DOCSLIB.ORG

Electron Transport and Adenosine Triphosphatase Activities in Turnip

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Electron Transport and Adenosine Triphosphatase Activities in Turnip rfr/rr TRANSPORT AÀID ÂDENOS,I.NE* TRIPHOSPHATASE ACTIVITIES IN TURNIP AND FRACTIONS A thesís submitted to the univetsitg of Adelaide as a teqtJitement for the degtee of DOCTOR OF PHILOSOPHY by ,toHN MTCHAEL RUNcrE, B.sc" (Hons) Botang Depattment universitg of Adelaìde November 7971 CONTENTS Page SUMMARY DECLARATION ACKNOVüLEDGEMENTS ABBREVTATIONS CHAPTER I . GENERALINTRODUCTION I-38 A" TNTRODUCTTON I B. DEFTNTTTON OF POST-MTTOCHONDRTAL FRACTTONS 2 C " ANTMAL MTCROSOT'IAT.' ANÐ SOLUBLE ACTTVÏTTES 4 7. AnimaT microsomal electlon transpott 4 (a) NADE-specific eJ-ectton transport 5 (b) NADPH-specific eJectron transport 8 (c) rnteraction between the t¡o chains 11 2" Animal soluble eJectron transport, L2 3" AnimaL micrgsomal and soTuble phosphatases L4 (a) Acid and aJ-kaline phosphatases L4 (b ) G Tucose-6- phosphatase I5 (c) Nucleoside phosphatases L6 4" Induced and deveTopmentaJ- ehanges ín ëhe anímaL L7 microsomâ.I and solubTe sgstems D" PLANT MTCROSOMAL AND SOLUBLE ACTTVTTTES 18 7" PLant mictosomaL electton ttans¡ott 18 (a) NADV-specific eTectron transport I9 (b) NADPV-specific eJ-ectron transport 20 2" Plant mictosomaT'and soLuble peroxidases 22 3. PJ-ant sofuble eþctton trans¡nrt 24 4" Plant microsomaL and soluble phosphatases 25 (a) ecid and aL:,kaline phosphatases 25 (b) Glucose-6- phosphatase 26 (c) Nucleoside phosphatases 26 5" Induced and deveTopmentaT changes in the plant 2A mictosomal and solubTe sgstems (a) lnduced phgsioTogical changes 29 (b) Induced RIVÀ and protein sgnthesis changes 32 (c) Induced changes in enzgme activities 34 (d) Induaed uLtrasttuctutal changes 36 EO THE PRESENT STUDY 37 7" General charactetization 37 2. Compatison with cawesponding animal f ractìons 38 3. Rol,e of the fractions in the overaTT energetics ot 38 èhe ce77 4 " Genetal membrane co.ncept 38 CHAPTER II - METHODS 39-5I A. PLANT MATERTAL 39 B" SLTETNG AIID AGTNG ROOT'STORAGE TTSSUE 39 C. TTSSUE PHYSTOLOGY 40 f" Manometrg 40 2" Conductivitg 40 D. PREPARATTON OF MTCROSOMES 4L E. SUBFRACTTONATTON OF MTCROSOMES 42 7. Method for Chapters rII , rv and vI 42 2. MethoQ for Chapter VII 43 3. Variations 43 F " .PREPENATTON OF SOLUBLE FRACTTON 43 G" SOLUBLE ENZYME PURTFTCATTON 44 (NHì 44 J. Z SO¿ fractÍonatìon 2 " Sephadex elution 44 H. ENZYME ASSAYS 45 7 " oxidation-teductíon activities 45 2. Mixed function oxidases 47 3" Peroxidase 48 4" AbsorpÈion spectra 48 5 " Phospltatases 49 T. CHEMTCA¿ .ASSAYS 50 J, ETJECTRON MICROSCOPY 50 K" CHEMTCALS 5I C H A P T E R I 1 I . MICROSOMAI, ELECTRON TRANSPORT 52-7L INTRODUCTION 52 RESULTS 52 A. GENERAL CHARACTERTZATTON 52 B, PROPERTTES OF REDUCTASE ACTTVTTTES 54 7. pH optima 54 2 " Inhibitor sensitivities 55 3. Concurrent reduction of two aaceptots 56 ' (a) FeCN and DCPIP 57 (b) FeCN and egtochrome c 57 (c) Cgtochrome c and DCPIP 58 C. MTCROSOMAL DTSRUPTTON 59 7" PhgsícaL 59 2 " Enzgmic 6L Ð " MTCROSOMAL SUBFRACTTONATTON 62 f, , DifferentiaT. centrifuging 62 2. Discontinuous sucrose gtadients 62 (a) Appeatance and ulttasttuctute of subftactions 63 (b) EnzVmic distribution 64 DISCUSSION 65 A. NADH ELECTRON TRANSPORT CHATN 65 B. NADPH ELECTRON TRANSPORT CHATN 68 C, MTCROSOI4AL SUBFRACTTONS 69 7" Enzgmic distribution 69 2. Origìn of ftaction 2 (niddTe) 70 D" PROPOSED ELECTRON TRANSPORT SCHEME 7T CH AP T E R I'V . CHANGES IN MICROSOMATELECTRON TRANSPORT 72-A9 TNDUCED BY SLICING AND AGING OF TISSUE INTRODUCTION 72 RESULTS 72 .72 A. EFFECT ON MTCROSOTTAL ACTTVTTTES TN VTVO 7" Changes in mícrosomaJ- h.ctivities induced bg sTicing and 72 agíng of tissue 2. ReTation between induced microsomaT changes and RNA and 73 protein sgnÈhesis in tutnip disks 3. Effect of disk size on inQuced microsomal changes 74 B" CHARACTERTZATTON OF LOSSES OF MTCROSOMAL ACTTVTTTES 75 7, rncubation of microsomes with sofuble fraction in vitro 75 2 " Effect of IAA on microsomal activities in vivo 78 3. Effect of sJicing and aging in isotonic sucrose 79 C, EFFECT ON DTSTRTBUTTON OF ACTTVTTIES WTTHTN MTCROSOMAL 80 SUBFRACTTONS DI SCUSS ION 83 A. RELATTON TO REPORTED BTOCHEMTCAL CHANGES TN MTCROSOMES IíTTH 83 AGTNG B. RELATTON TO REPORTED ULTRASTRUCTURAL CHANGES -r¡V ER WTTH AGTNG 84 C. NATURE OF TNDUCED T'TTCROSOMAL ACTTVTTY LOSSES 86 1,. iVature of sLicing-induced stimuTus 86 2 " Nature of the inactivation 8l 3. Nature of the developed insensitivittl 89 CHAP TE R V . SOLUBLEELECTRONTRANSPORT ACTIVITIES 90-106 TN FRESH AND AGED TISSUE INTRODUCTION 90 RESULTS 90 A" REDUCTASE ACTTVTTTES TN THE CRUDE SOLUBLE FNECTTON FROM FNNSH 90 ANÐ AGED TTSSUE B" SOLUBLE REDUCTASE PURTFTCATTON 92 (NH/ 7. Z So4 fractionation 92 2. Sephadex eLution 93 3 " Ptoperties 95 C. CHARACTERTZATTON OF THE DEVET,APMENT OF THE SOLUBLE REDUCTASE 96 ACTTVTTTES D. SOLUBLE REDUCTASE ACTTVTTTES TN OTHER TTSSUES 99 DISCUSSTON 101 A" SUMMARY OF THE SOLUBLE ACTTVTTTES 101 B" COMPARTSON WTTH REPORTED SOT'UBLE ACTTVTTTES FROM PLANTS LO2 C" COMPARTSAN WTTH REPORTED SOLUBLE ACTTVTTTES FROM ANTMALS LO2 D. COMPARTSON WTTH SOLUBTLTZEÐ MEMBRANE-BOUND ACTTVTTTES LO2 E. FUNCTTONS OF THE SOLUBLE ACTTVTTTES r04 F" DEVELOPMENT OE THE ACTTVITTES WTTH AGTNG r05 C H A P T E R V T - MICROSOMAL AND SOLUBLE OXYGENASES LO7-L20 IN FRESH A}ÍD AGED TISSUE INTRODUCTION IO7 RESULTS 108 A" MTCROSOMAL NAD(P)E OXTDATTON, REQUTRTNG O AND ORGANTC 108 2 SUBSTRATE 7. Characterization of the reaction 108 2 " Sensitivitg to irùtibitors and actÍvators 109 (a) EJectron transpnzt effectors 109 (b) Cgtochrome P-450 effectots 110 (c) Peroxidase effectots 111 (d) othets lrl 3. Products lL2 (a) DinethgTaniTìne TL2 (b) AniTine LL2 (c) PhenoL 113 B" SOLT]BLE NAD(P)H OXTDATTON, REQUTRTNG O AND ORGANTC 113 2 SUBSTRATE C, MTCROSOMAL AND SOLUBT,E PEROXTDASE 113 l. Characterìzation 113 2. Specificitg of membrane binding 115 D" AGTNG TNÐUCED CHANGES TN MTCROSOMAL AND SOLUBLE NAD(P)H. 116 ANTLTNE OXTDASE AND PEROXTDASE DISCUSSTON 118 CHAPTER V]I MICROSOMAL AI{D SOLUBLE PHOSPHATASES 121-132 IN FRESH AND AGED TISSUE ]NTRODUCTION L2L RESULTS L2I " A. ACTD ÞHOSPHATASE L2L B " GLI]COSE -6- PHOSPHATASE L22 C. MTCROSOMAL ATPASE L23 l. Elinination of acid phosphatase activitg 123 2. Subftactionation of mictosomal membranes l-23 3. Conditions for optimum activítg L23 4. Speclficitg of salt stimulation L24 5" Effect of membtane distuptìon 125 6" Relatíon to other ATPases L25 7. Changes utith tjssue aging L26 DISCUSSION I26 A. RELATTON OT TURN|P MTCROSOMAL ATPASE TO OTHER PLAI{T 126 T'TTCROSOMAL ATPASES ,'MTTCHELI',, B" ,RET'ATTON TO ATPASE L28 C" ORTGTN OF THE ATPASE r31 C H A P T E R V I I I - SLICING AND AGING INDUCED 13 3-140 PHYSIOLOGICAL CHANGES TNTRODUCTION 133 RESULTS 133 DISCUSSTON 136 A. TNDUCED RESPTRATTON r36 B. DEVELOPMENT OF NOONO- At:lD ANTIMYCIN A-INSENSITM r37 RESPTRATTON 7. DeveTopment of a reductase 137 ..NveToprtent tesistance 138 2 " of a 3. Discussion of two hgPotheses 139 C. TNDUCED TON ACCUMUT'ATTON I40 CH APTE R I X - GENERÃL DISCUSSION ANDFUTURET,IORK LAL-L44 BIBLIOGRAPHY 145-161 SUMMARY l. Microsomes htere isolated from turnip (Btassica rapa L') rqot sÇorage,tissue. subfractíonation (using discontinuous sucrose gradients ín the presence of ions) resulted in (a) the separation of (b) the majority of the ribosomes from the membranous vesicles and the further separation of at Ieast two types of smoo€h vesicles - one conçidered to be deríved from the endoplasmic reticulum and the other from the plasmamembranes" 2. specific NADH and NADPH dehydrogenases (Ín the presence of a variety of acceptors), and cytochrome b555, but not cytoohrQme P-450, were detected in the microsome fraction. The NADH dehydrogenase aêtivities, NADPH-cytochrome o reductase and cytoehrome bUUU were concentrated in one,of the smooth membrane fractíons¡ while the other throughout NADPH dehydrogenase actiVities were distributed more evenly the subfractions. The structure of the electron transport system was determined. The resuLts indicated that, as ín animal microsomes¡ there were t\¡ro electron transport chainsi one specific for each red.uced pyridinç nucleotid,e. Attempts to detect mixed function oxi- dase activity. involving the NADPH electron transport chain were unsuccessful. oxygen consumptíon \^'as detected in the presence of NAD(P)Ha4dorganicsubstrate¡butwasshowntobeduetoperoxidase.' 3. The microsome,fraction also contained a salt-stimulated ++ Mg---activated ATPase which \^/as .concentrated in the smooth membrane fractions. Activity was assayed "a-t high pH {7'"8} to eliminate inter- fe.rence f,rom aci.d. phosphatase whic.h,wag .also present in the microsomes" There was a specif,icity for the anion but not oation in the salt- stimulation of, the Mg++-ATPase. This aetùui"ty was further stimulated by carbonyl cyanide m+eh.lorophenylhydrazone (CCCP) , 2,A-dinitrophenol , valinomycin, nigericin and NH4CI" with.. a sy.nerg.isÈic effect between cccP and valinomyc-in. Activåty was i¡rsensitive to oligomycin, phlorízin a¡d ouabain. Based ,on .simùJ"ar;iÈy 'to-the chloroplast ATPase, it'was proposed .that this ACPase was s.iÆuated on the outside of the vesicle and moved,H* to the insdde-, J.eanr.íng the outstde alkaline. IÈ -is sugges.ted tha"t .tlrís resuLted ;in movemÊrrt of- the anLon to maintain electrieal neutrality¡ and exch.ange o-f, tlre proton for the cation to maintain pH neutr.alitY. 4. The. soluble su¡rernatant fracùion con-ta-ined- two reductase acLÍví.ties. pa:rtial. purificatíon,showed.tl¡'atrone-.was- speeific for both 'NADH and FeCN but that the other showed little specificity for either donor or acceptor. It was suggested that the former may be solubil- ized from the microsomal membranes during homogenizing of the tissue and that the latter may be the equivalent of the a¡rimal DT diaphorase" 5. Slicing turnip tissue índuced 20-100% loss of microsomal- NADH dehydrogenase activities within IO minutes. partial recovery of some activities occurred on subsequent aging of the disks, and these recoveries l^lere sensitive to cycloheximide andr'in part¡ to 6'methyl- purine.
Load more

Recommended publications
  • Microsomal and Cytosolic Scaling Factors in Dog and Human Kidney Cortex and Application for in Vitro-In Vivo Extrapolation of Renal Metabolic Clearance S
    Supplemental material to this article can be found at: http://dmd.aspetjournals.org/content/suppl/2017/03/07/dmd.117.075242.DC1 1521-009X/45/5/556–568$25.00 https://doi.org/10.1124/dmd.117.075242 DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 45:556–568, May 2017 Copyright ª 2017 by The Author(s) This is an open access article distributed under the CC BY Attribution 4.0 International license. Microsomal and Cytosolic Scaling Factors in Dog and Human Kidney Cortex and Application for In Vitro-In Vivo Extrapolation of Renal Metabolic Clearance s Daniel Scotcher, Sarah Billington, Jay Brown, Christopher R. Jones, Colin D. A. Brown, Amin Rostami-Hodjegan, and Aleksandra Galetin Centre for Applied Pharmacokinetic Research, University of Manchester, Manchester (D.S., A.R.-H., A.G.); Newcastle University, Newcastle (S.B., C.D.A.B.); Biobank, Central Manchester University Hospitals NHS Foundation Trust, Manchester (J.B.); DMPK, Oncology iMed, AstraZeneca R&D, Alderley Park, Macclesfield (C.R.J.); and Simcyp Limited (a Certara Company), Blades Enterprise Centre, Sheffield (A.R.-H.), United Kingdom Received January 26, 2017; accepted February 27, 2017 Downloaded from ABSTRACT In vitro-in vivo extrapolation of drug metabolism data obtained in in dog liver (n = 17), using P450 content and G6Pase activity, enriched preparations of subcellular fractions rely on robust esti- respectively. No trends were noted between kidney, liver, and mates of physiologically relevant scaling factors for the prediction of intestinal scalars from the same animals. Species differences were clearance in vivo. The purpose of the current study was to measure evident, as human MPPGK and CPPGK were 26.2 and 53.3 mg/g in the microsomal and cytosolic protein per gram of kidney (MPPGK kidney cortex (n = 38), respectively.
    [Show full text]
  • Microsome Isolation Kit 3/15 (Catalog # K249-50; 50 Isolations; Store at -20°C) I
    FOR RESEARCH USE ONLY! Microsome Isolation Kit 3/15 (Catalog # K249-50; 50 isolations; Store at -20°C) I. Introduction: Microsomes are spherical vesicle-like structures formed from membrane fragments following homogenization and fractionation of eukaryotic cells. The microsomal subcellular fraction is prepared by differential centrifugation and consists primarily of membranes derived from the endoplasmic reticulum (ER) and Golgi apparatus. Microsomes isolated from liver tissue are used extensively in pharmaceutical development, toxicology and environmental science to study the metabolism of drugs, organic pollutants and other xenobiotic compounds by the cytochrome P450 monooxidase (CYP) enzyme superfamily. Microsomal preparations are an affordable and convenient in vitro system for assessing Phase I biotransformation reactions, as they contain all of the xenobiotic-metabolizing CYP isozymes and the membrane-bound flavoenzymes (such as NADPH P450-Reductase and cytochrome b5) required for function of the multicomponent P450 enzyme system. BioVision’s Microsome Isolation Kit enables preparation of active microsomes in about one hour, without the need for ultracentrifugation or sucrose gradient fractionation. The kit contains sufficient reagents for 50 isolation procedures, yielding microsomes from roughly 25 grams of tissue or cultured cells. II. Applications: Convenient and fast isolation of microsomal fraction from animal tissues Assessment of CYP-mediated drug metabolism and xenobiotic biotransformation Protein profiling of microsomal membrane proteins by SDS-PAGE and Western blot III. Sample Type: Mammalian glands and soft tissues such as liver, spleen, lungs etc. Cultured eukaryotic cell lines such as HepG2 human hepatic carcinoma cells IV. Kit Contents: Components K249-50 Cap Code Part Number Homogenization Buffer 80 ml NM K249-50-1 Storage Buffer 20 ml WM K249-50-2 Protease Inhibitor Cocktail 1 vial Red K249-50-3 V.
    [Show full text]
  • The Extraction of Intracisternal A-Particles from a Mouse Plasma-Cell Tumor1
    (CANCER RESEARCH 28, 2137-2148,October1968] The Extraction of Intracisternal A-Particles from a Mouse Plasma-Cell Tumor1 Edward 1.Kuff, Nelson A. Wivel, and Kira K. Lueders Laboratory of Biochemistry and Viral Leukemia and Lymphoma Branch, National Cancer lnstitute, NIH, Department of Health, Education, and Welfare, USPHS, Bethesda, Maryland ?A%114 SUMMARY 29, A. J. Dalton, M. Potter, and H. B. Andervont, personal communication). Plasma-cell tumors in BALB/c mice contain numerous in The other type, with which the present study deals, has been tracistemnal A-particles which remain localized within micro found in myeloma lines arising both in C3H mice (carrying somal vesicles when the tumor cells are disrupted by homoge the mammary tumor agent) and in mice of the agent-free nization. Liberation of the particles has been achieved by sub BALB/c strain. Measuring between 70 and 100 m@iin diameter, jecting microsome suspensions to mechanical shear in the pres these particles consist of two concentric shells surrounding a ence of an optimal concentration of Triton X-100. The particles relatively electron-lucent core and are characterized by an cx were concentrated by two cycles of sedimentation in sucrose clusive localization within the cistemnae of the endoplasmic potassium citrate solutions, pH 72, and finally banded iso reticulum of the tumor cells. They appear to form by budding pycnically in a sucrose density gradient containing dilute at the reticulum membranes and may be very numerous in th,@ potassium citrate. Most of the particles were recovered in a neoplastic plasma cells. Particles of similar morphology and density range of 1.20.-i .24 gm/cu cm.
    [Show full text]
  • Association of Newly Synthesized Islet Prohormones with Intracellular Membranes
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by PubMed Central Association of Newly Synthesized Islet Prohormones with Intracellular Membranes BRYAN D. NOE and MICHAEL N. MORAN Department ofAnatomy, Emory University of School of Medicine, Atlanta, Georgia 30322; The Marine Biological Laboratory, Woods Hole, Massachusetts 02543 ABSTRACT Results from recent studies have indicated that pancreatic islet prohormone converting enzymes are membrane-associated in islet microsomes and secretory granules. This observation, along with the demonstration that proglucagon is topologically segregated to the periphery within alpha cell secretory granules in several species, led us to investigate the possibility that newly synthesized islet prohormones might be associated with intracellular membranes. Anglerfish islets were incubated with [3 H]tryptophan and [ 14C]isoleucine for 3 h, then fractionated by differential and density gradient centrifugation. Microsome (M) and secretory granule (SG) fractions were halved, sedimented, and resuspended in the presence or absence of dissociative reagents. After membrane lysis by repeated freezing and thawing, the membranous and soluble components were separated by centrifugation . Extracts of supernatants and pellets were chromatographed by gel filtration ; fractions were collected and counted . A high proportion (77-79%) of the newly synthesized proinsulin and insulin was associated with both M and SG membranes. Most of the newly synthesized proglucagons and prosomatostatins (12,000-mol-wt precursors) were also membrane-associated (86-88%) in M and SG . In contrast, glucagon- and somatostatin-related peptides exhibited much less mem- brane-association in SG (24-31%) . Bacitracin, bovine serum albumin EDTA, RNAse, a-meth- ylmannoside, N-acetylglucosamine, and dithiodipyridine had no effect on prohormone asso- ciation with membranes.
    [Show full text]
  • Method for Linking Nucleic Acids in a Microsome from Endoplasmatic
    (19) TZZ ¥¥_T (11) EP 2 626 433 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12Q 1/68 (2006.01) C12N 15/62 (2006.01) 12.04.2017 Bulletin 2017/15 C12N 15/13 (2006.01) C07K 16/00 (2006.01) (21) Application number: 12154726.9 (22) Date of filing: 09.02.2012 (54) Method for linking nucleic acids in a microsome from endoplasmatic reticuli Verfahren zum Koppeln von Nukleinsäuren in einem Mikrosom aus den endoplasmatischen Retikuli. Procédé pour lier les acides nucléiques dans un microsome provenant du réticulum endoplasmique (84) Designated Contracting States: • ANGENENDT P ET AL: "CELL-FREE PROTEIN AL AT BE BG CH CY CZ DE DK EE ES FI FR GB EXPRESSION AND FUNCTIONAL ASSAY IN GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO NANOWELL CHIP FORMAT", ANALYTICAL PL PT RO RS SE SI SK SM TR CHEMISTRY, AMERICAN CHEMICAL SOCIETY, US, vol. 76, no. 7, 1 April 2004 (2004-04-01), pages (43) Date of publication of application: 1844-1849, XP001196723, ISSN: 0003-2700, DOI: 14.08.2013 Bulletin 2013/33 10.1021/AC035114I • DATABASE MEDLINE [Online] US NATIONAL (73) Proprietors: LIBRARY OF MEDICINE (NLM), BETHESDA, MD, • Max-Planck-Gesellschaft zur Förderung US; December 2001 (2001-12), WANG M ET AL: der Wissenschaften e.V. "[Sequence analysis of the late region of human 80539 München (DE) papillomavirus type 6 genome].", XP002678283, • Alacris Theranostics GmbH Database accession no. NLM12901100 & 14195 Berlin (DE) ZHONGGUO YI XUE KE XUE YUAN XUE BAO.
    [Show full text]
  • Subproteomic Analysis of Soluble Proteins of the Microsomal Fraction from Two Leishmania Species
    Comparative Biochemistry and Physiology, Part D 1 (2006) 300–308 www.elsevier.com/locate/cbpd Subproteomic analysis of soluble proteins of the microsomal fraction from two Leishmania species Arthur H.C. de Oliveira a, Jerônimo C. Ruiz b, Angela K. Cruz b, ⁎ Lewis J. Greene b,c, José César Rosa b,c, Richard J. Ward a, a Departamento de Química, FFCLRP-USP, Universidade de São Paulo, Ribeirão Preto-SP, Brazil b Departamento de Biologia Celular e Molecular e Celular e Bioagentes Patogênicos, FMRP-USP, Ribeirão Preto-SP, Brazil c Centro de Química de Proteínas, FMRP-USP, Ribeirão Preto-SP, Brazil Received 17 November 2005; received in revised form 26 May 2006; accepted 27 May 2006 Available online 3 June 2006 Abstract Parasites of the genus Leishmania are the causative agents of a range of clinical manifestations collectively known as Leishmaniasis, a disease that affects 12 million people worldwide. With the aim of identifying potential secreted protein targets for further characterization, we have applied two-dimensional gel electrophoresis and mass spectrometry methods to study the soluble protein content of the microsomal fraction from two Leishmania species, Leishmania L. major and L. L. amazonensis. MALDI-TOF peptide mass fingerprint analysis of 33 protein spots from L. L. amazonensis and 41 protein spots from L. L. major identified 14 proteins from each sample could be unambiguously assigned. These proteins include the nucleotide diphosphate kinase (NDKb), a calpain-like protease, a tryparedoxin peroxidase (TXNPx) and a small GTP-binding Rab1-protein, all of which have a potential functional involvement with secretion pathways and/or environmental responses of the parasite.
    [Show full text]
  • Characterization of Electron Transport in Turnip Microsomes
    CHARACTERIZATION OF ELECTRON TRANSPORT IN TURNIP MICROSOMES By J. M. RUNGIE* and J.T. WISKICH* [Manuscript received 2 July 1971] Ab8tract Electron transport activities in the turnip microsome fraction were char­ acterized. Cytochrome c, 2,6-dichlorophenolindophenol (DCPIP), ferricyanide (FeCN), and neotetrazolium were reduced in the presence of NADH and NADPH, NADPH supporting a rate usually less than 10% that with NADH. Cytochrome ba was present and implicated in cytochrome c reduction. However, cytochrome P-450 was not detected. The effects of treatment of the microsomes with Triton X-I00, trypsin, and Naja naja venom on the reductase activities were studied. The treatments resulted in loss of NADH-cytochrome c reductase activity but had variable effects on the NADH-DCPIP and -FeCN reductase activities. The effects on the NADPH dehydro­ genase activities were also variable, but usually NADPH-cytochrome c reductase was inhibited while NADPH-DCPIP and -FeCN reductases were stimulated. Fractionation of the microsomes by differential centrifuging and centrifuging through sucrose gradients in the presence of ions yielded a fraction rich in NADH dehydrogenases, NADPH-cytochrome c reductase, and cytochrome b3 • There was a broader distribution of the other NADPH dehydrogenase activities. The results indicated the presence of two distinct electron transport chains on the turnip microsomal membranes, one specific for NADH and the other for NADPH. However, the activity of the latter may be partially due to soluble fraction contamination. I. INTRODUCTION Characterization of microsomal electron transport systems has concentrated on fractions isolated from animal tissue (Dallner 1963; Siekevitz 1963). It is now well established that there are two distinct electron transport schemes on the microsomal membranes.
    [Show full text]
  • Development of a Selective and Stable Reactive Oxygen Species-Activated Anti-Acute Myeloid Leukemia Agent and Localizing DNA Aptamer
    Development of a selective and stable Reactive Oxygen Species-activated anti-Acute Myeloid Leukemia agent and localizing DNA Aptamer a dissertation submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirements of the degree of DOCTOR OF PHILOSOPHY (Ph.D.) In the Department of Chemistry of McMicken College of Arts and Sciences by Kaylin Grace Earnest Bachelor of Science (B.S.), Chemical Science and Biochemistry, Xavier University, Cincinnati, OH Dissertation Advisor: Edward J. Merino, PhD Abstract Anticancer agents that modify DNA are a mainstay of chemotherapy regimens, but development of new classes of these agents has slowed because of the modifications of DNA in non-cancerous cells. This is what gives rise to serious side effects via poor selectivity. The Merino Lab has developed a pro-drug strategy to achieve specificity by translating the finding that levels of reactive oxygen species (ROS) are elevated in cancers, such as Acute Myeloid Leukemia (AML). This pro-drug approach allows cellular ROS to oxidize the pro-drug into its active form to achieve selective cytotoxicity. Our current lead agent (A100) is shown to have 10-fold selectivity between AML cells over normal CD34+ blood cells in vitro and showed some efficacy in the in vivo AML mouse model; however, it did not perform as highly as expected. It was hypothesized that the poor in vivo results were due to poor solubility and susceptibility to metabolic enzymes. This work started with computational analysis to determine what parts of the molecule were a target for metabolic enzymes. The first step taken to improve the molecule was to add polyethylene glycol (PEG) to the free phenol, increasing both its solubility and metabolic stability.
    [Show full text]
  • Discovery of Industrially Relevant Oxidoreductases
    DISCOVERY OF INDUSTRIALLY RELEVANT OXIDOREDUCTASES Thesis Submitted for the Degree of Master of Science by Kezia Rajan, B.Sc. Supervised by Dr. Ciaran Fagan School of Biotechnology Dublin City University Ireland Dr. Andrew Dowd MBio Monaghan Ireland January 2020 Declaration I hereby certify that this material, which I now submit for assessment on the programme of study leading to the award of Master of Science, is entirely my own work, and that I have exercised reasonable care to ensure that the work is original, and does not to the best of my knowledge breach any law of copyright, and has not been taken from the work of others save and to the extent that such work has been cited and acknowledged within the text of my work. Signed: ID No.: 17212904 Kezia Rajan Date: 03rd January 2020 Acknowledgements I would like to thank the following: God, for sending me angels in the form of wonderful human beings over the last two years to help me with any- and everything related to my project. Dr. Ciaran Fagan and Dr. Andrew Dowd, for guiding me and always going out of their way to help me. Thank you for your patience, your advice, and thank you for constantly believing in me. I feel extremely privileged to have gotten an opportunity to work alongside both of you. Everything I’ve learnt and the passion for research that this project has sparked in me, I owe it all to you both. Although I know that words will never be enough to express my gratitude, I still want to say a huge thank you from the bottom of my heart.
    [Show full text]
  • The Biology of Microsomes
    i7?1/olitiCuiiijmt~y's model of a human cell. 24 feet wide and l.(10(l,O(10.0O(ltimes larger than an actual cell logy of Mierosomes by James Bonner These are stirring times in the world of biologj. We It appears toddy that proteins are synthesized upon are beginning to find out a little bit about how pro- the surfaces of a particular kind of subcellular entity teins are synthesized by living creatures. -the microsoine. The microsome, in turn appears to In recent years we have come to know a great deal be made in the nucleus. The microsome is not only the about the structure of proteins-as, for example. that engine ut protein synthesis but also the device where- proteins consist of 'imino acids linked into long pep- by the coded information of the chromosome is car- tide chains. and that these chains are wound in heli- ried to and utilized by the protoplasm of the growing cal form. cell in the synthesis of its individual enzymes. Now we are beginning to understand why individ- Our new knowledge of protein synthesis has been ual proteins are different from one another, and why made possible by the development during the past 10 they have different enzymatic activities-even though years of methods for separating the cell into its coni- they are all composed of the same 20 amino acid ponent parts. These methods have, in part, been building blocks. We know that the indhidualit) of a Iexised at Caltech b) Samuel G. Wildman (now pro- protein resides in part in the sequence in which its fessor of botdny at UCLA ) ; George Laties, senior amino acid units are put together to form the protein researcli fellow at Caltecli; and others.
    [Show full text]
  • Proteomic and Bioinformatics Analyses of Mouse Liver Microsomes
    Hindawi Publishing Corporation International Journal of Proteomics Volume 2012, Article ID 832569, 24 pages doi:10.1155/2012/832569 Research Article Proteomic and Bioinformatics Analyses of Mouse Liver Microsomes Fang Peng,1 Xianquan Zhan,1 Mao-Yu Li,1 Fan Fang,1 Guoqing Li,1, 2 Cui Li,1 Peng-Fei Zhang,1 and Zhuchu Chen1 1 Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital, Central South University, Hunan, Changsha 410008, China 2 Department of Biology, School of Pharmacy and Life Science, University of South China, Hengyang 421001, China Correspondence should be addressed to Xianquan Zhan, [email protected] and Zhuchu Chen, [email protected] Received 28 July 2011; Revised 9 November 2011; Accepted 20 November 2011 Academic Editor: Visith Thongboonkerd Copyright © 2012 Fang Peng et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Microsomes are derived mostly from endoplasmic reticulum and are an ideal target to investigate compound metabolism, membrane-bound enzyme functions, lipid-protein interactions, and drug-drug interactions. To better understand the molecular mechanisms of the liver and its diseases, mouse liver microsomes were isolated and enriched with differential centrifugation and sucrose gradient centrifugation, and microsome membrane proteins were further extracted from isolated microsomal fractions by the carbonate method. The enriched microsome proteins were arrayed with two-dimensional gel electrophoresis (2DE) and carbonate-extracted microsome membrane proteins with one-dimensional gel electrophoresis (1DE). A total of 183 2DE-arrayed proteins and 99 1DE-separated proteins were identified with tandem mass spectrometry.
    [Show full text]
  • The Role of Microsomes and Nuclear Envelope in the Metabolic Activation of Benzo(A)Pyrene Leading to Binding with Nuclear Macromolecules1
    [CANCER RESEARCH 37, 3427-3433, September 1977] The Role of Microsomes and Nuclear Envelope in the Metabolic Activation of Benzo(a)pyrene Leading to Binding with Nuclear Macromolecules1 John M. Pezzuto, Michael A. Lea, and Chung S. Yang2 Department of Biochemistry, College of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07013 SUMMARY spread atmospheric pollutant that has also been shown to be a component of cigarette smoke (7). Like many chemical In an attempt to resolve existing conflicting reports and carcinogens, unmetabolized BP does not covalently react further substantiate the roles of microsomes and the with macromolecules (10, 12). Metabolic activation results nuclear envelope in the metabolic activation of in binding with several cellular nucleophilic centers (13,14, benzo(a)pyrene (BP), factors affecting the binding of BP to 22, 33), and such binding will presumably lead to carcino- the DNA, RNA, histone, and nonhistone proteins of isolated genesis. Recent evidence suggests that metabolism by the nuclei were investigated. Examination of the spectra and cytochrome P-450-containing mixed-function oxidase catalytic properties of the mixed-function oxidase systems (AHH) in conjunction with epoxide hydrase results in the of nuclei and microsomes indicated that they are similar. formation of a diol-epoxide that is probably the ultimate Regard less of the BP concentration used, microsomes from carcinogen (4, 8, 34, 36, 38, 41). The relatively short lifetime control or 3-methylcholanthrene-treated rats increased the of this species (36, 38) and the existence of cytoplasmic binding of BP to the components of control nuclei.
    [Show full text]