DOCSLIB.ORG

Heme Peroxidase Activity Measured in Single Mosquitoes Identifies Individuals Expressing an Elevated Oxidase for Insecticide Resistance

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Heme Peroxidase Activity Measured in Single Mosquitoes Identifies Individuals Expressing an Elevated Oxidase for Insecticide Resistance Journal of the American Mosquito Control Association, 13(3):233-237, 1997 Copyright @ 1997 by the American Mosquito Control Association, Inc. HEME PEROXIDASE ACTIVITY MEASURED IN SINGLE MOSQUITOES IDENTIFIES INDIVIDUALS EXPRESSING AN ELEVATED OXIDASE FOR INSECTICIDE RESISTANCE WILLIAM G. BROGDON,' JANET C. McALLISTERI rNr JOHN VULULE? ABSTRACT. Optimum conditions are described for a simple, rapid, microplate-based assay that indirectly measures the differences in oxidase levels between individual susceptible, resistant, or induced mosquitoes. A small proportion (0.01-0.1) of a single mosquito is used, allowing multiple replicates of the oxidase assay. Cytochrome C is used as a positive control. The levels of oxidase found in sample populations of pyrethroid- susceptible, pyrethroid-resistant, and phenobarbital-indtced Anopheles albimanus mosquitoes are characterized with the assay. INTRODUCTION al. 1977); aldrin epoxidation to dieldrin (Yu et al. 1971, Feyereisen 1983); or aryl hydrocarbon hy- Xenobiotic detoxification in organisms is accom- droxylase (Dehnen et al. 1973). All of these meth- plished by families of enzymes that hydrolyze, ox- ods suffer from the drawback that pooled micro- idize, and conjugate injurious compounds into less somal fractions from a number of insects must be toxic, more water-soluble products that can be used; none of these methods can be used to mea- readily excreted. Among the most important detox- sure differences in oxidase levels in single mos- iflcation enzymes are the mixed-function, or P450, quitoes. oxidases (also termed oxygenases). These enzymes An indirect alternative is to measure the level of are present throughout the tissues of plants and an- heme-containing enzymes (including the cyto- imals and catalyze an enorrnous range of biochem- chrome oxidase enzymes) in single insects. The ical reactions essential to life (Agosin 1985, Hodg- levels of these enzymes should be correlated with son 1985). Our particular interest in oxidase en- the peroxidase activity of the heme group. Such a zymes involves detoxification of insecticides by technique would provide a useful means for mea- disease vectors. In biochemical detection of insec- suring large-scale differences in oxidase levels ticide resistance (Brogdon 1989), it is necessary characteristic of insecticide resistance and oxidase that enzyme levels be determined in single mos- induction, provided that the heme peroxidase levels quitoes, because populations often consist of are low but measurable in susceptible, noninduced mixtures of susceptible and resistant individuals. mosquitoes. Methods have been developed for the measurement The present study had 3 objectives. The first was of levels of esterases and glutathione s-transferases to develop a method for measuring basal levels of (Brogdon and Dickinson 1983, Brogdon and Barber heme peroxidase activity, thus, hemoprotein levels, l99oa) in single mosquitoes. A simple method for in susceptible populations of anopheline mosqui- detecting elevated levels of oxidase in single insects toes. Second, sample populations were compared has not been available. using the assay to determine if enzyme levels are There have been 2 approaches to measurement enhanced by induction or by the presence of insec- and study of oxidase activity in insects (Agosin ticide resistance. Third, reliability of the technique 1976). The first approach has been to evaluate spec- was evaluated through analysis of within-run and tral and catalytic properties of oxidases associated between-run variability of results. The means for with the membrane, using techniques such as the running a standard curve of enzyme activity and an determination of carbon monoxide inhibition of en- internal standard were also detailed. The standard zyme spectral characteristics (Omura and Sato curve was designed to show the linearity of the 1964). The second approach has been to attempt heme peroxidase assay with hemoprotein concen- the solubilization, resolution, purification and re- tration. Cytochrome C was used as an internal stan- constitution of the enzyme system. This has re- dard for each microplate. Cytochrome C was cho- quired measurement using specific substrates of sen because of its wide availability and low cost. known oxidase enzyme functions, such as: O-de- This method is intended to provide a simple means alkylation using 7-methoxy-4-methylcoumarin of quantifying oxidase activity in mixed popula- (Feyereisen and Vincent 1984), 7-ethoxycoumarin tions of resistant and susceptible mosquitoes. (Patil et al. 1990), or methoxyresorufin (Mayer et 'Entomology Branch F-22, Division of Parasitic Dis- MATERIALS AND METHODS eases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Public Health Service, Mosquitoes: Reference strains of Anopheles al- U.S. Department of Health and Human Services, Cham- bimanus Wied. used in this study are maintained at blee, GA 30341. the Centers for Disease Control and Prevention in 2 Kenya Medical Research Institute, Nairobi, Kenya. Atlanta, GA. Resistant mosquitoes were from a 233 234 JounNar- oF THE AMERTcANMosetlro CoNrRoL Assocnrrox Vor. 13,No. -? strain of An. albimanu.s from Guatemala that con_ CytochromeC tains oxidase resistance to permethrin, but not the high esterase resistance that also occurs in the 1.6 country (Brogdon and Barber 1990b). This colony is periodically selected to preserve the mechanism. 14 The association of the elevated oxidase measured in these sample populations with insecticide resis- E tance has been confirmed via a single-family isolate from the parent strain through bioassay (resistance O E ln level >100X based on dose-response), complete synergism by piperonyl butoxide, and induction ex- 3 og periments. Details of these experiments will be re- ported elsewhere. .i oo Sample preparation: Adult 3- to 4-day-old, non- bloodfed female mosquitoes were frozen and ho- mogenized in 100 pl assay buffer in plastic micro- centrifuge tubes using plastic pestles. Late 4th-in- 0.2 star larvae and pupae were transferred alive into assay buffer for homogenization. Homogenates 0.0 were diluted to I ml with additional buffer. For 0 5 10 15 20 25 30 35 bloodfed mosquitoes, the abdomen was removed before homogenization. Time (Minutes) Enzyme assays: The method for enzyme assays Fig. 1. Linearity of TMBZ substrate peroxidation for was an adaptation of that for detecting heme per- O.03 to 1.06 pg of cytochrome C. n : 8 replicates. oxidase activify on electrophoresis gels (Thomas et al. 1976) using the substrate 3,3'5,5'-tetra- methylbenzidine (TMBZ). Both the free base and (PB), an oxidase inducer, that was provided over- dihydrochloride forms of TMBZ were obtained night in a l07o sucrose solution provided to nourish from Sigma Chemical Company, St. Louis, MO. the mosquito colony. Sucrose/PB was replaced with The TMBZ working solution was prepared fresh normal lOTo sucrose the next morning to prevent daily by dissolving 50 mg TMBZ free base or mortality from PB. Mosquitoes were collected 24 h dihydrochloride in 25 ml absolute methanol and after initial exposure to PB along with mosquitoes adding 75 ml O.25 M sodium acetate buffer (pH from the same sample population that had not been 5.0; pH adjusted with acetic acid). induced. Another sample population from the To 100 pl of mosquito homogenate, 200 pl of mixed resistant colony was selected at the LDro lev- '|MBZ solution were added in assay wells, fol- el with O.O25Vopermethrin-impregnated resistance lowed by 25 1t"lof 37o hydrogen peroxide (com- test papers (World Health Organization, Geneva, mercial grade). The plate was read in a microplate- Switzerland). A sample population of the F, gen- reading spectrophotometer at test wavelengths of eration from survivors was compared for oxidase 630-690 nm at time T : 0 and at intervals there- activity with the original population values for the after, depending on the purpose of the experiment. parent strain. Mosquitoes were conveniently assayed in sample sizes of 32, since 3 replicates of 32 mosquitoes fill RESULTS a single microplate. Standard curves (8 replicates) for heme peroxi- Standard cunes: The cytochrome C standard dase were prepared using dilutions of a commercial showed linear peroxidation kinetics for at least 30 preparation of cytochrome C (Sigma Chemical Co., min at concentrations below 0.5 pg €ig. l). Above St. Louis, MO). Mosquito protein was measured on that concentration, linearity was lost after 5 min. 50-pl aliquots of assay homogenates using the Therefore, initial enzyme rates measured at T : 5 Bradford assay (Bradford 1976; Brogdon 1984a, min were used for all microassays. 1984b). This was used as a general indicator of size The complete standard curve for 0.03-1.066 ng and to test for differences in size among different of cytochrome C is accurately described by an ex- populations. ponential equation ! : aeb' (Fig. 2a). However, for Population comparisons : Individual mosquitoes practical purposes, the data may be adequately de- from 4 populations were compared. A permethrin- scribed using 2 linear equations of the form y : susceptible control population was compared with mx * b (Figs. 2b--c). The first describes the curve a resistant sffain (LD.u > 100X the control popu- from 0 to l4O ng (l : 0.99), and the second de- lation) with elevated oxidase levels. A portion of a scribes the curve from 0.150 to over I pg (l : population sample of a mixed resistant colony 0.99). (ranging from susceptible individuals to highly re- Comparison of sample populations: The control sistant) was then induced with O.lVo phenobarbital and resistant sample populations were significantly Srprnagrn 1997 Itornrcr OXIDASE AssAY 235 't6 a 0.s 't4 D SusceptibleMosquitoesI m Mixed.Popul8l.ion I 0.4 Susceptiblemd I 7tz ResistantMosquit@s l Nl Single Fmily nesistant I !? o 1n Y"'19 i F o.s .F o.z 24 0.1 0.5 0.6 0.7 0.8 0.9 1.0 1.'l 1.2 1.3 'l.4 Absorbance 629 1''' b o.oo Fig. 3. Comparison of permethrin-susceptible and -re- sistant sample populations of Anopheles albimanus.
Load more

Recommended publications
  • Protein Engineering of a Dye Decolorizing Peroxidase from Pleurotus Ostreatus for Efficient Lignocellulose Degradation
    Protein Engineering of a Dye Decolorizing Peroxidase from Pleurotus ostreatus For Efficient Lignocellulose Degradation Abdulrahman Hirab Ali Alessa A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy The University of Sheffield Faculty of Engineering Department of Chemical and Biological Engineering September 2018 ACKNOWLEDGEMENTS Firstly, I would like to express my profound gratitude to my parents, my wife, my sisters and brothers, for their continuous support and their unconditional love, without whom this would not be achieved. My thanks go to Tabuk University for sponsoring my PhD project. I would like to express my profound gratitude to Dr Wong for giving me the chance to undertake and complete my PhD project in his lab. Thank you for the continuous support and guidance throughout the past four years. I would also like to thank Dr Tee for invaluable scientific discussions and technical advices. Special thanks go to the former and current students in Wong’s research group without whom these four years would not be so special and exciting, Dr Pawel; Dr Hossam; Dr Zaki; Dr David Gonzales; Dr Inas,; Dr Yomi, Dr Miriam; Jose; Valeriane, Melvin, and Robert. ii SUMMARY Dye decolorizing peroxidases (DyPs) have received extensive attention due to their biotechnological importance and potential use in the biological treatment of lignocellulosic biomass. DyPs are haem-containing peroxidases which utilize hydrogen peroxide (H2O2) to catalyse the oxidation of a wide range of substrates. Similar to naturally occurring peroxidases, DyPs are not optimized for industrial utilization owing to their inactivation induced by excess amounts of H2O2.
    [Show full text]
  • Peroxidase (P6782)
    Peroxidase from horseradish Sigma Type VI-A Catalog Number P6782 Storage Temperature 2–8 C EC 1.11.1.7 HRP is a widely used label for immunoglobulins in CAS RN 9003-99-0 many different immunochemistry applications including Synonym: Hydrogen peroxide oxidoreductase, HRP ELISA, immunoblotting, and immunohistochemistry. HRP can be conjugated to antibodies by several Product Description different methods, including glutaraldehyde, periodate Horseradish peroxidase (HRP) is isolated from oxidation, through disulfide bonds, and also via amino horseradish roots (Amoracia rusticana) and belongs to and thiol directed cross-linkers. HRP is the most the ferroprotoporphyrin group of peroxidases. HRP desired label for antibodies, since it is the smallest and readily combines with hydrogen peroxide (H2O2), and most stable of the three most popular enzyme labels the resultant [HRP-H2O2] complex can oxidize a wide (HRP, -galactosidase, and alkaline phosphatase), and variety of hydrogen donors. its glycosylation leads to lower non-specific binding.6 A review of glutaraldehyde and periodate conjugation 7 Donor + H2O2 Oxidized Donor + 2 H2O methods has been published. Peroxidase will oxidize a variety of substrates (see Peroxidase is also utilized for the determination of Table 2): chromogenic, chemiluminescent (luminol and glucose8 and peroxides9 in solution. isoluminol), and fluorogenic (tyramine, homovanillic acid, and 4-hydroxyphenyl acetic acid). This product is supplied as an essentially salt free, lyophilized powder. HRP is a single chain polypeptide containing four disulfide bridges. It is a glycoprotein containing 18% Specific Activity: carbohydrate. The carbohydrate composition consists 250 units/mg solid (pyrogallol as substrate) of galactose, arabinose, xylose, fucose, mannose, 950–2,000 units/mg solid (ABTS as substrate) mannosamine, and galactosamine, depending upon the Note: Using 2,2-Azino-bis(3-ethylbenzthiazoline- 1 specific isozyme.
    [Show full text]
  • The Role of Peroxiredoxin 6 in Cell Signaling
    antioxidants Review The Role of Peroxiredoxin 6 in Cell Signaling José A. Arevalo and José Pablo Vázquez-Medina * Department of Integrative Biology, University of California, Berkeley, CA, 94705, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-510-664-5063 Received: 7 November 2018; Accepted: 20 November 2018; Published: 24 November 2018 Abstract: Peroxiredoxin 6 (Prdx6, 1-cys peroxiredoxin) is a unique member of the peroxiredoxin family that, in contrast to other mammalian peroxiredoxins, lacks a resolving cysteine and uses glutathione and π glutathione S-transferase to complete its catalytic cycle. Prdx6 is also the only peroxiredoxin capable of reducing phospholipid hydroperoxides through its glutathione peroxidase (Gpx) activity. In addition to its peroxidase activity, Prdx6 expresses acidic calcium-independent phospholipase A2 (aiPLA2) and lysophosphatidylcholine acyl transferase (LPCAT) activities in separate catalytic sites. Prdx6 plays crucial roles in lung phospholipid metabolism, lipid peroxidation repair, and inflammatory signaling. Here, we review how the distinct activities of Prdx6 are regulated during physiological and pathological conditions, in addition to the role of Prdx6 in cellular signaling and disease. Keywords: glutathione peroxidase; phospholipase A2; inflammation; lipid peroxidation; NADPH (nicotinamide adenine dinucleotide phosphate) oxidase; phospholipid hydroperoxide 1. Introduction Peroxiredoxins are a ubiquitous family of highly conserved enzymes that share a catalytic mechanism in which a redox-active (peroxidatic) cysteine residue in the active site is oxidized by a peroxide [1]. In peroxiredoxins 1–5 (2-cys peroxiredoxins), the resulting sulfenic acid then reacts with another (resolving) cysteine residue, forming a disulfide that is subsequently reduced by an appropriate electron donor to complete a catalytic cycle [2,3].
    [Show full text]
  • GRAS Notice 665, Lactoperoxidase System
    GRAS Notice (GRN) No. 665 http://www.fda.gov/Food/IngredientsPackagingLabeling/GRAS/NoticeInventory/default.htm ORIGINAL SUBMISSION 000001 Mo•·gan Lewis Gf<N Ob()&h5 [R1~~~~~~[Q) Gary L. Yingling Senior Counsel JUL 1 8 2016 + 1.202. 739 .5610 gary.yingling@morganlewis .com OFFICE OF FOO~ ADDITIVE SAFETY July 15, 2016 VIA FEDERAL EXPRESS Dr. Antonia Mattia Director Division of Biotechnology and GRAS Notice Review Office of Food Additive Safety (HFS-200) Center for Food Safety and Applied Nutrition Food and Drug Administration 5100 Paint Branch Parkway College Park, MD 20740-3835 Re: GRAS Notification for the Lactoperoxidase System Dear Dr. Mattia: On behalf of Taradon Laboratory C'Taradon"), we are submitting under cover of this letter three paper copies and one eCopy of DSM's generally recognized as safe ("GRAS'') notification for its lactoperoxidase system (''LPS''). The electronic copy is provided on a virus-free CD, and is an exact copy of the paper submission. Taradon has determined through scientific procedures that its lactoperoxidase system preparation is GRAS for use as a microbial control adjunct to standard dairy processing procedures such as maintaining appropriate temperatures, pasteurization, or other antimicrobial treatments to extend the shelf life of the products. In many parts of the world, the LPS has been used to protect dairy products, particularly in remote areas where farmers are not in close proximity to the market. In the US, the LPS is intended to be used as a processing aid to extend the shelf life of avariety of dairy products, specifically fresh cheese including mozzarella and cottage cheeses, frozen dairy desserts, fermented milk, flavored milk drinks, and yogurt.
    [Show full text]
  • Investigation of Glutathione Peroxidase Activity in Chicken Meat
    Ciência e Tecnologia de Alimentos ISSN 0101-2061 Investigation of glutathione peroxidase activity in chicken meat under different experimental conditions Original Investigação da atividade de glutationa peroxidase em carne de frango submetida a diferentes condições experimentais Alexandre José CICHOSKI1*, Renata Bezerra ROTTA2, Gerson SCHEUERMANN3, Anildo CUNHA JUNIOR3, Juliano Smanioto BARIN1 Abstract Due to the fact that previous studies on the enzymatic activity of Glutathione peroxidase (GSH-Px) diverge widely in their methodology and results, this study aimed to investigate the influence of different analytical conditions on GSH-Px activity in chicken thighs from broilers that were fed different diets with different sources and concentrations of selenium. GSH-Px activity was evaluated six hours after slaughter and 120 days after frozen storage at –18 °C. The different analytical conditions included time of pre-incubation (0, 10 and 30 minutes), reaction medium, types of substrate (H2O2 (0.72 mM, 7.2 mM, and 72 mM) and Terc-butil hydroperoxide 15 mM), and different buffer concentrations (buffer 1, potassium phosphate 50 mM pH 7.0 + EDTA 1 mM + mercaptoethanol 1 mM, and buffer 2, tris-HCl 50 mM pH 7.6 + EDTA 1 mM + mercapthanol 5 mM). The results show that the highest GSH-Px activity was observed when enzyme and substrate were in contact at 22 °C without any pre-incubation, and that, when used at concentrations above 0.72 mM, hydrogen peroxide saturated the GSH-Px enzyme and inhibited its activity. The enzyme presented higher affinity to hydrogen peroxide when compared to terc-butil peroxide, and the addition of a buffer containing mercaptoethanol did not increase GSH-Px enzymatic activity.
    [Show full text]
  • Translational Regulation of Glutathione Peroxidase 4 Expression Through Guanine- Rich Sequence-Binding Factor 1 Is Essential for Embryonic Brain Development
    Downloaded from genesdev.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press Translational regulation of glutathione peroxidase 4 expression through guanine- rich sequence-binding factor 1 is essential for embryonic brain development Christoph Ufer,1,2,6 Chi Chiu Wang,3,4,6 Michael Fähling,5 Heike Schiebel,1 Bernd J. Thiele,5 E. Ellen Billett,2 Hartmut Kuhn,1,7 and Astrid Borchert1 1Institute of Biochemistry, University Medicine Berlin–Charité, D-10117 Berlin, F.R. Germany; 2School of Science and Technology, Nottingham Trent University, Nottingham NG11 8NS, United Kingdom; 3Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong; 4Department of Obstetrics and Gynaecology, The Chinese University of Hong Kong, Shatin, Hong Kong; 5Institute of Physiology, University Medicine Berlin–Charité, D-10117 Berlin, F.R. Germany Phospholipid hydroperoxide glutathione peroxidase (GPx4) is a moonlighting selenoprotein, which has been implicated in basic cell functions such as anti-oxidative defense, apoptosis, and gene expression regulation. GPx4-null mice die in utero at midgestation, and developmental retardation of the brain appears to play a major role. We investigated post-transcriptional mechanisms of GPx4 expression regulation and found that the guanine-rich sequence-binding factor 1 (Grsf1) up-regulates GPx4 expression. Grsf1 binds (to a defined target sequence in the 5؅-untranslated region (UTR) of the mitochondrial GPx4 (m-GPx4 mRNA, up-regulates UTR-dependent reporter gene expression, recruits m-GPx4 mRNA to translationally active polysome fractions, and coimmunoprecipitates with GPx4 mRNA. During embryonic brain development, Grsf1 and m-GPx4 are coexpressed, and functional knockdown (siRNA) of Grsf1 prevents embryonic GPx4 expression.
    [Show full text]
  • Peroxidase Activity of De Novo Heme Proteins Immobilized on Electrodes
    Available online at www.sciencedirect.com InorganicJOURNAL OF Biochemistry Journal of Inorganic Biochemistry 101 (2007) 1820–1826 www.elsevier.com/locate/jinorgbio Peroxidase activity of de novo heme proteins immobilized on electrodes Aditi Das 1, Michael H. Hecht * Department of Chemistry, Princeton University, Princeton NJ 08544-1009, United States Received 26 June 2007; received in revised form 12 July 2007; accepted 13 July 2007 Available online 27 July 2007 This article is dedicated to the memory of Ed Stiefel. As his student and his colleague, the authors were educated by his intellect and inspired by his exuberance. Abstract De novo proteins from designed combinatorial libraries were bound to heme terminated gold electrodes. The novel heme proteins were shown to possess peroxidase activity, and this activity was compared to that of horseradish peroxidase and bovine serum albumin when immobilized in a similar fashion. The various designed proteins from the libraries displayed distinctly different levels of peroxidase activ- ity, thereby demonstrating that the sequence and structure of a designed protein can exert a substantial effect on the peroxidase activity of immobilized heme. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Protein design; Binary patterning; Cyclic voltammetry; Redox protein; Heme protein; Peroxidase activity; Horseradish peroxidase; Chronocoulometry 1. Introduction bilized on a heme modified electrode is compared to HRP immobilized in a similar fashion. The design of novel proteins that fold into native-like The de novo proteins described in the current study were structures capable of molecular recognition and catalytic chosen from a library of binary patterned sequences activity is emerging as an important goal of protein chem- designed to fold into four-helix bundles [15–17].
    [Show full text]
  • Catalysis of Oxygen Reduction by Catalase and HRP on Glassy
    Catalysis of Oxygen Reduction by Catalase and HRP according to the thermodynamic data available in the on Glassy Carbon Electrodes : Comparison of the literature. To our knowledge, such a catalysis of oxygen Mechanisms reduction by catalase [2] and HRP [3] represent Alain BERGEL, Maria Elena LAI innovative results, which may give some pertinent Laboratoire de Génie Chimique - CNRS contribution to the explanation of aerobic biocorrosion Université Paul Sabatier, 118 route de Narbonne, under marine biofilms [4]. 31062 Toulouse, France 2 H+ Catalase and horseradish peroxidase (HRP) are both O2 1/2 O2 H2O hemic enzymes containing Fe(III)-protoporphyrin as 2 e prosthetic group. Both enzymes usually work with hydrogen peroxide as substrate; catalase catalyses the 2 H+ H O Cat disproportionation of hydrogen peroxide into oxygen and 2 2 2 e water: 2 H2O2 O2 + 2 H2O and HRP catalyses the oxidation of numerous substrates 2 H2O Scheme I (noted SH2) by hydrogen peroxide, following the general reaction : 2 SH2 + H2O2 2 SH° + 2 H2O O2 4 H+ 4 e The glassy carbon electrodes (GCE) were used either Cat Scheme II clean, or after a preliminary electrochemical treatment. -1 Current-potential curves were plotted at 20 mVs from 2 H2O +0.10 to –1.10 V/SCE with catalase and HRP either dissolved in solution (0.15M NaCl - 0.1M potassium Fig. 1. Proposed mechanisms of the catalase-catalyzed phosphate buffer pH 8.0), or adsorbed on the GCE electrochemical reduction of oxygen. surface. Adsorption was achieved thanks a 15-minute immersion of the electrode in a DMSO solution that ! ¤ contained the enzyme.
    [Show full text]
  • Significance of Peroxidase in Eosinophils
    SIGNIFICANCE OF PEROXIDASE IN EOSINOPHILS M a rg a ret A . K e lsa ll Peroxidase-bearing granules are the primary component and product of eosino­ phils. The physiological significance of eosinophils is, therefore, considered to be related to the ability of this cell to synthesize, store, and transport peroxidase and to release the peroxidase-positive granules into body fluids by a lytic process that is controlled by hormones, by variations in the histamine-epinephrine balance, and by several other stimuli. Peroxidase occurs not only in eosinophils, but also in neutrophils and blood platelets; but it is not present in most cells of animal tissues. The purpose of this work is to consider, as a working hypothesis, that the function of eosinophils is to produce, store, and transport peroxidase to catalyze oxidations. Many of the aerobic dehydrogenases that catalyze reactions in which hydrogen peroxide is produced are involved in protein catabolism. Therefore, relations between eosinophils and several normal and pathological conditions of increased protein catabolism are emphasized, and also the significance of peroxi­ dase in eosinophils and other leukocytes to H 20 2 produced by irradiation is considered. The functions of eosinophils and peroxidases are difficult to establish experi­ mentally, because other leukocytes and platelets contain peroxidase, and because several peroxidatic reactions can be catalyzed by hemoglobin and cytochromes (335), and because catalase, which decomposes H 20 2, is present in erythrocytes and other cells, thereby preventing accumulation of H20 2 (616). In addition, catalase can act as a peroxidase in oxidation of alcohols (247) and large amounts of catalase cause purely oxidative effects, in contrast to the predominance of peroxidase activity when only small amounts of catalase are present (558).
    [Show full text]
  • Molecular Biology of Glutathione Peroxidase 4: from Genomic Structure to Developmental Expression and Neural Function
    View metadata, citation and similar papers at core.ac.ukArticle in press - uncorrected proof brought to you by CORE provided by RERO DOC Digital Library Biol. Chem., Vol. 388, pp. 1007–1017, October 2007 • Copyright ᮊ by Walter de Gruyter • Berlin • New York. DOI 10.1515/BC.2007.126 Review Molecular biology of glutathione peroxidase 4: from genomic structure to developmental expression and neural function Nicolai E. Savaskan1,2,*, Christoph Ufer3, Introduction Hartmut Ku¨hn3 and Astrid Borchert3 Molecular biology and bioinformatics have recently 1 Division of Cellular Biochemistry, The Netherlands revealed at least 25 selenoproteins, five of which are glu- Cancer Institute, NL-1066 CX Amsterdam, tathione peroxidases (GPxs) (Kryukov et al., 2003; Utomo The Netherlands et al., 2004). All selenoproteins with known enzymatic 2 Brain Research Institute, Department of activity bear a selenocysteine at their active center that Neuromorphology, ETH and University of Zurich, is essential for catalytic activity and cannot simply be CH-8057 Zurich, Switzerland replaced by a cysteine residue. The first mammalian sele- 3 Institute of Biochemistry, University Medicine Berlin – noprotein was identified by Mills (1957) as the 85-kDa Charite´ , D-10117 Berlin, Germany classical GPx or GPx1. Subsequently, additional GPx iso- * Corresponding author zymes were uncovered and today the GPx family com- e-mail: [email protected]; [email protected] prises seven well-characterized members, all of which except two (GPx5 in humans and mice and GPx7) con- tain a selenocysteine as the catalytically active amino acid. The presence of selenocysteine at the active site confers a more rapid reaction with peroxide substrates, which make peroxidase catalysis much more efficient Abstract (Nauser et al., 2006).
    [Show full text]
  • Expression and Characterization of a Dye-Decolorizing Peroxidase from Pseudomonas Fluorescens Pf0-1
    catalysts Article Expression and Characterization of a Dye-Decolorizing Peroxidase from Pseudomonas Fluorescens Pf0-1 1,2, 3 4 2, 3 Nikola Lonˇcar *, Natalija Draškovi´c , Nataša Boži´c , Elvira Romero y, Stefan Simi´c , Igor Opsenica 3, Zoran Vujˇci´c 3 and Marco W. Fraaije 2 1 GECCO Biotech, Nijenborgh 4, 9747AG Groningen, The Netherlands 2 Molecular Enzymology group, University of Groningen, Nijenborgh 4, 9747AG Groningen, The Netherlands; [email protected] (E.R.); [email protected] (M.W.F.) 3 Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia; [email protected] (N.D.); [email protected] (S.S.); [email protected] (I.O.); [email protected] (Z.V.) 4 ICTM-Center of Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia; [email protected] * Correspondence: [email protected] Current address: AstraZeneca R&D Gothenburg, Discovery Sciences, S-431 83 Mölndal, Sweden. y Received: 7 April 2019; Accepted: 16 May 2019; Published: 20 May 2019 Abstract: The consumption of dyes is increasing worldwide in line with the increase of population and demand for clothes and other colored products. However, the efficiency of dyeing processes is still poor and results in large amounts of colored effluents. It is desired to develop a portfolio of enzymes which can be used for the treatment of colored wastewaters. Herein, we used genome sequence information to discover a dye-decolorizing peroxidase (DyP) from Pseudomonas fluorescens Pf-01. Two genes putatively encoding for DyPs were identified in the respective genome and cloned for expression in Escherichia coli, of which one (Pf DyP B2) could be overexpressed as a soluble protein.
    [Show full text]
  • Peroxidase in Pueraria Montana 1
    PEROXIDASE IN PUERARIA MONTANA 1 The Discovery and Analysis of Peroxidase Enzyme in Pueraria montana Kathryn Briley A Senior Thesis submitted in partial fulfillment of the requirements for graduation in the Honors Program Liberty University Spring 2021 PEROXIDASE IN PUERARIA MONTANA 2 Acceptance of Senior Honors Thesis This Senior Honors Thesis is accepted in partial fulfillment of the requirements for graduation from the Honors Program of Liberty University. ______________________________ Gregory M. Raner, Ph.D. Thesis Chair ______________________________ Michael Bender, Ph.D. Committee Member ______________________________ David Schweitzer, Ph.D. Assistant Honors Director ______________________________ Date PEROXIDASE IN PUERARIA MONTANA 3 Abstract Peroxidase enzymes are used for a variety of industrial and biotechnological applications because of their ease of purification, broad range of chemical activities, and low cost of use. Identification of quality peroxidase sources that are convenient for enzymatic isolation and give way to high yields of product is a desirable pursuit in biochemical research. Kudzu is an excellent candidate for this pursuit as it displays high catalytic activity in screening assays and is found in abundance. This project seeks to determine the enzyme stability, optimal pH conditions, and possible novel chemical activities of peroxidase isolated from kudzu leaves. The methods for this project include standard protein purification protocols, analytical techniques for evaluation of chemical activities, and purity of the resulting preparations. Keywords: peroxidase, kudzu, enzyme, biochemical research, chemical activities PEROXIDASE IN PUERARIA MONTANA 4 The Discovery and Analysis of Peroxidase Enzyme in Pueraria montana Background Peroxidases are a group of enzymes belonging to the catalase family that are able to oxidize organic compounds using hydrogen peroxide in order to generate chemical reactions.
    [Show full text]