autoimmunity are antibodies to thyroid peroxidase. Objective: The aim of this study was to analyze the relationship between antibodies anti peroksidaznih ii thyroid malignancies in patients with thyroid altered thyroid. Material and Methods: We retrospectively reviewed the 248 reports of thyroid FNA cytology of samples obtained by puncture of patients with nodular thyroid goiter, at Department of thyroid gland, Department of Endocrinology, Diabetes and Metabolic Diseases, Clinical Center of Serbia in the period from October 2007. by January 2010. year. We analyzed the relationship between findings of cytopathological diagnostic categories and serum concentrations of anti peroxidase antibodies. The data were statistically processed using the computer program SPSS 12.0 software package. Results: Of total 248 patients, 148 patients (59.7% had anti-TPO antibody values in a reference limits (30 IU/ml, and 40.3% elevated anti-TPO antibodies (over 30 IU/ml. In the group of patients with elevated values of anti-TPO antibodies 7% (7/100 of patients had malignant cytologic findings, and in the group of patients with anti-TPO antibody values in the normal range only 1.4% (2/148 of patients had malignant cytologic findings. Conclusion: Defining the relationship between thyroid autoimmune disease and differentiated thyroid cancer, providing new insights in the field of immunotherapy of thyroid carcinoma. Enlightening the molecular mechanisms link autoimmune thyroid disease and thyroid cancer development in patients with thyroid nodule help find new therapeutic strategies against thyroid cancer.

  19. Mechanistic Insights into Dye-Decolorizing Peroxidase Revealed by Solvent Isotope and Viscosity Effects.

    Science.gov (United States)

    Shrestha, Ruben; Huang, Gaochao; Meekins, David A; Geisbrecht, Brian V; Li, Ping

    2017-09-01

    Dye-decolorizing peroxidases (DyPs) are a family of H 2 O 2 -dependent heme peroxidases, which have shown potential applications in lignin degradation and valorization. However, the DyP kinetic mechanism remains underexplored. Using structural biology and solvent isotope (sKIE) and viscosity effects, many mechanistic characteristics have been uncovered for the B-class El DyP from Enterobacter lignolyticus . Its structure revealed that a water molecule acts as the sixth axial ligand with two channels at diameters of ~3.0 and 8.0 Å leading to the heme center. A conformational change of ERS * to ERS, which have identical spectral characteristics, was proposed as the final step in DyPs' bisubstrate Ping-Pong mechanism. This step is also the rate-determining step in ABTS oxidation. The normal KIE of wild-type El DyP with D 2 O 2 at pH 3.5 suggested that cmpd 0 deprotonation by the distal aspartate is rate-limiting in the formation of cmpd I, which is more reactive under acidic pH than under neutral or alkaline pH. The viscosity effects and other biochemical methods implied that the reducing substrate binds with cmpd I instead of the free enzyme. The significant inverse sKIEs of k cat / K M and k ERS* suggested that the aquo release in DyPs is mechanistically important and may explain the enzyme's adoption of two-electron reduction for cmpd I. The distal aspartate is catalytically more important than the distal arginine and plays key roles in determining DyPs' acidic pH optimum. The kinetic mechanism of D143H- El DyP was also briefly studied. The results obtained will pave the way for future protein engineering to improve DyPs' lignolytic activity.

  20. Ablation of ferroptosis regulator glutathione peroxidase 4 in forebrain neurons promotes cognitive impairment and neurodegeneration

    Directory of Open Access Journals (Sweden)

    William Sealy Hambright

    2017-08-01

    Full Text Available Synaptic loss and neuron death are the underlying cause of neurodegenerative diseases such as Alzheimer's disease (AD; however, the modalities of cell death in those diseases remain unclear. Ferroptosis, a newly identified oxidative cell death mechanism triggered by massive lipid peroxidation, is implicated in the degeneration of neurons populations such as spinal motor neurons and midbrain neurons. Here, we investigated whether neurons in forebrain regions (cerebral cortex and hippocampus that are severely afflicted in AD patients might be vulnerable to ferroptosis. To this end, we generated Gpx4BIKO mouse, a mouse model with conditional deletion in forebrain neurons of glutathione peroxidase 4 (Gpx4, a key regulator of ferroptosis, and showed that treatment with tamoxifen led to deletion of Gpx4 primarily in forebrain neurons of adult Gpx4BIKO mice. Starting at 12 weeks after tamoxifen treatment, Gpx4BIKO mice exhibited significant deficits in spatial learning and memory function versus Control mice as determined by the Morris water maze task. Further examinations revealed that the cognitively impaired Gpx4BIKO mice exhibited hippocampal neurodegeneration. Notably, markers associated with ferroptosis, such as elevated lipid peroxidation, ERK activation and augmented neuroinflammation, were observed in Gpx4BIKO mice. We also showed that Gpx4BIKO mice fed a diet deficient in vitamin E, a lipid soluble antioxidant with anti-ferroptosis activity, had an expedited rate of hippocampal neurodegeneration and behavior dysfunction, and that treatment with a small-molecule ferroptosis inhibitor ameliorated neurodegeneration in those mice. Taken together, our results indicate that forebrain neurons are susceptible to ferroptosis, suggesting that ferroptosis may be an important neurodegenerative mechanism in diseases such as AD. Keywords: Ferroptosis, Neurodegeneration, Cognitive impairment, Alzheimer's disease, Glutathione peroxidase 4, Transgenic mice