Nitrogen Regulation of Catabolic Enzymes of Neurospora Crassa

Total Page:16

File Type:pdf, Size:1020Kb

Nitrogen Regulation of Catabolic Enzymes of Neurospora Crassa I ! 77-2398 FACKLAM, Thomas John, 1950- NITROGEN REGULATION OF CATABOLIC ENZYMES OF NEUROSPORA CRASSA. The Ohio State University, Ph.D., 1976 Chemistry, biological Xerox University MicrofilmsAnn , Arbor, Michigan 48106 NITROGEN REGULATION OF CATABOLIC ENZYMES OF Neurospora crassa DISSERTATION Presented in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy in the Graduate School of The Ohio State University By Thomas John Facklam, B.S. The Ohio State University 1976 Reading Committee Approved by G. A. Marzluf, Ph.D. L. F. Johnson, Ph.D. T. J. Byers, Ph.D. Advisor / / \ (J Developmental Biology Progra ACKNOWLEDGEMENTS I wish to thank Dr. George Marzluf for his patient assistance and guidance during my graduate training. I extend special appreciation to my wife, Nancy, for her support and encouragement during the last four years. I gratefully acknowledge the financial support of the Developmental Biology Program. ii VITA June 28, 1950 Born - Buffalo, New York 1972 B.S., Cornell University, Ithaca, New York. 1973-1974 Graduate Teaching Associate, Department of Zoology, The Ohio State University, Columbus, Ohio. 1974-1976 N.I.H. Developmental Biology Traineeship The Ohio State University, Columbus, Ohio. r PUBLICATIONS "Nitrogen Regulation of Amino Acid Metabolism in Neurospora crassa." Genetics 80:s29 (1975). iii TABLE OF CONTENTS ACKNOWLEDGEMENTS ii VITA, iii LIST OF TABLES v LIST OF FIGURES vi INTRODUCTION, 1 METHODS AND MATERIALS 26 Growth of organism Enzyme assays Allantoinase isolation Affinity column Polyacrylamide gel electrophoresis Molecular weight determination RESULTS................................................................ 38 Growth of Neurospora on various nitrogen sources Amino acid uptake Growth of amino acid transport mutants Smino acid transport in pm-g and pm-n Regulation of arginine, ornithine and proline catabolic enzymes Allantoinase stabilization Isolation of allantoinase Molecular weight Multi-enzyme complex Michaelis constant Effects of cations on activity Presence of metals in allantoinase Effect of sulfhydryl reagents Feedback inhibition Thermal stability Sensitivity to proteases Characteristics of type II activity In vitro turnover DISCUSSION............................................................. 110 BIBLIOGRAPHY 127 iv LIST OF TABLES 1. Growth of wild-type and amr on various nitrogen sources. 2. Growth of amino acid transport mutants on various amino acids. 3. Arginase specific activity. 4. Ornithine transaminase specific activity. 5. Pyrroline-5-carboxylate dehydrogenase specific activity. 6. Proline oxidase specific activity. 7. Fractionation of cell-free extracts by (NH^^SO^. precipitation. 8. Lack of association of allantoicase and uricase with allantoinase. 9. Effect of cations on allantoinase activity. 10. Effect of chelators on allantoinase activity. 11. Effect of reducing substances on allantoinase. 12. Effect of potential feedback. 13. Allantoinase sensitivity to proteases. 14. Properties of type II activity. 15. SDS treatment of allantoinase. 16. Protease activity of allantoinase preparation. 17. Treatment of allantoinase by aln extract. 18. Summary of nitrogen control. v LIST OF FIGURES 1. Degradative pathway of purines. 2. Degradative pathway of arginine, ornithine and proline. 3. Flow diagram of allantoinase isolation. 4. Amino acid uptake of wild-type and amr. 5. Uptake of arginine, phenylalanine and aspartate by pm-n. 6. Uptake of arginine, phenylalanine and aspartate by pm-g. 7. Allantoinase stability in vivo. 8. Allantoinase stability in vivo in the presence of cyclohemimide. 9. Allantoinase stability in vitro. 10. Allantoinase stability in vitro in the presence of protease inhibitors. 11. Allantoinase stability in vitro in the presence of sulfhydryl reagents. 12. Elution of Sephadex G-150 column. 13. Elution of Sephadex G-100 column. 14. Polyacrylamide gels of G-150 and G-100 allantoinase preparations, 15. Molecular weight determination of allantoinase. 16. Molecular weight determination by SDS gel electrophoresis. 17. Lineweaver-Burk plot of allantoinase. 18. Thermal stability of allantoinase I and II. 19. Thermal stability with and without presence of substrate. 20. Stability of G-150 allantoinase. 21. Stability of G-100 allantoinase. 22. Molecular weight determination of type II. vi 23. Gel-filtration of SDS dialyzed allantoinase. 24. Degradation of allantoinase. 25. Model of nitrogen regulation. vii INTRODUCTION Neurospora crassa, a member of the fungal class Ascomycetes, is a typical eukaryote and can provide a model for studying regulatory mechanisms in higher organisms. Neurospora is a typical eukaryote in that it con­ tains mitochondria, a nucleus, ribosomes, seven chromosomes, poly A-con- taining mRNA (1), histones, and other structures and metabolic functions attributable to higher eukaryotes. As a heterotroph, Neurospora can utilize such simple compounds as acetate, glycerol, and glucose as carbon sources. Nitrate, ammonium, and various amino acids are able to provide the cells with nitrogen (2). Neurospora crassa is the best genetically characterized eukaryote, second only to Drosophila melanogaster. The combination of a well defined genetics, plus the ease with which it grows on completely defined media make Neurospora crassa an ideal organism with which to study regulation. Neurospora crassa is able to utilize most amides, amines, purines, and many amino acids as nitrogen sources. One aspect of my research was to examine nitrogen control of amino acid catabolism, with particular attention to arginine and proline metabolic degradation. I have also studied the closely related complex regulation of the purine catabolic enzyme, allantoinase, whose synthesis requires simultaneous induction and catabolic derepression. I will first review salient features of the regulatory mechanisms possessed by Neurospora crassa and related eukaryote organisms, and related aspects of nitrogen metabolism in these forms. 1 2 Then, my specific research objectives will be described and major conclusions will be summarized. Davis and his coworkers have studied extensively the control of arginine and ornithine metabolism in Neurospora. Their work has lead to a description of a regulatory mechanism where metabolites and enzymes are channeled and compartmentalized. The effect of this arrangement is to maintain high local concentrations of metabolites as well as to protect them from their catabolic enzymes. A second effect is to allow the regulation of independent metabolic pathways containing identical inter­ mediates. In Neurospora crassa the arginine and pyrimidine biosynthetic pathways utilize such regulatory mechanisms. The common intermediate shared by both the arginine and pyrimidine pathways is carbamyl phosphate (CAP). Carbamyl phosphate is synthesized by two carbamyl phosphate synthetases (CPSase), one for pyrimidines (CPSase P) and one for the arginine pathway (CPSase A). CPSase A is located in the mitochondria and is fully repressible by arginine (4,5). CPSase P has been localized in the nucleus complexed to arginine trans- carbamylase (ATCase)(6). The bifunctional CPSase P within the nucleus is feedback inhibited by UTP and is derepressed under the condition of low uridine (7,8). The enzyme, by being compartmentalized within the nucleus, is situated where the nucleoside triphosphates are pooled and quickly used, thus providing for a most rapid and sensitive regulation. Both CPSases provide for a separate pool of CAP, but an accumulation of CAP in one pathway can result in an overflow into the other pathway (4). This overflow effect is particularly evident with mutations lacking a particular CPSase; this deprives one pathway of CAP, but the defficiency is relieved if the second pool overflows (9, 10). 3 The channeling of the pyrimidine CAP pool could be accomplished by binding CAP to the CPSase-ATCase aggregate (4,5). This form of molecu­ lar compartmentation (11) would commit the CAP to the pyrimidine path­ way via ATCase tp synthesize ureidosuccinate. In the arginine pathway, CAP is believed to be confined in the mito­ chondria along with CPSase A (12,13). The intramitochondrial concentra­ tion of CAP is sufficiently high for efficient use by the next enzyme, ornithine carbamyltransferase, also located within the mitochondria (4,7). For arginine biosynthesis CAP is compartmentalized to increase its local concentration as well as keep it segregated from being metabo­ lized by ATCase. CPSase A, which is very sensitive to repression by arginine, is active in the presence of large concentrations of intracellular arginine. CPSase A is insensitive to feedback inhibition by arginine (5). Along with CPSase A and ornithine carbamyltransferase, ornithine acetyltrans- ferase and two other enzymes which synthesize ornithine, are located within the mitochondria (12). The product of ornithine carbamyltrans­ ferase, citrulline, is exported from the mitochondria into the cytoplasm where the remainder of the arginine biosynthetic enzymes reside. The catabolic enzymes are also found in the cytoplasm (12). Arginine, both exogenous and biosynthetic, goes directly towards protein synthesis, bypassing a large sequestered cellular pool. The arginine which fails to be incorporated into protein enters this pool (14). Labeling studies indicate that once exogenous arginine enters the intracellular pool it remains there and is exchanged only very slowly. This maintains a low arginine concentration within the cytoplasm insufficient to maintain catabolism. Weiss (15) has
Recommended publications
  • Generated by SRI International Pathway Tools Version 25.0, Authors S
    An online version of this diagram is available at BioCyc.org. Biosynthetic pathways are positioned in the left of the cytoplasm, degradative pathways on the right, and reactions not assigned to any pathway are in the far right of the cytoplasm. Transporters and membrane proteins are shown on the membrane. Periplasmic (where appropriate) and extracellular reactions and proteins may also be shown. Pathways are colored according to their cellular function. Gcf_000238675-HmpCyc: Bacillus smithii 7_3_47FAA Cellular Overview Connections between pathways are omitted for legibility.
    [Show full text]
  • Discovery of an Alternate Metabolic Pathway for Urea Synthesis in Adult Aedes Aegypti Mosquitoes
    Discovery of an alternate metabolic pathway for urea synthesis in adult Aedes aegypti mosquitoes Patricia Y. Scaraffia*†‡, Guanhong Tan§, Jun Isoe*†, Vicki H. Wysocki*§, Michael A. Wells*†, and Roger L. Miesfeld*† Departments of §Chemistry and *Biochemistry and Molecular Biophysics and †Center for Insect Science, University of Arizona, Tucson, AZ 85721-0088 Edited by Anthony A. James, University of California, Irvine, CA, and approved December 4, 2007 (received for review August 27, 2007) We demonstrate the presence of an alternate metabolic pathway We previously reported that mosquitoes dispose of toxic for urea synthesis in Aedes aegypti mosquitoes that converts uric ammonia through glutamine (Gln) and proline (Pro) synthesis, acid to urea via an amphibian-like uricolytic pathway. For these along with excretion of ammonia, uric acid, and urea (20). By studies, female mosquitoes were fed a sucrose solution containing using labeled isotopes and mass spectrometry techniques (21), 15 15 15 15 15 NH4Cl, [5- N]-glutamine, [ N]-proline, allantoin, or allantoic we have recently determined how the N from NH4Cl is acid. At 24 h after feeding, the feces were collected and analyzed incorporated into the amide side chain of Gln, and then into Pro, in a mass spectrometer. Specific enzyme inhibitors confirmed that in Ae. aegypti (22). In the present article we demonstrate that the 15 15 15 mosquitoes incorporate N from NH4Cl into [5- N]-glutamine nitrogen of the amide group of Gln contributes to uric acid and use the 15N of the amide group of glutamine to produce synthesis in mosquitoes and, surprisingly, that uric acid can be 15 labeled uric acid.
    [Show full text]
  • Yeast Genome Gazetteer P35-65
    gazetteer Metabolism 35 tRNA modification mitochondrial transport amino-acid metabolism other tRNA-transcription activities vesicular transport (Golgi network, etc.) nitrogen and sulphur metabolism mRNA synthesis peroxisomal transport nucleotide metabolism mRNA processing (splicing) vacuolar transport phosphate metabolism mRNA processing (5’-end, 3’-end processing extracellular transport carbohydrate metabolism and mRNA degradation) cellular import lipid, fatty-acid and sterol metabolism other mRNA-transcription activities other intracellular-transport activities biosynthesis of vitamins, cofactors and RNA transport prosthetic groups other transcription activities Cellular organization and biogenesis 54 ionic homeostasis organization and biogenesis of cell wall and Protein synthesis 48 plasma membrane Energy 40 ribosomal proteins organization and biogenesis of glycolysis translation (initiation,elongation and cytoskeleton gluconeogenesis termination) organization and biogenesis of endoplasmic pentose-phosphate pathway translational control reticulum and Golgi tricarboxylic-acid pathway tRNA synthetases organization and biogenesis of chromosome respiration other protein-synthesis activities structure fermentation mitochondrial organization and biogenesis metabolism of energy reserves (glycogen Protein destination 49 peroxisomal organization and biogenesis and trehalose) protein folding and stabilization endosomal organization and biogenesis other energy-generation activities protein targeting, sorting and translocation vacuolar and lysosomal
    [Show full text]
  • The Structure of Allophanate Hydrolase from Granulibacter Bethesdensis Provides Insights Into Substrate Specificity in the Amidase Signature Family
    Marquette University e-Publications@Marquette Biological Sciences Faculty Research and Publications Biological Sciences, Department of 2013 The Structure of Allophanate Hydrolase from Granulibacter bethesdensis Provides Insights into Substrate Specificity in the Amidase Signature Family Yi Lin Marquette University, [email protected] Martin St. Maurice Marquette University, [email protected] Follow this and additional works at: https://epublications.marquette.edu/bio_fac Part of the Biochemistry, Biophysics, and Structural Biology Commons Recommended Citation Lin, Yi and St. Maurice, Martin, "The Structure of Allophanate Hydrolase from Granulibacter bethesdensis Provides Insights into Substrate Specificity in the Amidase Signature Family" (2013). Biological Sciences Faculty Research and Publications. 138. https://epublications.marquette.edu/bio_fac/138 Marquette University e-Publications@Marquette Biological Sciences Faculty Research and Publications/College of Arts and Sciences This paper is NOT THE PUBLISHED VERSION; but the author’s final, peer-reviewed manuscript. The published version may be accessed by following the link in the citation below. Biochemistry, Vol. 54, No. 4 (January 29, 2013): 690-700. DOI. This article is © American Chemical Society Publications and permission has been granted for this version to appear in e- Publications@Marquette. American Chemical Society Publications does not grant permission for this article to be further copied/distributed or hosted elsewhere without the express permission from American Chemical Society Publications. The Structure of Allophanate Hydrolase from Granulibacter bethesdensis Provides Insights into Substrate Specificity in the Amidase Signature Family Yi Lin Department of Biological Sciences, Marquette University, Milwaukee, WI Martin St. Maurice Department of Biological Sciences, Marquette University, Milwaukee, WI Abstract Allophanate hydrolase (AH) catalyzes the hydrolysis of allophanate, an intermediate in atrazine degradation and urea catabolism pathways, to NH3 and CO2.
    [Show full text]
  • Nfletffillfl Sm of Nuelieotfl Dles
    Nfletffillflsm of Nuelieotfl dles ucleotides \f consistof a nitrogenousbase, a | \ pentose and a phosphate. The pentose sugaris D-ribosein ribonucleotidesof RNAwhile in deoxyribonucleotides(deoxynucleotides) of i Aspariaie--'N.,,,t .J . DNA, the sugaris 2-deoxyD-ribose. Nucleotides t participate in almost all the biochemical processes/either directly or indirectly.They are the structuralcomponents of nucleicacids (DNA, Y RNA), coenzymes, and are involved in tne Glutamine regulationof severalmetabolic reactions. Fig. 17.1 : The sources of individuat atoms in purine ring. (Note : Same colours are used in the syntheticpathway Fig. lZ.2). n T. C4, C5 and N7 are contributedby glycine. Many compoundscontribute to the purine ring of the nucleotides(Fig.t7.l). 5. C6 directly comes from COr. 1. purine N1 of is derivedfrom amino group It should be rememberedthat purine bases of aspartate. are not synthesizedas such,but they are formed as ribonucleotides. The purines 2. C2 and Cs arise from formate of N10- are built upon a formyl THF. pre-existing ribose S-phosphate. Liver is the major site for purine nucleotide synthesis. 3. N3 and N9 are obtainedfrom amide group Erythrocytes,polymorphonuclear leukocytes and of glutamine. brain cannot producepurines. 388 BIOCHEMISTF|Y m-gg-o-=_ |l Formylglycinamide ribosyl S-phosphate Kn H) Glutam H \-Y OH +ATt OH OH Glutame cl-D-Ribose-S-phosphate + ADP orr-l t'1 PRPPsYnthetase ,N o"t*'] \cH + Hrcl-itl HN:C-- O EO-qn2-O.- H -NH l./ \l KH H) I u \.]_j^/ r,\-iEl-/^\-td Ribose5-P II Formylglycinamidineribosyl-s-phosphate
    [Show full text]
  • Arginase Specific Activity and Nitrogenous Excretion of Penaeus Japonicus Exposed to Elevated Ambient Ammonia
    MARINE ECOLOGY PROGRESS SERIES Published July 10 Mar Ecol Prog Ser Arginase specific activity and nitrogenous excretion of Penaeus japonicus exposed to elevated ambient ammonia Jiann-Chu Chen*,Jiann-Min Chen Department of Aquaculture. National Taiwan Ocean University. Keelung, Taiwan 20224, Republic of China ABSTRACT: Mass-specific activity of arginase and nitrogenous excretion of Penaeus japonicus Bate (10.3 * 3.7 g) were measured for shrimps exposed to 0.029 (control), 1.007 and 10.054 mg 1-' ammonia- N at 32%, S for 24 h. Arginase specific activity of gill, hepatopancreas and midgut increased directly with ambient ammonia-N, whereas arginase specific activity of muscle was inversely related to ambient ammonia-N. Excretion of total-N (total nitrogen), organic-N and urea-N increased, whereas excretion of ammonia-N, nitrate-N and nitrite-N decreased significantly with an increase of ambient ammonia- N. In the control solution, japonlcus excreted 68.94% ammonia-N, 25.39% organic-N and 2.87% urea-N. For the shrimps exposed to 10 mg 1" ammonia-N, ammonia-N uptake occurred, and t.he con- tribution of organic-N and urea-N excretion increased to 90.57 and 8.78%, respectively, of total-N. High levels of arginase specific activity in the gill, midgut and hepatopancreas suggest that there is an alternative route of nitrogenous waste for P. japonicus under ammonia exposure. KEY WORDS: Penaeus japonicus - Ammonia . Arginase activity . Nitrogenous excretion . Metabolism INTRODUCTION processes. Therefore, accumulation of ammonia and its toxicity are of primary concern. Kuruma shrimp Penaeus japonicus Bate, which is Ammonia has been reported to increase molting fre- distributed in Pacific rim countries, is also found in the quency, reduce growth, and even cause mortality of Mediterranean.
    [Show full text]
  • Quinic Acid-Mediated Induction of Hypovirulence and a Hypovirulence-Associated Double-Stranded RNA in Rhizoctonia Solani Chunyu Liu
    The University of Maine DigitalCommons@UMaine Electronic Theses and Dissertations Fogler Library 8-2001 Quinic Acid-Mediated Induction of Hypovirulence and a Hypovirulence-Associated Double-Stranded RNA in Rhizoctonia Solani Chunyu Liu Follow this and additional works at: http://digitalcommons.library.umaine.edu/etd Part of the Biochemistry Commons, and the Molecular Biology Commons Recommended Citation Liu, Chunyu, "Quinic Acid-Mediated Induction of Hypovirulence and a Hypovirulence-Associated Double-Stranded RNA in Rhizoctonia Solani" (2001). Electronic Theses and Dissertations. 332. http://digitalcommons.library.umaine.edu/etd/332 This Open-Access Dissertation is brought to you for free and open access by DigitalCommons@UMaine. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of DigitalCommons@UMaine. QUlNlC ACID-MEDIATED INDUCTION OF HYPOVIRULENCE AND A HY POVlRULENCE-ASSOCIATED DOUBLESTRANDED RNA IN RHIZOCTONIA SOLANI BY Chunyu Liu B.S. Wuhan University, 1989 MS. Wuhan University. 1992 A THESIS Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy (in Biochemistry and Molecular Biology) The Graduate School The Universrty of Maine August, 2001 Advisory Committee: Stellos Tavantzis, Professor of Plant Pathology, Advisor Seanna Annis, Assistant Professor of Mycology Robert Cashon, Assistant Professor of Biochemistry, Molecular Biology Robert Gundersen, Associate Professor of Biochemistry, Molecular Biology John Singer, Professor of Microbiology QUlNlC ACID-MEDIATED INDUCTION OF HYPOVIRULENCE AND A HYPOVIRULENCE-ASSOCIATED DOUBLE-STRANDED RNA (DSRNA) IN RHIZOCTONIA SOLANI By Chunyu Liu Thesis Advisor: Dr. Stellos Tavantzis An Abstract of the Thesis Presented in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy (in Biochemistry and Molecular Biology) August, 2001 This study is a part of a project focused on the relationship between dsRNA and hypovirulence in R.
    [Show full text]
  • Microbial Degradation of the Morphine Alkaloids Purification and Characterization of Morphine Dehydrogenase from Pseudomonas Putida M10
    Biochem. J. (1991) 274, 875-880 (Printed in Great Britain) 875 Microbial degradation of the morphine alkaloids Purification and characterization of morphine dehydrogenase from Pseudomonas putida M10 Neil C. BRUCE,* Clare J. WILMOT, Keith N. JORDAN, Lauren D. Gray STEPHENS and Christopher R. LOWE Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QT, U.K. The NADP+-dependent morphine dehydrogenase that catalyses the oxidation of morphine to morphinone was detected in glucose-grown cells of Pseudomonas putida M 10. A rapid and reliable purification procedure involving two consecutive affinity chromatography steps on immobilized dyes was developed for purifying the enzyme 1216-fold to electrophoretic homogeneity from P. putida M 10. Morphine dehydrogenase was found to be a monomer of Mr 32000 and highly specific with regard to substrates, oxidizing only the C-6 hydroxy group of morphine and codeine. The pH optimum of morphine dehydrogenase was 9.5, and at pH 6.5 in the presence of NADPH the enzyme catalyses the reduction of codeinone to codeine. The Km values for morphine and codeine were 0.46 mm and 0.044 mm respectively. The enzyme was inhibited by thiol-blocking reagents and the metal-complexing reagents 1, 10-phenanthroline and 2,2'-dipyridyl, suggesting that a metal centre may be necessary for activity of the enzyme. INTRODUCTION EXPERIMENTAL The morphine alkaloids have attracted considerable attention Materials owing to their analgaesic properties and, consequently, much Mimetic Orange 3 A6XL and Mimetic Red A6XL were effort in the past has been directed at the production of new obtained from Affinity Chromatography Ltd., Freeport, morphine alkaloids by micro-organisms (lizuka et al., 1960, Ballasalla, Isle of Man, U.K.
    [Show full text]
  • Generated by SRI International Pathway Tools Version 25.0, Authors S
    Authors: Pallavi Subhraveti Ron Caspi Quang Ong Peter D Karp An online version of this diagram is available at BioCyc.org. Biosynthetic pathways are positioned in the left of the cytoplasm, degradative pathways on the right, and reactions not assigned to any pathway are in the far right of the cytoplasm. Transporters and membrane proteins are shown on the membrane. Ingrid Keseler Periplasmic (where appropriate) and extracellular reactions and proteins may also be shown. Pathways are colored according to their cellular function. Gcf_000725805Cyc: Streptomyces xanthophaeus Cellular Overview Connections between pathways are omitted for legibility.
    [Show full text]
  • Effects of Feeding and Confinement on Nitrogen Metabolism and Excretion in the Gulf Toadfish Opsanus Beta
    The Journal of Experimental Biology 198, 1559–1566 (1995) 1559 Printed in Great Britain © The Company of Biologists Limited 1995 EFFECTS OF FEEDING AND CONFINEMENT ON NITROGEN METABOLISM AND EXCRETION IN THE GULF TOADFISH OPSANUS BETA PATRICK J. WALSH1 AND C. LOUISE MILLIGAN2 1Division of Marine Biology and Fisheries, Rosenstiel School of Marine and Atmospheric Science, University of Miami, Miami, FL 33149, USA and 2Department of Zoology, University of Western Ontario, London, Ontario, Canada N6A 5B7 Accepted 14 March 1995 Summary In order to elucidate further the cues for, and the nitrogenous waste as ammonia, and excretion of excess biochemical mechanisms of, the transition to ureogenesis in dietary nitrogen was completed by 24 h. Elevations of the gulf toadfish Opsanus beta, experiments on the effects hepatic glutamine synthetase (GNS) activities accompanied of feeding (i.e. nitrogen loading) were carried out. Baseline confinement and were shown to be almost exclusively in the nitrogen excretion rates were first measured on solitary cytosolic compartment and to be correlated with a decrease toadfish in large water volumes (i.e. unconfined conditions). in the ratio of hepatic levels of glutamate:glutamine. These These nitrogen excretion rates were higher, and had a GNS activity increases also appear to account in part for higher proportion as ammonia (61 %), than previously the decrease in the percentage of ammoniotely in toadfish published ‘control’ measurements. Feeding of unconfined under conditions of nitrogen loading after confinement. toadfish elevated total nitrogen excretion approximately However, additional means of regulating total nitrogen threefold, with little change in the proportion of urea versus excretion (e.g.
    [Show full text]
  • Downloaded As a Text File, Is Completely Dynamic
    BMC Bioinformatics BioMed Central Database Open Access ORENZA: a web resource for studying ORphan ENZyme activities Olivier Lespinet and Bernard Labedan* Address: Institut de Génétique et Microbiologie, CNRS UMR 8621, Université Paris-Sud, Bâtiment 400, 91405 Orsay Cedex, France Email: Olivier Lespinet - [email protected]; Bernard Labedan* - [email protected] * Corresponding author Published: 06 October 2006 Received: 25 July 2006 Accepted: 06 October 2006 BMC Bioinformatics 2006, 7:436 doi:10.1186/1471-2105-7-436 This article is available from: http://www.biomedcentral.com/1471-2105/7/436 © 2006 Lespinet and Labedan; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Despite the current availability of several hundreds of thousands of amino acid sequences, more than 36% of the enzyme activities (EC numbers) defined by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) are not associated with any amino acid sequence in major public databases. This wide gap separating knowledge of biochemical function and sequence information is found for nearly all classes of enzymes. Thus, there is an urgent need to explore these sequence-less EC numbers, in order to progressively close this gap. Description: We designed ORENZA, a PostgreSQL database of ORphan ENZyme Activities, to collate information about the EC numbers defined by the NC-IUBMB with specific emphasis on orphan enzyme activities.
    [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]