DOCSLIB.ORG

Copurification of Chicken Liver Soluble Thiamine Monophosphatase and Low Molecular Weight Acid Phosphatase

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Copurification of Chicken Liver Soluble Thiamine Monophosphatase and Low Molecular Weight Acid Phosphatase ISSN 2409-4943. Ukr. Biochem. J., 2017, Vol. 89, N 6 UDC 577.164.11:577.152.3 doi: https://doi.org/10.15407/ubj89.06.013 COPURIFICATION OF CHICKEN LIVER SOLUBLE THIAMINE MONOPHOSPHATASE AND LOW MOLECULAR WEIGHT ACID PHOSPHATASE I. K. KOLAS, A. F. MAKARCHIKOV Grodno State Agrarian University; Institute of Biochemistry of Biologically Active Compounds, National Academy of Sciences of Belarus; e-mail: [email protected] Thiamine monophosphatase (ThMPase) is an enzyme of thiamine metabolism in animals whose mo- lecular nature has still to be elucidated. In this study we have achieved a 714-fold purification of a soluble enzyme possessing ThMPase activity from a chicken liver extract. In addition to ThMPase, acid phosphatase activity was traced during purification. Both activities proved to have coincident elution profiles at all chro- matographic steps implying the same enzyme involved. The molecular weight of the enzyme was 18 kDa as estimated by gel filtration. Along with ThMP and p-nitrophenyl phosphate, the purified enzyme was capable of hydrolyzing flavin mononucleotide as well as phosphotyrosine. Subcellular distribution of ThMPase activity was also explored indicating its cytosolic localization. The results of the present work imply the involvement of low molecular weight acid phosphatase in thiamine metabolism in the chicken liver. K e y w o r d s: thiamine monophosphatase, low molecular weight acid phosphatase, HPLC, chicken liver. hiamine (vitamin B1) fulfills vital biochemi‑ teria, yeast and plants this compound was found to cal functions in all living organisms studied be an intermedia te on thiamine biosynthesis path‑ T to date. In animals, thiamine diphosphate way, the enzyme thiamine phosphate synthase (ThDP) is well known to serve as a coenzyme (EC 2.5.1.3) joining 4­amino­5­hydroxymethyl­ for at least five enzyme systems involved in ener­ 2­methylpyrimidine pyrophosphate and 4­methyl­5­ gy producing and metabolism of carbohydrates, β­hydroxyethylthiazole monophosphate to produce branched­chain amino acids as well as 3­methyl­ ThMP. Some bacterial species, like Escherichia coli branched fatty acids [1]. Usually, ThDP accounts for and S. typhimurium, are also capable of direct phos‑ about 80% of the total thiamine content in the cell. phorylation of thiamine to ThMP by the enzyme Apart from its coenzyme form, several other phos‑ thiamine kinase (EC 2.7.1.89) [10]. In animal cells, phorylated deriva tives of vitamin B1 are present in ThMP is believed to be produced exclusively by the biological objects , namely thiamine monophosphate hydrolysis of ThDP. In addition, ThMP can be trans‑ (ThMP), thiamine triphosphate (ThTP) and thiamine ported into the cell from the blood plasma, where adenosine triphosphate (AThTP). Quantitatively, it constitutes up to 80% of total thiamine, through the amount of these compounds, with some excep‑ reduced folate carrier, RFC1 [11]. The hydrolysis tions in the case of ThTP, varies from less than 1% to thiamine is then a necessary step for ThMP to for AThTP and ThTP to 2­15% for ThMP in terms serve as a substrate in coenzyme synthesis by thia‑ of tissue total thiamine content [2­7]. While evi‑ mine pyrophosphokinase (EC 2.7.6.2) in eukaryo­ dence exists indicating both ThMP and AThTP are tic organisms [1, 12]. Little knowledge is available likely to be involved in mechanisms of short­term from the literature concer ning the nature of ThMP biochemical adaptation [8, 9], a possible biological hydrolyzing enzymes in animal tissues. It is only role of ThMP has not been recog nized yet. In bac‑ known that this compound could be a substrate for © 2017 Kolas I. K., Makarchikov A. F. This is an open­access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 13 ISSN 2409-4943. Ukr. Biochem. J., 2017, Vol. 89, N 6 alkaline phosphatase (ALKP, EC 3.1.3.1) [13, 14] and to a final concentration of 15 mM. In two hours, the fluoride­resistant (prostatic) acid phosphatase (PAP, microsomal fraction was obtained as a precipitate EC 3.1.3.2) [15, 16], both enzymes being membrane­ (14 000 g, 10 min), while the remaining supernatant bound proteins. Quite recently, we have revealed a represented the fraction of cytosol. The particular soluble ThMPase activity in chicken liver and hy‑ fractions were suspended in 50 mM Tris­HCl buffer, pothesized that this activity might belong to a low­ pH 7.3, containing 0.15 M KCl, 0.2 mM EDTA and molecular­weight acid phosphatase (LMW­AP), also frozen at –80 °C along with the cytosol for storage. known as a low­molecular­weight protein phospho‑ Lactate dehydrogenase (LDH, EC 1.1.1.27), tyrosine phosphatase (LMW­PTP) [17]. The present succinate dehydrogenase (EC 1.3.5.1) and glucose­ work is to study ThMPase subcellular distribution as 6­phosphatase (EC 3.1.3.9) activities, as well as well as to purify and characterize its basic properties DNA content, were assayed in subcellular fractions in chicken liver. by conventional methods [18­21]. The standard mixture for ThMPase activity de‑ Materials and Methods termination contained 25 mM Tris­maleate buffer, Fresh livers of broiler chicken ROSS 308 were pH 6.0, 1 mM ThMP, 20 µg of bovine serum albu‑ obtained from local poultry farm, placed on ice min (BSA) and an aliquot of enzyme solution in a and frozen during about 1 h after the animals were total volume of 0.2 ml. The reaction was carried out killed. The tissues were stored at –20 °C before use. for 20­60 min at 37 °C and stopped by adding 0.1 ml Sephadex G­50, Sephadex G­75, SP Sephadex C­50 of 10% TCA. Then the mixture was centrifuged and Sephacryl S­200 were purchased from Phar‑ (10 min, 2500 rpm), and the amount of inorganic macia (Uppsala, Sweden), Servacel P­23, ThMP, phosphate (Pi) liberated during the reaction was phenylmethylsulfonyl fluoride (PMSF), pyridoxal­ determined in accordance to the method of Lanzet‑ 5­phosphate and protein molecular weight markers ta P. A.et al. [22]. Kinetic experiments required some were from Serva (Heidelberg, Germany), dithio­ alterations in the assay with respect to pH, substrate threitol (DTT), p­nitrophenyl phosphate (p­NPP), concentration or metal composition; it is indicated flavin mononucleotide (FMN), phosphotyrosine, as necessary. During ion­exchange chromatography, β­glycerophosphate, glucose­6­phosphate, fruc‑ when the enzyme elution was carried out with phos‑ tose­1,6­diphosphate, inosine 5′­monophosphate, phate, ThMPase activity was monitored by HPLC. In adenosine 5′­monophosphate, 2­morpholinoethane‑ this case, TCA was extracted prior to analysis with sulfonic acid (MES) and trichloroacetic acid (TCA) 9 volumes of diethyl ether. Then a 50 µl aliquot was were from Sigma (St. Louis, Mo., USA). All other oxidized with 30 µl of 4.3 mM potassium ferricya‑ chemicals were of analytical grade. nide in 12.5% NaOH, diluted with 0.9 ml of 50 mM All operations on preparing subcellular frac‑ Na­phosphate buffer, pH 7.2, and adjusted to pH ~8.0 tions were performed at 0­4 °C (in an ice bath). A adding 35 µl of 1 M HCl. A 10­µl sample was in‑ sample of fresh liver tissue was forced through a jected into the chromatographic system (Agilent 1­mm pore press and homogenized by 10 strokes in 1200) and the separation was performed at 25 °C at a teflon/glass Potter­Elvehjem device at 600 rpm in a flow rate of 1 ml/min on a Zorbax SB­C18 column 10 volumes of 10 mM Tris­HCl buffer, pH 7.4, con‑ (3×100 mm) in 50 mM NaH2PO4, pH 8.0, contai ning taining 0.25 M sucrose, 0.5 mM EDTA and 0.1 mM 2.5 % tetrahydrofuran. Thiochrome derivatives of thiamine and ThMP were quantified using a fluoro‑ MgCl2. The homogenate was passed through four layers of wet gauze and centrifuged at 600 g for metric detector (λex = 365 nm, λem = 433 nm). 10 min. The pellet was washed by suspending in 10 Acid phosphatase (AP) activity was assayed volumes of the initial buffer and centrifuging (600 g, by incubating an aliquot of enzyme solution for 10 min) to give a crude nuclear fraction. To prepare 5­10 min in a total volume of 0.1 ml of 25 mM Tris­ mitochondria the post­nuclear supernatants were maleate buffer, pH 6.0, containing 10 µg of BSA pooled and subjected to further centrifugation at and 0.5 mM p­NPP. The reaction was terminated 12 000 g for 10 min. The particles were washed once by addition of 1.0 ml of 0.1 M NaOH, and the ab‑ with the same medium yielding a crude mitochon‑ sorbance of the solution was measured at 405 nm drial fraction. Post­mitochondrial supernatants were after centrifugation (10 min, 2500 rpm). The amount then combined and adjusted to pH 7.5 with 0.5 M of p­nitrophenol formed was calculated from ε = 18 300 M‑1cm–1. Tris­HCl, pH 8.0, followed by the addition of CaCl2 405 14 I. K. Kolas, A. F. Makarchikov When studying the enzyme substrate specifici­ concern ThMP cleavage inside the cell. So the mo‑ ty, the amounts of Pi liberated were measured under lecular nature of TMPase is currently unknown. In conditions of ThMPase assay. the search for an enzyme which would be a con‑ A unit of activity (U) was defined as the stituent of the intracellular thiamine metabolizing system in birds, we started studying some proper‑ amount catalyzing the formation of 1 µmole of Pi per min.
Load more

Recommended publications
  • Progressive Increase in Mtdna 3243A>G Heteroplasmy Causes Abrupt
    Progressive increase in mtDNA 3243A>G PNAS PLUS heteroplasmy causes abrupt transcriptional reprogramming Martin Picarda, Jiangwen Zhangb, Saege Hancockc, Olga Derbenevaa, Ryan Golhard, Pawel Golike, Sean O’Hearnf, Shawn Levyg, Prasanth Potluria, Maria Lvovaa, Antonio Davilaa, Chun Shi Lina, Juan Carlos Perinh, Eric F. Rappaporth, Hakon Hakonarsonc, Ian A. Trouncei, Vincent Procaccioj, and Douglas C. Wallacea,1 aCenter for Mitochondrial and Epigenomic Medicine, Children’s Hospital of Philadelphia and the Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104; bSchool of Biological Sciences, The University of Hong Kong, Hong Kong, People’s Republic of China; cTrovagene, San Diego, CA 92130; dCenter for Applied Genomics, Division of Genetics, Department of Pediatrics, and hNucleic Acid/Protein Research Core Facility, Children’s Hospital of Philadelphia, Philadelphia, PA 19104; eInstitute of Genetics and Biotechnology, Warsaw University, 00-927, Warsaw, Poland; fMorton Mower Central Research Laboratory, Sinai Hospital of Baltimore, Baltimore, MD 21215; gGenomics Sevices Laboratory, HudsonAlpha Institute for Biotechnology, Huntsville, AL 35806; iCentre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, East Melbourne, VIC 3002, Australia; and jDepartment of Biochemistry and Genetics, National Center for Neurodegenerative and Mitochondrial Diseases, Centre Hospitalier Universitaire d’Angers, 49933 Angers, France Contributed by Douglas C. Wallace, August 1, 2014 (sent for review May
    [Show full text]
  • Branched Chain A-Ketoacid Dehydrogenase to Thiamine and Thiamine Pyrophosphate
    Pediat. Res. 12: 235-238 (1978) Branched chain amino acids thiamine maple syrup urine disease vitamin responsiveness mitochondrial membranes In Vivo and in Vitro Response of Human Branched Chain a-Ketoacid Dehydrogenase to Thiamine and Thiamine Pyrophosphate DEAN J. DANNER,'27' FRANCES B. WHEELER, SANDRA K. LEMMON, AND LOUIS J. ELSAS I1 Department of Pediatrics, Division of Medical Genetics, Emory University School of Medicine, Atlanta, Georgia, USA Summary lability at 37"; and failure to respond to added NAD+, CoASH, and MgZ+. In a homozygous affected patient with maple syrup urine We propose that "excess" thiamine led to increased available disease, pharmacologic doses of thiamine lowered urinary excre- thiamine pyrophosphate which stabilized the branched chain a- tion of branched chain a-ketoacids and stimulated branched ketoacid dehydrogenase, decreased biologic turnover, increased chain a-ketoacid dehydrogenase (BCKAD) in his peripheral enzyme specific activity and produced in vivo tolerance to blood leukocvtes. Suvvlementation of his branched chain ami- branched chain aminoacids in these patients with maple syrup noacid restricted diei hth 100 mglday of thiamine eliminated urine disease. recurrent episodes of ketoacidosis.~heseclinical responses were Speculation studied in vitro using mitochondria1 inner membranes prepared from his cultured skin fibroblasts and those from another By studying the partially purified normal and mutant branched thiamine-responsive patient from Canada. BCKAD in both chain a-ketoacid dehydrogenases from cultured human fibro- mutant cell lines had similarities to normal enzyme including: blasts, direct in vitro effects of thiamine pyrophosphate can be identical apparent K,,, value for thiamine pyrophosphate; similar measured and related to in vivo clinical responses.
    [Show full text]
  • Supplementary Information
    Supplementary information (a) (b) Figure S1. Resistant (a) and sensitive (b) gene scores plotted against subsystems involved in cell regulation. The small circles represent the individual hits and the large circles represent the mean of each subsystem. Each individual score signifies the mean of 12 trials – three biological and four technical. The p-value was calculated as a two-tailed t-test and significance was determined using the Benjamini-Hochberg procedure; false discovery rate was selected to be 0.1. Plots constructed using Pathway Tools, Omics Dashboard. Figure S2. Connectivity map displaying the predicted functional associations between the silver-resistant gene hits; disconnected gene hits not shown. The thicknesses of the lines indicate the degree of confidence prediction for the given interaction, based on fusion, co-occurrence, experimental and co-expression data. Figure produced using STRING (version 10.5) and a medium confidence score (approximate probability) of 0.4. Figure S3. Connectivity map displaying the predicted functional associations between the silver-sensitive gene hits; disconnected gene hits not shown. The thicknesses of the lines indicate the degree of confidence prediction for the given interaction, based on fusion, co-occurrence, experimental and co-expression data. Figure produced using STRING (version 10.5) and a medium confidence score (approximate probability) of 0.4. Figure S4. Metabolic overview of the pathways in Escherichia coli. The pathways involved in silver-resistance are coloured according to respective normalized score. Each individual score represents the mean of 12 trials – three biological and four technical. Amino acid – upward pointing triangle, carbohydrate – square, proteins – diamond, purines – vertical ellipse, cofactor – downward pointing triangle, tRNA – tee, and other – circle.
    [Show full text]
  • Supplementary Informations SI2. Supplementary Table 1
    Supplementary Informations SI2. Supplementary Table 1. M9, soil, and rhizosphere media composition. LB in Compound Name Exchange Reaction LB in soil LBin M9 rhizosphere H2O EX_cpd00001_e0 -15 -15 -10 O2 EX_cpd00007_e0 -15 -15 -10 Phosphate EX_cpd00009_e0 -15 -15 -10 CO2 EX_cpd00011_e0 -15 -15 0 Ammonia EX_cpd00013_e0 -7.5 -7.5 -10 L-glutamate EX_cpd00023_e0 0 -0.0283302 0 D-glucose EX_cpd00027_e0 -0.61972444 -0.04098397 0 Mn2 EX_cpd00030_e0 -15 -15 -10 Glycine EX_cpd00033_e0 -0.0068175 -0.00693094 0 Zn2 EX_cpd00034_e0 -15 -15 -10 L-alanine EX_cpd00035_e0 -0.02780553 -0.00823049 0 Succinate EX_cpd00036_e0 -0.0056245 -0.12240603 0 L-lysine EX_cpd00039_e0 0 -10 0 L-aspartate EX_cpd00041_e0 0 -0.03205557 0 Sulfate EX_cpd00048_e0 -15 -15 -10 L-arginine EX_cpd00051_e0 -0.0068175 -0.00948672 0 L-serine EX_cpd00054_e0 0 -0.01004986 0 Cu2+ EX_cpd00058_e0 -15 -15 -10 Ca2+ EX_cpd00063_e0 -15 -100 -10 L-ornithine EX_cpd00064_e0 -0.0068175 -0.00831712 0 H+ EX_cpd00067_e0 -15 -15 -10 L-tyrosine EX_cpd00069_e0 -0.0068175 -0.00233919 0 Sucrose EX_cpd00076_e0 0 -0.02049199 0 L-cysteine EX_cpd00084_e0 -0.0068175 0 0 Cl- EX_cpd00099_e0 -15 -15 -10 Glycerol EX_cpd00100_e0 0 0 -10 Biotin EX_cpd00104_e0 -15 -15 0 D-ribose EX_cpd00105_e0 -0.01862144 0 0 L-leucine EX_cpd00107_e0 -0.03596182 -0.00303228 0 D-galactose EX_cpd00108_e0 -0.25290619 -0.18317325 0 L-histidine EX_cpd00119_e0 -0.0068175 -0.00506825 0 L-proline EX_cpd00129_e0 -0.01102953 0 0 L-malate EX_cpd00130_e0 -0.03649016 -0.79413596 0 D-mannose EX_cpd00138_e0 -0.2540567 -0.05436649 0 Co2 EX_cpd00149_e0
    [Show full text]
  • Imaging Metabolite Dynamics in Living Cells Using a Spinach-Based
    Imaging metabolite dynamics in living cells using a PNAS PLUS Spinach-based riboswitch Mingxu You1, Jacob L. Litke1, and Samie R. Jaffrey2 Department of Pharmacology, Weill Medical College, Cornell University, New York, NY 10065 Edited by Jennifer A. Doudna, University of California, Berkeley, CA, and approved April 24, 2015 (received for review March 6, 2015) Riboswitches are natural ligand-sensing RNAs typically that are 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) (6, 7). found in the 5′ UTRs of mRNA. Numerous classes of riboswitches Similar to the manner by which GFP induces fluorescence in HBI, have been discovered, enabling mRNA to be regulated by diverse Spinach binds to the otherwise nonfluorescent DFHBI and and physiologically important cellular metabolites and small mol- switches it to a highly fluorescent state. This property has allowed ecules. Here we describe Spinach riboswitches, a new class of ge- Spinach to be used to tag and image specific RNAs in living cells (6). netically encoded metabolite sensor derived from naturally occur- We recently converted the constitutively fluorescent Spinach– ring riboswitches. Drawing upon the structural switching mechanism DFHBI complex into sensors that fluoresce in proportion to the of natural riboswitches, we show that Spinach can be swapped for concentration of specific metabolites (5). These sensors comprise the expression platform of various riboswitches, allowing metabo- two domains: Spinach and a target-binding aptamer. The target- lite binding to induce Spinach fluorescence directly. In the case of the binding aptamer is inserted into a structurally critical stem of thiamine 5′-pyrophosphate (TPP) riboswitch from the Escherichia Spinach (5). Because the target-binding aptamer is unstructured coli thiM gene encoding hydroxyethylthiazole kinase, we show without the target molecule, the critical stem is disrupted, pre- that insertion of Spinach results in an RNA sensor that exhibits venting Spinach from folding and binding DFHBI.
    [Show full text]
  • 12) United States Patent (10
    US007635572B2 (12) UnitedO States Patent (10) Patent No.: US 7,635,572 B2 Zhou et al. (45) Date of Patent: Dec. 22, 2009 (54) METHODS FOR CONDUCTING ASSAYS FOR 5,506,121 A 4/1996 Skerra et al. ENZYME ACTIVITY ON PROTEIN 5,510,270 A 4/1996 Fodor et al. MICROARRAYS 5,512,492 A 4/1996 Herron et al. 5,516,635 A 5/1996 Ekins et al. (75) Inventors: Fang X. Zhou, New Haven, CT (US); 5,532,128 A 7/1996 Eggers Barry Schweitzer, Cheshire, CT (US) 5,538,897 A 7/1996 Yates, III et al. s s 5,541,070 A 7/1996 Kauvar (73) Assignee: Life Technologies Corporation, .. S.E. al Carlsbad, CA (US) 5,585,069 A 12/1996 Zanzucchi et al. 5,585,639 A 12/1996 Dorsel et al. (*) Notice: Subject to any disclaimer, the term of this 5,593,838 A 1/1997 Zanzucchi et al. patent is extended or adjusted under 35 5,605,662 A 2f1997 Heller et al. U.S.C. 154(b) by 0 days. 5,620,850 A 4/1997 Bamdad et al. 5,624,711 A 4/1997 Sundberg et al. (21) Appl. No.: 10/865,431 5,627,369 A 5/1997 Vestal et al. 5,629,213 A 5/1997 Kornguth et al. (22) Filed: Jun. 9, 2004 (Continued) (65) Prior Publication Data FOREIGN PATENT DOCUMENTS US 2005/O118665 A1 Jun. 2, 2005 EP 596421 10, 1993 EP 0619321 12/1994 (51) Int. Cl. EP O664452 7, 1995 CI2O 1/50 (2006.01) EP O818467 1, 1998 (52) U.S.
    [Show full text]
  • Imaging Metabolite Dynamics in Living Cells Using a Spinach-Based
    Imaging metabolite dynamics in living cells using a PNAS PLUS Spinach-based riboswitch Mingxu You1, Jacob L. Litke1, and Samie R. Jaffrey2 Department of Pharmacology, Weill Medical College, Cornell University, New York, NY 10065 Edited by Jennifer A. Doudna, University of California, Berkeley, CA, and approved April 24, 2015 (received for review March 6, 2015) Riboswitches are natural ligand-sensing RNAs typically that are 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) (6, 7). found in the 5′ UTRs of mRNA. Numerous classes of riboswitches Similar to the manner by which GFP induces fluorescence in HBI, have been discovered, enabling mRNA to be regulated by diverse Spinach binds to the otherwise nonfluorescent DFHBI and and physiologically important cellular metabolites and small mol- switches it to a highly fluorescent state. This property has allowed ecules. Here we describe Spinach riboswitches, a new class of ge- Spinach to be used to tag and image specific RNAs in living cells (6). netically encoded metabolite sensor derived from naturally occur- We recently converted the constitutively fluorescent Spinach– ring riboswitches. Drawing upon the structural switching mechanism DFHBI complex into sensors that fluoresce in proportion to the of natural riboswitches, we show that Spinach can be swapped for concentration of specific metabolites (5). These sensors comprise the expression platform of various riboswitches, allowing metabo- two domains: Spinach and a target-binding aptamer. The target- lite binding to induce Spinach fluorescence directly. In the case of the binding aptamer is inserted into a structurally critical stem of thiamine 5′-pyrophosphate (TPP) riboswitch from the Escherichia Spinach (5). Because the target-binding aptamer is unstructured coli thiM gene encoding hydroxyethylthiazole kinase, we show without the target molecule, the critical stem is disrupted, pre- that insertion of Spinach results in an RNA sensor that exhibits venting Spinach from folding and binding DFHBI.
    [Show full text]
  • Semi-Automated Curation of Metabolic Models Via Flux Balance Analysis: a Case Study with Mycoplasma Gallisepticum Eddy J
    University of Connecticut OpenCommons@UConn Open Access Author Fund Awardees' Articles UConn Library 9-5-2013 Semi-automated Curation of Metabolic Models via Flux Balance Analysis: A Case Study with Mycoplasma Gallisepticum Eddy J. Bautista University of Connecticut - Storrs Joseph Zinski University of Connecticut - Storrs Steven M. Szczepanek University of Connecticut - Storrs Erik L. Johnson University of Connecticut - Storrs Edan R. Tulman University of Connecticut - Storrs See next page for additional authors Follow this and additional works at: https://opencommons.uconn.edu/libr_oa Part of the Engineering Commons, and the Medicine and Health Sciences Commons Recommended Citation Bautista, Eddy J.; Zinski, Joseph; Szczepanek, Steven M.; Johnson, Erik L.; Tulman, Edan R.; Geary, Steven J.; and Srivastava, Ranjan, "Semi-automated Curation of Metabolic Models via Flux Balance Analysis: A Case Study with Mycoplasma Gallisepticum" (2013). Open Access Author Fund Awardees' Articles. 19. https://opencommons.uconn.edu/libr_oa/19 Authors Eddy J. Bautista, Joseph Zinski, Steven M. Szczepanek, Erik L. Johnson, Edan R. Tulman, Steven J. Geary, and Ranjan Srivastava This article is available at OpenCommons@UConn: https://opencommons.uconn.edu/libr_oa/19 Semi-automated Curation of Metabolic Models via Flux Balance Analysis: A Case Study with Mycoplasma gallisepticum Eddy J. Bautista1., Joseph Zinski1.¤a, Steven M. Szczepanek2,3, Erik L. Johnson1¤b, Edan R. Tulman2,4, Wei-Mei Ching5, Steven J. Geary2,4, Ranjan Srivastava1,6* 1 Department of Chemical
    [Show full text]
  • Characterization of Thiamin Phosphate Kinase in the Hyperthermophilic Archaeon Pyrobaculum Calidifontis
    J Nutr Sci Vitaminol, 61, 369–374, 2015 Characterization of Thiamin Phosphate Kinase in the Hyperthermophilic Archaeon Pyrobaculum calidifontis Maria HAYASHI and Kazuto NOSAKA* 2nd Department of Biochemistry, School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women’s University, Nishinomiya, Hyogo 663–8179, Japan (Received May 8, 2015) Summary Thiamin pyrophosphate is an essential cofactor in all living systems. In its biosynthesis, the thiamin structure is initially formed as thiamin phosphate from a thia- zole and a pyrimidine moiety, and then thiamin pyrophosphate is synthesized from thia- min phosphate. Many eubacterial cells directly synthesize thiamin pyrophosphate by the phosphorylation of thiamin phosphate by thiamin phosphate kinase (ThiL), whereas this final step occurs in two stages in eukaryotic cells and some eubacterial cells: hydrolysis of thiamin phosphate to free thiamin and its pyrophosphorylation by thiamin pyrophosphoki- nase. In addition, some eubacteria have thiamin kinase, a salvage enzyme that converts the incorporated thiamin from the environment to thiamin phosphate. This final step in thiamin biosynthesis has never been experimentally investigated in archaea, although the putative thiL genes are found in their genome database. In this study, we observed thiamin phosphate kinase activity in the soluble fraction of the hyperthermophilic archaeon Pyrobaculum calidi- fontis. On the other hand, neither thiamin pyrophosphokinase nor thiamin kinase activity was detected, suggesting that in this archaeon the phosphorylation of thiamin phosphate is only way to synthesize thiamin pyrophosphate and it cannot use exogenous thiamin for the salvage synthesis of thiamin pyrophosphate. We also investigated the kinetic properties of thiamin phosphate kinase activity using the recombinant ThiL protein from P.
    [Show full text]
  • Investigations Into How Bacteria Influence Nutrient Availability in Their Environment
    INVESTIGATIONS INTO HOW BACTERIA INFLUENCE NUTRIENT AVAILABILITY IN THEIR ENVIRONMENT A Dissertation Presented to the Faculty of the Graduate School of Cornell University In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy by David Robert Sannino December 2017 © 2017 David Robert Sannino INVESTIGATIONS INTO HOW BACTERIA INFLUENCE NUTRIENT AVAILABILITY IN THEIR ENVIRONMENT David Robert Sannino, Ph. D. Cornell University 2017 Bacteria shape many of their interactions with other organisms through the manipulation of nutrient availability, whether through cooperation by providing nutrients, or through competition for nutrients. Thiamin is an essential vitamin necessary for all life, however, how bacteria shape the ecological interactions between organisms for this nutrient is not well understood. We employed a Drosophila melanogaster-microbiota model utilizing a chemically defined diet to understand the interaction between how the host is influenced by the microbiota’s interaction with the dietary component thiamin. We found that the Drosophila melanogaster microbiota provisions thiamin to its host in a low thiamin environment. This provision rescued development of Drosophila on a no thiamin diet, as axenic flies were unable to develop on this diet. Our study was a clear demonstration supporting the long standing hypothesis that animal microbiotas function to provision thiamin and other vitamins to their host. A small subset of bacteria produce the enzyme thiaminase I, which degrades thiamin to its two moieties. The biological function of this enzyme is not understood. We used a genomic approach to investigate a potential function of this enzyme and found that it is located in a conserved operon in three thiaminase I producing Paenibacillus species, with other genes involved in thiamin salvage and production of the thiamin antimetabolite bacimethrin, suggesting it may play a role in thiamin salvage and competition for this nutrient.
    [Show full text]
  • Hypoxia Regulates Endogenous Double-Stranded RNA Production Via Reduced Mitochondrial DNA Transcription Esther Arnaiz1,2
    bioRxiv preprint doi: https://doi.org/10.1101/2020.07.31.230300; this version posted July 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Title: Hypoxia regulates endogenous double-stranded RNA production via reduced 2 mitochondrial DNA transcription 3 Esther Arnaiz1,2#, Ana Miar1,3#, Antonio Gregorio Dias Junior4,5, Naveen Prasad3, Ulrike 4 Schulze6, Dominic Waithe6, Jan Rehwinkel4, Adrian L. Harris1* 5 1Department of Medical Oncology, Molecular Oncology Laboratories, Weatherall Institute of 6 Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DS, UK 7 2Current address: Cambridge Institute for Therapeutic Immunology & Infectious Disease. 8 Jeffrey Cheah Biomedical Centre. Puddlecombe Way, Cambridge, CA CB20AW, UK 9 3Department of Oncology, Old Road Campus Research Building, University of Oxford, 10 Oxford, OX3 7DQ, UK 11 4Medical Research Council Human Immunology Unit, Medical Research Council Weatherall 12 Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, 13 Oxford OX3 9DS, UK. 14 5Current address: Division of Infectious Diseases and Vaccinology, School of Public Health, 15 University of California, Berkeley, USA. 16 6Radcliffe Department of Medicine, Weatherall Institute of Molecular Medicine, University of 17 Oxford, John Radcliffe Hospital, Oxford, OX3 9DS, UK 18 19 #equal contribution 20 *To whom correspondence should be addressed: Adrian L. Harris Department of Oncology, 21 Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, 22 OX3 9DS, UK. E-mail: [email protected]. Phone: +44 (0)1865 222457 (PA). 23 Short title: Hypoxia effect on dsRNA in cancer 24 This work has been supported by Breast Cancer Now (grant reference 2015MayPR479) and 25 Breast Cancer Research Foundation.
    [Show full text]
  • A Combined Growth Factor-Deleted and Thymidine Kinase-Deleted Vaccinia Virus Vector
    (19) & (11) EP 2 325 321 A1 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 25.05.2011 Bulletin 2011/21 C12N 15/863 (2006.01) A61K 48/00 (2006.01) (21) Application number: 10179286.9 (22) Date of filing: 26.05.2000 (84) Designated Contracting States: • Bartlett, David L. AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU Darnestown, MD 20878 (US) MC NL PT SE • Moss, Bernard Bethesda, MD 20814 (US) (30) Priority: 28.05.1999 US 137126 P (74) Representative: Donald, Jenny Susan (62) Document number(s) of the earlier application(s) in Forrester & Boehmert accordance with Art. 76 EPC: Pettenkoferstrasse 20-22 00939374.5 / 1 180 157 80336 München (DE) (71) Applicant: THE GOVERNMENT OF THE UNITED Remarks: STATES OF AMERICA as •This application was filed on 24-09-2010 as a represented by the SECRETARY OF THE divisional application to the application mentioned DEPARTMENT OF under INID code 62. HEALTH AND HUMAN SERVICES •Claims filed after the date of filing of the application Rockville, MD 20852 (US) / after the date of receipt of the divisional appliaction (Rule 68(4) EPC). (72) Inventors: • McCart, Andrea J. Silver Springs, MD 20910 (US) (54) A combined growth factor-deleted and thymidine kinase-deleted vaccinia virus vector (57) A composition of matter comprising a vaccinia virus expression vector with a negative thymidine kinase phe- notype and a negative vaccinia virus growth factor phenotype. EP 2 325 321 A1 Printed by Jouve, 75001 PARIS (FR) EP 2 325 321 A1 Description Background of the Invention 5 Field of the Invention [0001] The present invention relates to mutant vaccinia virus expression vectors The mutant expression vectors of the present invention show substantially no virus replication in non dividing cells and as such are superior to previous vaccinia virus expression vectors.
    [Show full text]