DOCSLIB.ORG

Information to Users

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Information to Users Functional characterization of hepatic microsomal and heterologousely expressed rabbit cytochrome P450 2B enzymes Item Type text; Dissertation-Reproduction (electronic) Authors Grimm, Scott Wayne. Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 06/10/2021 12:38:56 Link to Item http://hdl.handle.net/10150/186952 INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely. event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. UMJ[ A Bell & Howell Informallon Company 300 North Zeeb Road. Ann Arbor. M148106·1346 USA 313.'761·4700 800:521·0600 Order Number 9517564 Functional characterization of hepatic microsomal and heterologous ely expressed rabbit cytochrome P450 2B enzymes Grimm, Scott Wayne, Ph.D. The University of Arizona, 1994 U·M·K 300 N. Zeeb Rd. Ann Arbor, MI 48106 FUNCTIONAL CHARACTERIZATION OF HEPATIC MICROSOMAL AND HETEROLOGOUSLY EXPRESSED RABBIT CYTOCHROME P450 2B ENZYMES by Scott Wayne Grimm A Dissertation Submitted to the Faculty of the COMMITTEE ON PHARMACOLOGY AND TOXICOLOGY (GRADUATE) In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY In the Graduate College THE UNIVERSITY OF ARIZONA 1994 2 THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE As members of the Final Examination Committee, we certify that we have read the dissertation prepared bY~S~c~o~t~t~W~ayYun~e~G~r_jwmwm~ _____________________ entitled FUNCTIONAl CHARACTERIZATION OF HEPATIC MICROSOMAl AND HETEROLOGOUSLY EXPRESSED RABBITT CYTOCHROME P4S0 2B EXZYMES and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy ----------------~~~-------------------- Date l II f/ /4'1 Date /I~//c;-~ Date ; I/,/:IAI/ Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copy of the dissertation to the Graduate College. I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement. JIV)veJ) )2, ) I e"'1)u>b' Date James R. Halpert 3 STATEMENT BY AUTHOR This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under the rules of the Library. Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgement of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgement the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author. 4 ACKNOWLEDGMENTS My sincere gratitude is due to Dr. James Halpert for willingly taking me under his direction as my research advisor, for challenging me, encouraging me, and mentoring me. I would also like to thank the other members of my advisory committee, Dr. Martin Dyroff, Dr. Daniel Liebler, Dr. Hugh E. Laird II, and Dr. Josephine Lai for encouraging me and helping me keep a straight course in the somewhat unorthodox situation of performing much of my research 2300 miles away from the University of Arizona. I thank ZENECA Pharmaceuticals for financial support for my family during my leave of absence to the University of Arizona in 1991. A special thanks is due to Dr. Martin Dyroff and Dr. Chris Rhodes at ZENECA Pharmaceuticals for supporting my ambitions to return to graduate school, for winning the approval of the upper management at ZENECA to pursue this goal, and generously giving me time to perform some of my research during work hours. I gratefully acknowledge the time spent by Dr. Karen Kedzie and Dr. Jeff Stevens to teach me new techniques in molecular biology and cytochrome P450 purification and analysis. I also acknowledge Dr. Richard Philpot and Dr. John Bend for supplying me with some of the materials to conduct this research and for their helpful advice during these investigations and during the preparation of the manuscripts for publication. I sincerely appreciate the prayers, encouragement, and support of my parents, family, and friends during my stressed and tired moments. 5 DEDICATION This dissertation is dedicated to my wife and children. To Kathy, who supported me with her love and patience and unselfishly dedicated the last 4Y2 years of her life to raising our two young sons, Evan and Jeremy. I also dedicate this to Evan and Jeremy, who unknowingly gave up much of our precious time together during their early years. 6 TABLE OF CONTENTS LIST OF FIGURES ......................................................................................................... 9 LIST OF TABLES ........................................................................................................... 10 ABSTRACT .................................................................................................................... 11 1. INTRODUCTION ...................................................................................................... 12 Xenobiotic Metabolism ............................................................................................ 12 The Cytochrome P450 Dependent Monooxygenase System ................................... 13 The Molecular Biology of Cytochromes P450 ........................................................ 14 Cytochrome P450 Nomenclature ..................................................................... 15 Molecular Aspects of Cytochrome P450 Regulation and Function ................. 16 Structure-Function Relationships of Cytochromes P450 ......................................... 17 Characterizing Substrate Specificities of Cytochromes P450 .................................. 19 Heterologous Expression of Cytochromes P450 ............................................ 20 Cytochrome P450 Inhibition ............................................................................ 21 Mechanisms of Cytochrome P450 Inhibition .......................................... 21 Inhibitors as Tools to Study the Function of Individual Cytochrome P450 Forms .............................................................................................. 23 Cytochrome P450 Induction .................................................................................... 24 Polymorphic Expression of Cytochrome P450 Genes ............................................. 26 Microheterogeneity in the Cytochrome P450 2B Subfamily ................................... 28 Cytochromes P450 2B in Rats ......................................................................... 29 Cytochromes P450 2B in Rabbits .................................................................... 30 Research Problem Addressed in This Dissertion ..................................................... 33 Significance and Implications .................................................................................. 33 Organization of Dissertation .................................................................................... 35 2. FUNCTIONAL COMPARISONS OF PURIFIED AND HETEROLOGOUSL Y EXPRESSED RABBIT CYTOCHROMES P450 2B4, 2B-BX, 2B-B 1, AND 2B5 ......................................................................................... 36 Background .............................................................................................................. 36 Materials and Methods ......................... " .................................................................. 38 Materials .......................................................................................................... 38 Animals, Treatments, and Microsome Preparation ......................................... 39 Strains and Media .......... , ................................................................................. 39 COS-7 Cell Expression Plasmids ..................................................................
Load more

Recommended publications
  • Lanosterol 14Α-Demethylase (CYP51)
    463 Lanosterol 14-demethylase (CYP51), NADPH–cytochrome P450 reductase and squalene synthase in spermatogenesis: late spermatids of the rat express proteins needed to synthesize follicular fluid meiosis activating sterol G Majdicˇ, M Parvinen1, A Bellamine2, H J Harwood Jr3, WWKu3, M R Waterman2 and D Rozman4 Veterinary Faculty, Clinic of Reproduction, Cesta v Mestni log 47a, 1000 Ljubljana, Slovenia 1Institute of Biomedicine, Department of Anatomy, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland 2Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232–0146, USA 3Pfizer Central Research, Department of Metabolic Diseases, Box No. 0438, Eastern Point Road, Groton, Connecticut 06340, USA 4Institute of Biochemistry, Medical Center for Molecular Biology, Medical Faculty University of Ljubljana, Vrazov trg 2, SI-1000 Ljubljana, Slovenia (Requests for offprints should be addressed to D Rozman; Email: [email protected]) (G Majdicˇ is now at Department of Internal Medicine, UT Southwestern Medical Center, Dallas, Texas 75235–8857, USA) Abstract Lanosterol 14-demethylase (CYP51) is a cytochrome detected in step 3–19 spermatids, with large amounts in P450 enzyme involved primarily in cholesterol biosynthe- the cytoplasm/residual bodies of step 19 spermatids, where sis. CYP51 in the presence of NADPH–cytochrome P450 P450 reductase was also observed. Squalene synthase was reductase converts lanosterol to follicular fluid meiosis immunodetected in step 2–15 spermatids of the rat, activating sterol (FF-MAS), an intermediate of cholesterol indicating that squalene synthase and CYP51 proteins are biosynthesis which accumulates in gonads and has an not equally expressed in same stages of spermatogenesis. additional function as oocyte meiosis-activating substance.
    [Show full text]
  • Mrna Vaccine: a Potential Therapeutic Strategy Yang Wang† , Ziqi Zhang† , Jingwen Luo† , Xuejiao Han† , Yuquan Wei and Xiawei Wei*
    Wang et al. Molecular Cancer (2021) 20:33 https://doi.org/10.1186/s12943-021-01311-z REVIEW Open Access mRNA vaccine: a potential therapeutic strategy Yang Wang† , Ziqi Zhang† , Jingwen Luo† , Xuejiao Han† , Yuquan Wei and Xiawei Wei* Abstract mRNA vaccines have tremendous potential to fight against cancer and viral diseases due to superiorities in safety, efficacy and industrial production. In recent decades, we have witnessed the development of different kinds of mRNAs by sequence optimization to overcome the disadvantage of excessive mRNA immunogenicity, instability and inefficiency. Based on the immunological study, mRNA vaccines are coupled with immunologic adjuvant and various delivery strategies. Except for sequence optimization, the assistance of mRNA-delivering strategies is another method to stabilize mRNAs and improve their efficacy. The understanding of increasing the antigen reactiveness gains insight into mRNA-induced innate immunity and adaptive immunity without antibody-dependent enhancement activity. Therefore, to address the problem, scientists further exploited carrier-based mRNA vaccines (lipid-based delivery, polymer-based delivery, peptide-based delivery, virus-like replicon particle and cationic nanoemulsion), naked mRNA vaccines and dendritic cells-based mRNA vaccines. The article will discuss the molecular biology of mRNA vaccines and underlying anti-virus and anti-tumor mechanisms, with an introduction of their immunological phenomena, delivery strategies, their importance on Corona Virus Disease 2019 (COVID-19) and related clinical trials against cancer and viral diseases. Finally, we will discuss the challenge of mRNA vaccines against bacterial and parasitic diseases. Keywords: mRNA vaccine, Self-amplifying RNA, Non-replicating mRNA, Modification, Immunogenicity, Delivery strategy, COVID-19 mRNA vaccine, Clinical trials, Antibody-dependent enhancement, Dendritic cell targeting Introduction scientists are seeking to develop effective cancer vac- A vaccine stimulates the immune response of the body’s cines.
    [Show full text]
  • Characterization of Methylene Diphenyl Diisocyanate Protein Conjugates
    Portland State University PDXScholar Dissertations and Theses Dissertations and Theses Spring 6-5-2014 Characterization of Methylene Diphenyl Diisocyanate Protein Conjugates Morgen Mhike Portland State University Follow this and additional works at: https://pdxscholar.library.pdx.edu/open_access_etds Part of the Allergy and Immunology Commons, and the Chemistry Commons Let us know how access to this document benefits ou.y Recommended Citation Mhike, Morgen, "Characterization of Methylene Diphenyl Diisocyanate Protein Conjugates" (2014). Dissertations and Theses. Paper 1844. https://doi.org/10.15760/etd.1843 This Dissertation is brought to you for free and open access. It has been accepted for inclusion in Dissertations and Theses by an authorized administrator of PDXScholar. Please contact us if we can make this document more accessible: [email protected]. Characterization of Methylene Diphenyl Diisocyanate Protein Conjugates by Morgen Mhike A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry Dissertation Committee: Reuben H. Simoyi, Chair Paul D. Siegel Itai Chipinda Niles Lehman Shankar B. Rananavare Robert Strongin E. Kofi Agorsah Portland State University 2014 © 2014 Morgen Mhike ABSTRACT Diisocyanates (dNCO) such as methylene diphenyl diisocyanate (MDI) are used primarily as cross-linking agents in the production of polyurethane products such as paints, elastomers, coatings and adhesives, and are the most frequently reported cause of chemically induced immunologic sensitization and occupational asthma (OA). Immune mediated hypersensitivity reactions to dNCOs include allergic rhinitis, asthma, hypersensitivity pneumonitis and allergic contact dermatitis. There is currently no simple diagnosis for the identification of dNCO asthma due to the variability of symptoms and uncertainty regarding the underlying mechanisms.
    [Show full text]
  • Cytochrome Oxidase (A3) Heme and Copper Observed by Low- Temperature Fourier Transform Infrared Spectroscopy Ofthe CO Complex (Photolysis/Mitoehondria/Beef Heart) J
    Proc. Natl Acad. Sci. USA Vol. 78, No. 1, pp. 234-237, January 1981 Biochemistry Cytochrome oxidase (a3) heme and copper observed by low- temperature Fourier transform infrared spectroscopy ofthe CO complex (photolysis/mitoehondria/beef heart) J. 0. ALBEN, P. P. MOH, F. G. FIAMINGO, AND R. A. ALTSCHULD Department ofPhysiological Chemistry, The Ohio State University, Columbus, Ohio 43210 Communicated by Hans Frauenfelder, October 10, 1980 ABSTRACT Carbon monoxide bound to iron or copper in sub- chondria is bound reversibly to the a3 copper would appear to strate-reduced mitochondrial cytochrome c oxidase (ferrocyto- explain these results. Here, we extend the observations to lower chrome c:oxygen oxidoreductase, EC 1.9.3.1) from beefheart has beenused toexplore the structural interaction ofthea3 heme-copper temperatures, illustrate the structural differences between pocket at.15 K and 80 K in the dark and in the presence ofvisible heme and copper CO complexes in cytochrome a3, and show light. The vibrational absorptions of CO measured by a Fourier how this may be related to the functional contributions ofthese transform infrared interferometer occur in the dark at 1963 cm-' metal centers. with small absorptions near 1952 cm-', and aredue toa3 heme-CO complexes. These disappear in strong visible light and are re- placed by a major absorption at 2062 cm' and a minor one at 2043 MATERIALS AND METHODS cm' due to CU-CO. Relaxation in the dark is rapid and quantita- tive at 210 K, but becomes negligible below 140 K. The multiple Mitochondria, prepared from fresh beef heart by use of Nagarse absorptions indicate structural heterogeneity of.cytochrome oxi- (8), were kindly donated by G.
    [Show full text]
  • 17F8-Estradiol Hydroxylation Catalyzed by Human Cytochrome P450 Lbl (Catechol Estrogen/Indole Carbinol/Dioxin/Breast Cancer/Uterine Cancer) CARRIE L
    Proc. Natl. Acad. Sci. USA Vol. 93, pp. 9776-9781, September 1996 Medical Sciences 17f8-Estradiol hydroxylation catalyzed by human cytochrome P450 lBl (catechol estrogen/indole carbinol/dioxin/breast cancer/uterine cancer) CARRIE L. HAYES*, DAVID C. SPINKt, BARBARA C. SPINKt, JOAN Q. CAOt, NIGEL J. WALKER*, AND THOMAS R. SUTTER*t *Department of Environmental Health Sciences, Johns Hopkins University, School of Hygiene and Public Health, Baltimore, MD 21205-2179; and tWadsworth Center, New York State Department of Health, Albany, NY 12201-0509 Communicated by Paul Talalay, Johns Hopkins University, Baltimore, MD, June 11, 1996 (received for review April 24, 1996) ABSTRACT The 4-hydroxy metabolite of 178-estradiol of 4-hydroxyestradiol (4-OHE2), and lack of activity of 2-hy- (E2) has been implicated in the carcinogenicity of this hor- droxyestradiol (2-OHE2), (17-19), implicate the 4-hydroxy- mone. Previous studies showed that aryl hydrocarbon- lated metabolites in estrogen-induced carcinogenesis. Perti- receptor agonists induced a cytochrome P450 that catalyzed nent to elucidating the contribution of 4-OHE2 to the devel- the 4-hydroxylation of E2. This activity was associated with opment of human cancer is the identification of the enzyme(s) human P450 lBi. To determine the relationship of the human that produce this metabolite. P450 lBl gene product and E2 4-hydroxylation, the protein Previous studies demonstrated that treatment of MCF-7 was expressed in Saccharomyces cerevisiae. Microsomes from breast cancer cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin the transformed yeast catalyzed the 4- and 2-hydroxylation of (TCDD), an environmental pollutant and potent agonist of the E2 with Km values of 0.71 and 0.78 ,uM and turnover numbers aryl hydrocarbon (Ah)-receptor, resulted in greater than 10- of 1.39 and 0.27 nmol product min'l nmol P450-1, respec- fold increases in the rates of E2 4- and 2-hydroxylation (20).
    [Show full text]
  • Current Trends in Cancer Immunotherapy
    biomedicines Review Current Trends in Cancer Immunotherapy Ivan Y. Filin 1 , Valeriya V. Solovyeva 1 , Kristina V. Kitaeva 1, Catrin S. Rutland 2 and Albert A. Rizvanov 1,3,* 1 Institute of Fundamental Medicine and Biology, Kazan Federal University, 420008 Kazan, Russia; [email protected] (I.Y.F.); [email protected] (V.V.S.); [email protected] (K.V.K.) 2 Faculty of Medicine and Health Science, University of Nottingham, Nottingham NG7 2QL, UK; [email protected] 3 Republic Clinical Hospital, 420064 Kazan, Russia * Correspondence: [email protected]; Tel.: +7-905-316-7599 Received: 9 November 2020; Accepted: 16 December 2020; Published: 17 December 2020 Abstract: The search for an effective drug to treat oncological diseases, which have become the main scourge of mankind, has generated a lot of methods for studying this affliction. It has also become a serious challenge for scientists and clinicians who have needed to invent new ways of overcoming the problems encountered during treatments, and have also made important discoveries pertaining to fundamental issues relating to the emergence and development of malignant neoplasms. Understanding the basics of the human immune system interactions with tumor cells has enabled new cancer immunotherapy strategies. The initial successes observed in immunotherapy led to new methods of treating cancer and attracted the attention of the scientific and clinical communities due to the prospects of these methods. Nevertheless, there are still many problems that prevent immunotherapy from calling itself an effective drug in the fight against malignant neoplasms. This review examines the current state of affairs for each immunotherapy method, the effectiveness of the strategies under study, as well as possible ways to overcome the problems that have arisen and increase their therapeutic potentials.
    [Show full text]
  • Molecular Studies of Hemocyanin Expression in the Dungeness
    MOLECULAR STUDIES OF HEMOCYANIN EXPRESSION IN THE DUNGENESS CRAB by GREGOR DURSTEWITZ A DISSERTATION Presented to the Department of Biology and the Graduate School of the University of Oregon in partial fulfillment of the requirements for the degree of Doctor of Philosophy June 1996 II "Molecular Studies of Hemocyanin Expression in the Dungeness Crab," a dissertation prepared by Gregor Durstewitz in partial fulfillment of the requirements for the Doctor of Philosophy degree in the Department of Biology. This dissertation has been approved and accepted by: Dr. Nora B. Terwilliger, Chair of the Examining Committee Date committee in charge: Dr. Nora B. Terwilliger, Chair Dr. Roderick capaldi Dr. Ry Meeks-Wagner Eric Schabtach Dr. Kensal van Holde Dr. Tom Stevens Accepted by: Vice Provost and Dean of the Graduate School j i I 1 J iii c 1996 Gregor Durstewitz iv An Abstract of the Dissertation of Gregor Durstewitz for the degree of Doctor of Philosophy in the Department of Biology to be taken June 1996 Title: MOLECULAR STUDIES OF HEMOCYANIN EXPRESSION IN THE DUNGENESS CRAB Approved: Dr. Nora B. Terwilliger This study investigates developmentally regulated changes in the expression of the copper based respiratory protein hemocyanin (Hc) in the Dungeness crab (Cancer magister). Hc gene expression was studied by Northern blot analysis. All six protein subunits were purified and their amino-terminal sequences determined. SUbunit-specific oligonucleotide primers were designed based on these amino­ terminal sequences and on a conserved region near the active site. These primers were then used to PCR-amplify stretches of cDNA coding for developmentally regulated Hc subunit 6 to be used as sUbunit-specific probes in Northern blots.
    [Show full text]
  • Characterisation of the Unique Campylobacter Jejuni Cytochrome P450, CYP172A1
    Characterisation of the unique Campylobacter jejuni cytochrome P450, CYP172A1. A thesis submitted to The University of Manchester for the degree of Doctor of Philosophy in the Faculty of Life Sciences. 2013 Peter C. Elliott Preliminary Pages Table of Contents List of Figures ................................................................................................................................ 7 List of Tables ............................................................................................................................... 10 List of abbreviations .................................................................................................................... 11 Abstract ........................................................................................................................................ 14 Declaration ................................................................................................................................... 15 Copyright ..................................................................................................................................... 15 Acknowledgements ...................................................................................................................... 16 Chapter 1. Introduction ................................................................................................................ 17 1.1.1. The Campylobacter genus and related organisms ......................................................... 17 1.1.2 Campylobacter jejuni and
    [Show full text]
  • View Preprint
    Accessing and applying molecular history Joshua G. Stern1 and Eric A. Gaucher2 1Machine Learning Research, Silver Spring, MD 20910; [email protected] 2School of Biology, School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332; General Genomics, Atlanta, GA 30332 ABSTRACT Studying the evolutionary history of life’s molecules - DNA, RNA, and protein - reveals nature-based solutions to real-world problems. We discuss an approach to applied molecular evolution that is well-known within the field but may be unfamiliar to a wider audience. Using a case study at the intersection of molecular evolution and medicine, we introduce the fundamental concepts of orthology and paralogy. We also explain a practical entry point to molecular evolution named STORI: Selectable s Taxon Ortholog Retrieval Iteratively. STORI is a machine learning algorithm designed to clear a bottleneck t that researchers encounter when studying evolution. n i r Availability. Existing source code is available for download from GitHub (https://github. P com/jgstern/STORI_singlenode). e r Keywords: molecular evolution, machine learning, synthetic biology P INTRODUCTION In 2013, sickle-cell anemia killed more than 56,000 people [4]. This genetic disorder occurs when someone has a mutation in their beta-hemoglobin gene [6]. This gene is the DNA blueprint for actual beta-hemoglobin proteins: subcellular, nanometer-scale molecular machines made of yet smaller building blocks known as amino acids. Like a jeweler making necklaces from 20 different types of bead, life uses 20 different types of amino acid to build proteins. A single cell contains millions of proteins [38], although many of them share the same sequence of amino acids and thus have the same function.
    [Show full text]
  • Hemocyanin (H7017)
    Hemocyanin from Megathura crenulata (keyhole limpet) Catalog Number H7017 Storage Temperature 2–8 °C Synonyms: KLH, Keyhole Limpet Hemocyanin Precautions and Disclaimer This product is for R&D use only, not for drug, Product Description household, or other uses. Please consult the Safety Hemocyanins are multimeric, high molecular mass, Data Sheet for information regarding hazards and safe oxygen transport metalloproteins. Keyhole Limpet handling practices. Hemocyanin (KLH), from the hemolymph of the marine mollusc Megathura crenulata, is expressed as two Preparation Instructions subunit isoforms (KLH1 and KLH2) of 350–400 kDa. Reconstitution with deionized water to a KLH The KLH monomers each contain 7 or 8 functional unit concentration of 10 mg/mL yields a solution in 31 mM domains, each functional unit containing an oxygen sodium phosphate buffer, pH 7.4, containing 0.46 M binding site carrying two copper atoms. Both KLH NaCl, 2% PVP, and 41 mM sucrose. isoforms can assemble into multimeric forms containing native decamers of 4–8 ´ 106 Da. Higher multimeric Reconstituted protein solution may be stored up to forms have also been described. 2 months at –20 °C. KLH is often used as a carrier protein due to its highly Storage/Stability immunogenic properties and the large number of lysine Hemocyanin is stable as a dry solid at 2–8 °C for at residues available for modification. KLH is suitable for least two years. the preparation of immunogens for injection, or as a control protein for ELISA and dot blot immunoassay, After reconstitution, store aliquots at –20 °C for no because of its high solubility in water and aqueous longer than two months.
    [Show full text]
  • Bsc Chemistry
    Subject Chemistry 11. Inorganic Chemistry –III (Metal-Complexes and Metal Paper No and Title Clusters) Module 16 :Transport and storage of oxygen in animals by Module No and Title dioxygen complexes: Activation of dioxygen molecules in transition metal dioxygen complexes. Module Tag CHE_P11_M16 CHEMISTRY PAPER No. : 11. Inorganic Chemistry –III (Metal-Complexes and Metal Clusters) 18: Transport and storage of oxygen in animals… TABLE OF CONTENTS 1. Learning Outcomes 2. Introduction 3. Myoglobin and Hemoglobin 4. Hemerythrin 5. Hemocyanin 6. Enzymes Involved in Oxygen Activation 7. Summary CHEMISTRY PAPER No. : 11. Inorganic Chemistry –III (Metal-Complexes and Metal Clusters) 18: Transport and storage of oxygen in animals… 1. Learning Outcomes After studying this module, you shall be able to learn about: the natural proteins that carry dioxygen in various animals. the role of myoglobin and hemoglobin in carrying dioxygen in mammals. the role of hemerythrin in dioxygen transport in marine invertebrates. various enzymes involved in dioxygen activation. 2. Introduction Most organisms require molecular oxygen for their survival. For some small animals and for plants, where the surface-to-volume ratio is large, an adequate supply of dioxygen can be obtained from simple diffusion across cell membranes. The dioxygen may be extracted from air or water and for plants that produce dioxygen in photosynthesis it is also available endogenously. For other organisms, particularly those with non-passive lifestyles, from scorpions to whales, diffusion does not supply sufficient dioxygen for respiration. Therefore, in these organisms specialized molecules for the transport and storage of oxygen are necessary. These functions are carried out by a number of well-known iron and copper containing species which occur in the blood.
    [Show full text]
  • Molecular Cloning, Structure and Phylogenetic Analysis of a Hemocyanin Subunit from the Black Sea Crustacean Eriphia Verrucosa (Crustacea, Malacostraca)
    G C A T T A C G G C A T genes Article Molecular Cloning, Structure and Phylogenetic Analysis of a Hemocyanin Subunit from the Black Sea Crustacean Eriphia verrucosa (Crustacea, Malacostraca) Elena Todorovska 1 , Martin Ivanov 1, Mariana Radkova 1, Alexandar Dolashki 2 and Pavlina Dolashka 2,* 1 AgroBioInstitute, Agricultural Academy, 8 Dragan Tsankov, 1164 Sofia, Bulgaria; [email protected] (E.T.); [email protected] (M.I.); [email protected] (M.R.) 2 Institute of Organic Chemistry with Centre of Phytochemistry, BAS, Block 9 “Akademik Bonchev” Street, 1113 Sofia, Bulgaria; [email protected] * Correspondence: [email protected] Abstract: Hemocyanins are copper-binding proteins that play a crucial role in the physiological processes in crustaceans. In this study, the cDNA encoding hemocyanin subunit 5 from the Black sea crab Eriphia verrucosa (EvHc5) was cloned using EST analysis, RT-PCR and rapid amplification of the cDNA ends (RACE) approach. The full-length cDNA of EvHc5 was 2254 bp, consisting of a 50 and 30 untranslated regions and an open reading frame of 2022 bp, encoding a protein consisting of 674 amino acid residues. The protein has an N-terminal signal peptide of 14 amino acids as is expected for proteins synthesized in hepatopancreas tubule cells and secreted into the hemolymph. The 3D model showed the presence of three functional domains and six conserved histidine residues that participate in the formation of the copper active site in Domain 2. The EvHc5 is O-glycosylated and the glycan is exposed on the surface of the subunit similar to Panulirus interruptus.
    [Show full text]