DOCSLIB.ORG

Effect of Guanosine Diphosphate on Microtubule Assembly and Stability

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text

Load more

THE ~VURNALof BlOLoCICALCHEMISTRY Vol. 256. Nn. ti. hue of March 25. pp. 22%-2292, 1980 Prmtrd in USA. Effect of Guanosine Diphosphate on Microtubule Assemblyand Stability* (Received for publication, April 23, 1979, and in revised form, August 10, 1979) Larry Jameson andMichael Caplow From the Department of Biochemistry, University of North Carolina, ChapelHill, North Carolina 27514 Polymerization of microtubular protein is promoted formation, only guanine nucleotides are bound to tubulinwith by GTP but not GDP. GDP is shown to inhibit both the high affinity (2, 3). The tubulin dimer has two binding sites rate and extent of GTP-induced microtubule assembly. for guanine nucleotides. One site is nonexchangeable (N-site)’ The critical protein concentration for assembly was and releases GTP only upon denaturation. The other site(E- increased from 0.3 mg/ml with 200 JLM GTP to 0.8 mg/ site) readily exchanges bound nucleotide for GDP or GTPin ml with 200 JLM GTP and 600 JLM GDP. The relative the medium (3). The nonguanine nucleoside triphosphates binding constants for GDP and GTP to the tubulin have been proposed to induce tubulin polymerization indi- exchangeable binding site (E-site) are used to quanti- rectly by phosphorylating GDP througha nucleoside diphos- tatively analyze the mechanism by which GDP raises phokinase reaction (3-5). GTP promotes polymerization by the critical protein concentration and thereby reduces interacting directly with the tubulin E-site(3-5). the extent of microtubule assembly. Evidence that guaninenucleotides also interact with GTP hydrolysis accompanies microtubule polymerization microtubular protein at a previously unknown, low (6-8). However, it has been difficult to determine whether affinity binding site is supported by the following ob- GTP hydrolysis is required for microtubule assembly or dis- servations. assembly, if either. Isolated microtubules contain only GDP 1. At a constant ratioof GDP/GTP, a greater degree at the E-site (3, 4, 9, lo), indicating that phosphate bond of inhibition of the extentof microtubule assemblywas hydrolysis occurs after microtubule assembly but before dis- observed at higher concentrations of total nucleotide. assembly. Nevertheless, analogs of GTP which are resistant This result cannot be accounted for by binding at only to hydrolysis at the p-y phosphate bond (e.g. Gpp(NH)p) the E-site. have been shownto promote microtubuleassembly (11). Since 2. Inhibition of microtubule assembly by a high con- the microtubulesformed using these analogs exhibit unusual centration of GDP (3 mM) is not reversed by increasing stability in the presence of concentrations of calcium which the GTP concentration to 7 m. Based upon the disso- completely depolymerize microtubules formed with GTP, it ciation constants forGDP and GTP at theE-site, tubulin has been suggested that hydrolysis of the terminal phosphate would be largely saturated with GTP under these con- of GTP may be necessary before microtubule disassembly can ditions and should polymerize in the absence of addi- occur (4). However, studies using another analog of GTP tional nucleotide interactions. which is resistant to phosphate bond hydrolysis at the a-p 3. GTP specifically inhibits microtubule assembly at position but not thep-y position (Gp(CH2)pp)give somewhat concentrations greater than3 mM. Inhibition of assem- different results. Using a quantitative colchicine binding assay bly by high concentrations (7 m~)of ATP and CTP is for tubulin polymerization, Sandoval et al. (12) demonstrated shown to involve a differentmechanism. that Gp(CH2)pp promotespolymerization of 98% of the avail- 4. Microtubules are depolymerized by high concen- able tubulin into microtubules, while only 70% of the tubulin trations of GTP (>5 mM) or GDP (>2 mM). The ratefor this disassembly is greater than thatobserved during was polymerized in the presence of GTP. Although the ter- microtubule turnover following inhibition of polymer- minal phosphate of Gp(CH8)pp was hydrolyzed,the rnicrotu- ization, suggestingthat high Concentrations of guanine bules formed with thisanalog were still resistant todepolym- nucleotides destabilize microtubules by interacting at erization by calcium. This observation was supported by ad- a low affinity site. ditional studieswhich showed that microtubulescould also be formed using the nonhydrolyzable analog of GDP, Gp(CHz)p, in either thepresence or absenceof calcium (13).These results suggest that hydrolysis of the a-P phosphate bond may be Microtubules are assembled from the dimeric protein sub- important for microtubule disassembly. unit, tubulin. The mechanism by which the assembly and The role of the GDPin microtubule assembly and disassem- disassembly of microtubules are regulatedin vivo is unknown. bly must be further elucidated before definitive conclusions However, the characterization of conditions which favor as- can be drawn concerning the function of p-y or a-p phosphate semblyor disassembly in vitro has progressed rapidly in bond hydrolysis. A number of laboratories have reported that recent years (for review, see Ref. 1). In particular, studies of GDPdoes not promote tubulin polymerization (4, 6, 13). tubulin-nucleotide interactions have revealed that microtu- bule assembly requires nucleoside triphosphates. Whilea ’ The abbreviations used are: N-site, nonexchangeable binding site; variety of nucleoside triphosphates can induce microtubule E-site, exchangeable binding site; Gpp(NH)p, guanyl-5”yl imidiphos- phate; Gp(CH2)pp,guanyl-5’-yl methylene diphosphonate; Gp(CHn)p, * This work was supported by Grant DE03246 from the National guanosine 5”methylene diphosphonate; HB, reassembly buffer (0.1 Institute for Dental Research. The costs of publication of this article M 2-(N-morpholino)ethanesulfonicacid buffer, 0.5 m~ MgClz, 1.0 mM were defrayed in part by the payment of page charges. This article EGTA, pH 6.8); MAPS, microtubule-associated proteins; PEI, poly- musttherefore be hereby marked “advertisement” in accordance ethyleneimine; EGTA,ethylene glycol bis(8-aminoethyl ether)N,- with 18 U.S.C. Section 1734 solely to indicate this fact. N,N‘,N’-tetraacetic acid. 2284 This is an Open Access article under the CC BY license. M icro tubu le Assembly and Disassembly and AssemblyMicrotubule 2285 Furthermore, GDP has been shown to inhibit GTP-induced by thin layer chromatography on PEI-cellulose as described previ- microtubuleassembly (4, 6, 8). Carlier and Pantaloni (14) ously (19). GTP hydrolysis was determined by the decrease in ["PIGTP relative to recovered ['HIGTP. recently studied polymerization of phosphocellulose-purified Measurement of Transphosphorylase Activity-Nucleoside di- tubulin in the presence of 5 mM Mg2+ and 3.4 M glycerol and phosphokinase (transphosphorylase) activity was determined by fol- found that in contrast to earlier reports, the tubulin. GDP lowing the conversion of ['HIGDP to [:'H]GTP in the presence of complex could participatein microtubule elongation after the added ATP. Shortly before use, ["HIGDP was purified by thin layer nucleation stepwas carried out usingGTP. chromatography on PEI-cellulose in order to remove radioactive The purpose of this report is to clarify the role of GDP in contaminants. Reaction mixtures containing 15 pI of ATP (20 mM or 200mM), 35 of purified vH]GDP (8500 cpm/pl), and 400 1.11 of microtubuleassembly disassembly. find the pl and We that tubulin (31 p~)were incubated at either 4OC or 37°C. The reactions tubulin.GDP complex does not participate in either the nu- were stopped at different times by centrifuging 5O-pl aliquots through cleation or the elongation of microtubules. GDP inhibition of 1-ml columns of centrifuge-packed G-25 to separate the protein from the rate and extent of GTP-induced microtubule assembly is exogenous nucleotide (ATP) (20). In separate experiments, it was analyzed in terms of a mechanism in whichthe tubulin-GDP found that only guanine nucleotides remain protein-bound under complex is not competent to polymerize. Evidence for a low these conditions, and that ATP and ADP are entirely removed by this procedure. The protein was eluted into 2% perchloric acid and affinity binding site for guanine nucleotides is presented. the released nucleotides isolated and separated by thin layer chro- matography on PEI-cellulose (19). Transphosphorylase activity was EXPERIMENTALPROCEDURES determined from the relative amounts of"H in the resolved spots Methods containing GTP and GDP. The recoveries of [ 'HIGDP and [ 'H1GTP are identical in the purification procedure (determined in separate Preparation of Microtubular Protein-Microtubular protein was experiments), so that this method allows determination of the relative prepared by a modification of the Shelanski (15) assembly-disassem- radioactivity in the nucleoside di- andtriphosphates in reaction bly procedure. Fresh pig brains were homogenized with ice-cold mixtures. reassembly buffer (I ml/g of brain) (0.1 M 2-(N-morpholino)- ethanesulfonic acid buffer, 0.5 mM MgClz,1.0 mM EGTA, pH 6.8) Materials containing 0.1 mM ATP in a Waring Blendor for 30 s. The supernatant Guanyl-5"yl methylene diphosphonate (Gp(CHz)pp, which had obtained from centrifugation (30,000 rpm, 75 min, 4°C) in a Beckman been purifiedby high pressure liquid chromatography, was purchased 30 rotor was diluted with an equal volume of glycerol-reassembly from ICN. ["]GTP (specific activity 10.6 Ci/mmol) and [:'H]GDP buffer (60.2 ml of glycerol: 24 ml of reassembly buffer) containing 1 (specific activity, 8.91 Ci/mmol) and
Recommended publications
  • 2'-Deoxyguanosine Toxicity for B and Mature T Lymphoid Cell Lines Is Mediated by Guanine Ribonucleotide Accumulation

    2'-Deoxyguanosine Toxicity for B and Mature T Lymphoid Cell Lines Is Mediated by Guanine Ribonucleotide Accumulation

    2'-deoxyguanosine toxicity for B and mature T lymphoid cell lines is mediated by guanine ribonucleotide accumulation. Y Sidi, B S Mitchell J Clin Invest. 1984;74(5):1640-1648. https://doi.org/10.1172/JCI111580. Research Article Inherited deficiency of the enzyme purine nucleoside phosphorylase (PNP) results in selective and severe T lymphocyte depletion which is mediated by its substrate, 2'-deoxyguanosine. This observation provides a rationale for the use of PNP inhibitors as selective T cell immunosuppressive agents. We have studied the relative effects of the PNP inhibitor 8- aminoguanosine on the metabolism and growth of lymphoid cell lines of T and B cell origin. We have found that 2'- deoxyguanosine toxicity for T lymphoblasts is markedly potentiated by 8-aminoguanosine and is mediated by the accumulation of deoxyguanosine triphosphate. In contrast, the growth of T4+ mature T cell lines and B lymphoblast cell lines is inhibited by somewhat higher concentrations of 2'-deoxyguanosine (ID50 20 and 18 microM, respectively) in the presence of 8-aminoguanosine without an increase in deoxyguanosine triphosphate levels. Cytotoxicity correlates instead with a three- to fivefold increase in guanosine triphosphate (GTP) levels after 24 h. Accumulation of GTP and growth inhibition also result from exposure to guanosine, but not to guanine at equimolar concentrations. B lymphoblasts which are deficient in the purine salvage enzyme hypoxanthine guanine phosphoribosyltransferase are completely resistant to 2'-deoxyguanosine or guanosine concentrations up to 800 microM and do not demonstrate an increase in GTP levels. Growth inhibition and GTP accumulation are prevented by hypoxanthine or adenine, but not by 2'-deoxycytidine.
  • We Have Previously Reported' the Isolation of Guanosine Diphosphate

    We Have Previously Reported' the Isolation of Guanosine Diphosphate

    VOL. 48, 1962 BIOCHEMISTRY: HEATH AND ELBEIN 1209 9 Ramel, A., E. Stellwagen, and H. K. Schachman, Federation Proc., 20, 387 (1961). 10 Markus, G., A. L. Grossberg, and D. Pressman, Arch. Biochem. Biophys., 96, 63 (1962). "1 For preparation of anti-Xp antisera, see Nisonoff, A., and D. Pressman, J. Immunol., 80, 417 (1958) and idem., 83, 138 (1959). 12 For preparation of anti-Ap antisera, see Grossberg, A. L., and D. Pressman, J. Am. Chem. Soc., 82, 5478 (1960). 13 For preparation of anti-Rp antisera, see Pressman, D. and L. A. Sternberger, J. Immunol., 66, 609 (1951), and Grossberg, A. L., G. Radzimski, and D. Pressman, Biochemistry, 1, 391 (1962). 14 Smithies, O., Biochem. J., 71, 585 (1959). 15 Poulik, M. D., Biochim. et Biophysica Acta., 44, 390 (1960). 16 Edelman, G. M., and M. D. Poulik, J. Exp. Med., 113, 861 (1961). 17 Breinl, F., and F. Haurowitz, Z. Physiol. Chem., 192, 45 (1930). 18 Pauling, L., J. Am. Chem. Soc., 62, 2643 (1940). 19 Pressman, D., and 0. Roholt, these PROCEEDINGS, 47, 1606 (1961). THE ENZYMATIC SYNTHESIS OF GUANOSINE DIPHOSPHATE COLITOSE BY A MUTANT STRAIN OF ESCHERICHIA COLI* BY EDWARD C. HEATHt AND ALAN D. ELBEINT RACKHAM ARTHRITIS RESEARCH UNIT AND DEPARTMENT OF BACTERIOLOGY, THE UNIVERSITY OF MICHIGAN Communicated by J. L. Oncley, May 10, 1962 We have previously reported' the isolation of guanosine diphosphate colitose (GDP-colitose* GDP-3,6-dideoxy-L-galactose) from Escherichia coli 0111-B4; only 2.5 umoles of this sugar nucleotide were isolated from 1 kilogram of cells. Studies on the biosynthesis of colitose with extracts of this organism indicated that GDP-mannose was a precursor;2 however, the enzymatically formed colitose was isolated from a high-molecular weight substance and attempts to isolate the sus- pected intermediate, GDP-colitose, were unsuccessful.
  • Isolation and Analysis of Sugar Nucleotides Using Solid Phase Extraction and fluorophore Assisted Carbohydrate Electrophoresis

    Isolation and Analysis of Sugar Nucleotides Using Solid Phase Extraction and fluorophore Assisted Carbohydrate Electrophoresis

    MethodsX 3 (2016) 251–260 Contents lists available at ScienceDirect MethodsX journal homepage: www.elsevier.com/locate/mex Isolation and analysis of sugar nucleotides using solid phase extraction and fluorophore assisted carbohydrate electrophoresis Jarrod Barnesa,1, Liping Tiana,1, Jacqueline Loftisa, James Hiznayc, Suzy Comhaira, Mark Lauera,2, Raed Dweika,b,* a Departments of Pathobiology, Cleveland Clinic, 9500 Euclid Ave, Cleveland, OH 44195, USA b Pulmonary Critical Care Medicine, Respiratory Institute, Cleveland Clinic, 9500 Euclid Ave, Cleveland, OH 44195, USA c Molecular Medicine, Cleveland Clinic, 9500 Euclid Ave, Cleveland, OH 44195, USA GRAPHICAL ABSTRACT ABSTRACT The building blocks of simple and complex oligosaccharides, termed sugar nucleotides, are often overlooked for their role in metabolic diseases and may hold the key to the underlying disease pathogenesis. Multiple reasons may account for the lack of analysis and quantitation of these sugar nucleotides, including the difficulty in isolation and purification as well as the required expensive instrumentation such as a high performance liquid Abbreviations: HPLC, high performance liquid chromatography; SPE, solid phase extraction; FACE, fluorophore assisted carbohydrate electrophoresis; UDP, uridine diphosphate; GDP, guanosine diphosphate; CMP, cytosine monophosphate; TEAA, triethylamine acetate; APS, ammonium persulfate; Gal, galactose; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylgluco- samine; Man, Mannose; NeuAc, sialic acid; GlcUA, glucuronic acid; AMAC, 2-aminoacridone; TEMED, N0,N0,N0N0- tetramethylenediamine. * Corresponding author at: Departments of Pathobiology, Cleveland Clinic, 9500 Euclid Ave, Cleveland, Ohio 44195, USA. E-mail address: [email protected] (R. Dweik). 1 Authors contributed equally to this work. 2 In honor of Dr. Mark Lauer who passed away October 12, 2015. http://dx.doi.org/10.1016/j.mex.2016.03.010 2215-0161/ ã 2016 The Authors.
  • Central Nervous System Dysfunction and Erythrocyte Guanosine Triphosphate Depletion in Purine Nucleoside Phosphorylase Deficiency

    Central Nervous System Dysfunction and Erythrocyte Guanosine Triphosphate Depletion in Purine Nucleoside Phosphorylase Deficiency

    Arch Dis Child: first published as 10.1136/adc.62.4.385 on 1 April 1987. Downloaded from Archives of Disease in Childhood, 1987, 62, 385-391 Central nervous system dysfunction and erythrocyte guanosine triphosphate depletion in purine nucleoside phosphorylase deficiency H A SIMMONDS, L D FAIRBANKS, G S MORRIS, G MORGAN, A R WATSON, P TIMMS, AND B SINGH Purine Laboratory, Guy's Hospital, London, Department of Immunology, Institute of Child Health, London, Department of Paediatrics, City Hospital, Nottingham, Department of Paediatrics and Chemical Pathology, National Guard King Khalid Hospital, Jeddah, Saudi Arabia SUMMARY Developmental retardation was a prominent clinical feature in six infants from three kindreds deficient in the enzyme purine nucleoside phosphorylase (PNP) and was present before development of T cell immunodeficiency. Guanosine triphosphate (GTP) depletion was noted in the erythrocytes of all surviving homozygotes and was of equivalent magnitude to that found in the Lesch-Nyhan syndrome (complete hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficiency). The similarity between the neurological complications in both disorders that the two major clinical consequences of complete PNP deficiency have differing indicates copyright. aetiologies: (1) neurological effects resulting from deficiency of the PNP enzyme products, which are the substrates for HGPRT, leading to functional deficiency of this enzyme. (2) immunodeficiency caused by accumulation of the PNP enzyme substrates, one of which, deoxyguanosine, is toxic to T cells. These studies show the need to consider PNP deficiency (suggested by the finding of hypouricaemia) in patients with neurological dysfunction, as well as in T cell immunodeficiency. http://adc.bmj.com/ They suggest an important role for GTP in normal central nervous system function.
  • J. HOHORST Institut Fiir Physiologische Chemie, Klinikum Der Universitiit Frankfurt, 6 Frankfurt (Main), Germany

    J. HOHORST Institut Fiir Physiologische Chemie, Klinikum Der Universitiit Frankfurt, 6 Frankfurt (Main), Germany

    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Volume 28, number 2 FEBS LETTERS December 1972 GUANOSINE-DIPHOSPHATE CAUJlNG CHANGES 1N THE PHOSPHORYLATION PATTERN OF ADENl& NUCLEOTIDES IN MlTOCHONDRIA FROM BROWN ADIPOSE TISSUE J. RAFAEL, H.W. HELDT and H.-J. HOHORST Institut fiir Physiologische Chemie, Klinikum der Universitiit Frankfurt, 6 Frankfurt (Main), Germany and Institut ftir Physiologische Chemie und Physikalische Biochemie, Universitiit Miinchen, 8 Miinchen, Germany Received 20 October 1972 1. Introduction energy state of the mitochondria; recoupling of the oxidative phosphorylation stated earlier in polaro- Oxidative phosphorylation of mitochondria graphic experiments [ 1,6] should be reflected in a isolated from brown adipose tissue (BAT) was found rise of the ATP/ADP ratio in the mitochondria. to be more or less loosely coupled in the sense of a decreased or even missing ADP dependent respiratory control and reduced P/O ratios [ 1, 21. The “tightness” 2. Materials and methods of the coupling of mitochondrial respiration and phosphorylation appears to be correlated to the Mitochondria from BAT of newborn guinea pigs thermogenetic activity of the tissue [ 1,3,4]. Respira- were prepared as described previously [7]. The mito- tory control and ATP production of BAT mitochon- chondrial protein was determined by a modified dria can be restored in vitro by incubating the mito- biuret method [ 11. chondria in a medium containing 2% bovine serum The intramitochondrial adenine nucleotides were albumin [5] or certain nucleoside di- and triphos- determined by radioactivity measurements. For this phates, especially guanosine-di-phosphate [ 1, 3,6].
  • Guanosine-Based Nucleotides, the Sons of a Lesser God in the Purinergic Signal Scenario of Excitable Tissues

    Guanosine-Based Nucleotides, the Sons of a Lesser God in the Purinergic Signal Scenario of Excitable Tissues

    International Journal of Molecular Sciences Review Guanosine-Based Nucleotides, the Sons of a Lesser God in the Purinergic Signal Scenario of Excitable Tissues 1,2, 2,3, 1,2 1,2, Rosa Mancinelli y, Giorgio Fanò-Illic y, Tiziana Pietrangelo and Stefania Fulle * 1 Department of Neuroscience Imaging and Clinical Sciences, University “G. d’Annunzio” of Chieti-Pescara, 66100 Chieti, Italy; [email protected] (R.M.); [email protected] (T.P.) 2 Interuniversity Institute of Miology (IIM), 66100 Chieti, Italy; [email protected] 3 Libera Università di Alcatraz, Santa Cristina di Gubbio, 06024 Gubbio, Italy * Correspondence: [email protected] Both authors contributed equally to this work. y Received: 30 January 2020; Accepted: 25 February 2020; Published: 26 February 2020 Abstract: Purines are nitrogen compounds consisting mainly of a nitrogen base of adenine (ABP) or guanine (GBP) and their derivatives: nucleosides (nitrogen bases plus ribose) and nucleotides (nitrogen bases plus ribose and phosphate). These compounds are very common in nature, especially in a phosphorylated form. There is increasing evidence that purines are involved in the development of different organs such as the heart, skeletal muscle and brain. When brain development is complete, some purinergic mechanisms may be silenced, but may be reactivated in the adult brain/muscle, suggesting a role for purines in regeneration and self-repair. Thus, it is possible that guanosine-50-triphosphate (GTP) also acts as regulator during the adult phase. However, regarding GBP, no specific receptor has been cloned for GTP or its metabolites, although specific binding sites with distinct GTP affinity characteristics have been found in both muscle and neural cell lines.
  • Nucleotide Sugars in Chemistry and Biology

    Nucleotide Sugars in Chemistry and Biology

    molecules Review Nucleotide Sugars in Chemistry and Biology Satu Mikkola Department of Chemistry, University of Turku, 20014 Turku, Finland; satu.mikkola@utu.fi Academic Editor: David R. W. Hodgson Received: 15 November 2020; Accepted: 4 December 2020; Published: 6 December 2020 Abstract: Nucleotide sugars have essential roles in every living creature. They are the building blocks of the biosynthesis of carbohydrates and their conjugates. They are involved in processes that are targets for drug development, and their analogs are potential inhibitors of these processes. Drug development requires efficient methods for the synthesis of oligosaccharides and nucleotide sugar building blocks as well as of modified structures as potential inhibitors. It requires also understanding the details of biological and chemical processes as well as the reactivity and reactions under different conditions. This article addresses all these issues by giving a broad overview on nucleotide sugars in biological and chemical reactions. As the background for the topic, glycosylation reactions in mammalian and bacterial cells are briefly discussed. In the following sections, structures and biosynthetic routes for nucleotide sugars, as well as the mechanisms of action of nucleotide sugar-utilizing enzymes, are discussed. Chemical topics include the reactivity and chemical synthesis methods. Finally, the enzymatic in vitro synthesis of nucleotide sugars and the utilization of enzyme cascades in the synthesis of nucleotide sugars and oligosaccharides are briefly discussed. Keywords: nucleotide sugar; glycosylation; glycoconjugate; mechanism; reactivity; synthesis; chemoenzymatic synthesis 1. Introduction Nucleotide sugars consist of a monosaccharide and a nucleoside mono- or diphosphate moiety. The term often refers specifically to structures where the nucleotide is attached to the anomeric carbon of the sugar component.
  • Hplc Analysis of Nucleotides

    Hplc Analysis of Nucleotides

    HPLC ANALYSIS OF NUCLEOTIDES Vicrant Narayan Bachelor of Applied Science Major in Microbiology Supervisors: Associate Professor Christine Knox; Dr Mark Wellard; Dr John Duley; Dr Amitha Hewavitharana; Dr Saad Al Shehri. Submitted in fulfilment of the requirements for the degree of Master of Applied Science (Research) Institute of Health and Biomedical Innovation (IHBI) Faculty of Health Queensland University of Technology 2017 hplc analysis of nucleotides i Keywords HILIC Saliva HPLC-UV Nucleotides ii hplc analysis of nucleotides Abstract Nucleotides play central roles in cellular metabolism, for synthesis of nucleic acids (DNA and RNA), as carriers of chemical energy, and as secondary messengers. Inherited defects in nucleotide pathways thus cause a range of metabolic diseases, presenting as anaemia, immunodeficiency, renal disease or severe neuropathologies. Nucleotides are normally found only intracellularly, so cell death causing the release of nucleotides into the extracellular space results in ‘alarm signals’ that are widely recognised in the body. The importance of nucleotides in energy metabolism also makes them the metabolic focus of sports performance studies. The structure of nucleotides comprises three main components: a heterocyclic nitrogenous purine or pyrimidine base, a sugar moiety (ribose in nucleotides and RNA, deoxyribose sugar in DNA), and one or more phosphate groups. Purine and pyrimidine nucleotides are formed in cells by either de novo synthesis pathways or by ‘salvage’ (or ‘recycling’) of bases and nucleosides. Since the role of nucleotides is so important for cellular metabolism scientists have been encouraged to develop methods for their detection, identification and quantification in biological specimens. However, the highly-charged and polar characteristics of nucleotides make them unsuitable for reversed-phase HPLC.
  • We Have Previously Reported' the Isolation of Guanosine

    We Have Previously Reported' the Isolation of Guanosine

    VOL. 48, 1962 BIOCHEMISTRY: HEATH AND ELBEIN 1209 9 Ramel, A., E. Stellwagen, and H. K. Schachman, Federation Proc., 20, 387 (1961). 10 Markus, G., A. L. Grossberg, and D. Pressman, Arch. Biochem. Biophys., 96, 63 (1962). "1 For preparation of anti-Xp antisera, see Nisonoff, A., and D. Pressman, J. Immunol., 80, 417 (1958) and idem., 83, 138 (1959). 12 For preparation of anti-Ap antisera, see Grossberg, A. L., and D. Pressman, J. Am. Chem. Soc., 82, 5478 (1960). 13 For preparation of anti-Rp antisera, see Pressman, D. and L. A. Sternberger, J. Immunol., 66, 609 (1951), and Grossberg, A. L., G. Radzimski, and D. Pressman, Biochemistry, 1, 391 (1962). 14 Smithies, O., Biochem. J., 71, 585 (1959). 15 Poulik, M. D., Biochim. et Biophysica Acta., 44, 390 (1960). 16 Edelman, G. M., and M. D. Poulik, J. Exp. Med., 113, 861 (1961). 17 Breinl, F., and F. Haurowitz, Z. Physiol. Chem., 192, 45 (1930). 18 Pauling, L., J. Am. Chem. Soc., 62, 2643 (1940). 19 Pressman, D., and 0. Roholt, these PROCEEDINGS, 47, 1606 (1961). THE ENZYMATIC SYNTHESIS OF GUANOSINE DIPHOSPHATE COLITOSE BY A MUTANT STRAIN OF ESCHERICHIA COLI* BY EDWARD C. HEATHt AND ALAN D. ELBEINT RACKHAM ARTHRITIS RESEARCH UNIT AND DEPARTMENT OF BACTERIOLOGY, THE UNIVERSITY OF MICHIGAN Communicated by J. L. Oncley, May 10, 1962 We have previously reported' the isolation of guanosine diphosphate colitose (GDP-colitose* GDP-3,6-dideoxy-L-galactose) from Escherichia coli 0111-B4; only 2.5 umoles of this sugar nucleotide were isolated from 1 kilogram of cells. Studies on the biosynthesis of colitose with extracts of this organism indicated that GDP-mannose was a precursor;2 however, the enzymatically formed colitose was isolated from a high-molecular weight substance and attempts to isolate the sus- pected intermediate, GDP-colitose, were unsuccessful.
  • The Immediate Nucleotide Precursor, Guanosine Triphosphate, in the Riboflavin Biosynthetic Pathway1

    The Immediate Nucleotide Precursor, Guanosine Triphosphate, in the Riboflavin Biosynthetic Pathway1

    J. Nutr. Sci. Vitaminol., 23, 23-34, 1977 THE IMMEDIATE NUCLEOTIDE PRECURSOR, GUANOSINE TRIPHOSPHATE, IN THE RIBOFLAVIN BIOSYNTHETIC PATHWAY1 Hisateru MITSUDA,Kenji NAKAJIMA,and Tomonori NADAMOTO2 Laboratory of Nutritional Chemistry, Faculty of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606 (Received July 30, 1976) Summary In the present paper, the nucleotide precursor of riboflavin was investigated by experiments with labeled purines using non-growing cells of Eremothecium ashbyii. The added purines, at 10-4M, were effectively incorporated into ribo flavin at an early stage of riboflavin biosynthesis under the experimental conditions. In particular, both labeled xanthine and labeled guanine were specifically transported to guanosine nucleotides, GMP, GDP, GDP-Mannose and GTP, in the cource of the riboflavin biosynthesis. A comparison of specific activities of labeled guanosine nucleotides and labeled riboflavin indicated that the nucleotide precursor of riboflavin is guanosine triphosphate. From the results obtained, a biosynthetic pathway of riboflavin is proposed under "DISCUSSION." It has been established that purine is directly incorporated into riboflavin with the exception of C(8) of its imidazole ring (1-3). However, it remained unclear which purine is the most immediate precursor of riboflavin on the bio synthetic pathway, in spite of many laborious studies (1, 4-6). The present authors supposed that the different efficiencies of purines for flavinogenesis re sulted from the different activities of purine uptake and interconversions of purines and nucleotides in various microorganisms and even in a same microorganism(7,8). Accordingly, it is believed that added purines are introduced into the ribo flavin biosynthetic pathway through different pathways. This supposes that not a purine precursor but a nucleotide precursor should be determined in the bio synthetic pathway of riboflavin.
  • A New Case of Purine Nucleoside Phosphorylase Deficiency: Enzymologic, Clinical, and Immunologic Characteristics

    A New Case of Purine Nucleoside Phosphorylase Deficiency: Enzymologic, Clinical, and Immunologic Characteristics

    0031-3998/87/2102-0137$02.00/0 PEDIATRIC RESEARCH Vol. 21, No.2, 1987 Copyright© 1987 International Pediatric Research Foundation, Inc. Printed in U.S.A. A New Case of Purine Nucleoside Phosphorylase Deficiency: Enzymologic, Clinical, and Immunologic Characteristics GERT RIJKSEN, WIETSE KUIS, SYBE K. WADMAN, LEO J. M. SPAAPEN, MARINUS DURAN, B. S. VOORBROOD, GERARD E. J. STAAL, JAN W. STOOP, AND BEN J. M. ZEGERS Department ofHaematology, Division ofMedical Enzymology {G.R., G.E.J.S.j, University Hospital and University Hospital ji1r Children and Yowh "Het Wilhelmina Kinderziekenhuis," Utrecht, The Netherlands {W.K., S.K. W., L.J.M.S., M.D., B.J.M.Z.j and Juliana Ziekenhuis Department of Pediatrics, Rhenen, The Netherlands {B.S. V.] ABSTRACf. Deficiency of purine nucleoside phosphoryl­ SAH, S-adenosyl homocysteine hydrolase ase (PNP) was detected in a 3-yr-old boy who was admitted dGDP, deoxyguanosine diphosphate for investigation of a behavior disorder and spastic diplegia. The urinary excretion of purines, analyzed by high-per­ formance liquid chromatography, showed the presence of large amounts of (deoxy)inosine and (deoxy)guanosine and low uric acid levels. Analysis ofthe (deoxy)nucleotide pools Since the first description of PNP deficiency associated with of erythrocytes showed elevated levels of deoxyguanine cellular immunodeficiency (1), about 20 patients from more nucleotides and NAD and decreased guanine nucleotides. than 10 families have been described (2-5). Clinically, PNP PNP activity in red blood cells was 0.1-0.5% of normal on deficiency may manifest itself later in life than ADA deficiency, two occasions and undetectable on four later measure­ which affects cellular immune function and also humoral im­ ments.
  • Phosphate-Modified Cap Analogues Obtained Via Click Chemistry S

    Phosphate-Modified Cap Analogues Obtained Via Click Chemistry S

    Electronic Supplementary Material (ESI) for Chemical Science. This journal is © The Royal Society of Chemistry 2016 Electronic Supplementary Material (ESI) for Chemical Science. This journal is © The Royal Society of Chemistry 2016 A novel route for preparing 5cap mimics and capped RNAs: phosphate-modified cap analogues obtained via click chemistry S. Walczak,ab A. Nowicka,ac D. Kubacka,c K. Fac,ab P. Wanat,c S. Mroczek,de J. Kowalskac and J. Jemielitya aCentre of New Technologies, University of Warsaw, Banacha 2c, 02-097, Warsaw, Poland bCollege of Inter-Faculty Individual Studies in Mathematics and Natural Sciences, University of Warsaw, Banacha 2c, 02-097, Warsaw, Poland cDivision of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Zwirki i Wigury 93, 02-089, Warsaw, Poland dDepartment of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, 02-106 Warsaw, Poland eInstitute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland Supplementary Information Content Tables: ............................................................................................................................................................................................................. 4 Table S1. Yields of syntheses of triazole-modified dinucleotide cap analogues............................................................................................. 4 Table S2. Yields of the syntheses of azide- and alkyne-modified nucleotide analogues. ..............................................................................