DOCSLIB.ORG

Hplc Analysis of Nucleotides

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
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. Current methods available for nucleotide analysis are ion-pair and ion-exchange HPLC, but there are problems associated with these methods, particularly as they are unsuitable for mass spectrometry (MS), which uses extremes of pH and high concentrations of salts or ion-pairing reagents. Hydrophilic-interaction-HPLC (HILIC) is a relatively unexplored method, theoretically capable of separating highly polar molecules such as nucleotides. Nucleotides have been reported to be present at very low concentrations in adult saliva. The possibility of extracellular nucleotides occurring in a secretory fluid presented a biological ‘anomaly’ that was worthwhile investigating. Nucleosides, e.g. adenosine, have been shown to be present in high levels in neonatal saliva but not in adult saliva. We asked; are nucleotides present in neonatal saliva? hplc analysis of nucleotides iii Therefore the aim of this research project was to develop and validate a MS compatible HILIC method for the analysis of these very polar molecules, i.e. nucleotides, and to test the method with biological specimens, e.g. blood cells and saliva. The project aimed initially to separate adenosine monophosphate (AMP), adenosine diphosphate (ADP) and adenosine triphosphate (ATP), but this was later expanded to encompass twelve different nucleotides. Five different HILIC columns were assessed; the best performing was a Cosmosil column. A series of eluting buffers (ammonium acetate, ammonium carbonate and ammonium citrate) were tested. A simple, MS-compatible method based on elution by ammonium citrate and methanol was then developed. This method separated twelve nucleotides within 36 minutes. Futhermore, the method was applied to red blood cell extracts (RBCX), white blood cell extracts (WBCX), adult saliva and neonatal saliva. The method was not specifically optimised for these specimens, but nucleotides were separated and identified from these extracts. This method also detected and identified the unusual metabolite 4-methylpyridonecarboxamide triphosphate (‘4PY-nucleotide’). Previous research has shown that neonatal saliva contains elevated concentrations of nucleosides and bases, but nucleotides have not yet been investigated. This study, for the first time, detected highly elevated concentrations of nucleotides in neonatal saliva, when compared to the concentrations present in adult saliva For this project Dr Amitha Hewavitharana provided a novel concept; she proposed that “competing ion” within the mobile phase could be utilised to elute nucleotides. Citrate buffer proved to be the most effective at eluting all of the nucleotides, due to the strong 3-negative charge. The other elution buffers tested, acetate (1-negative ion) and carbonate (2-negative ion), were able to elute the mono- and di-phosphates respectively. The lower limits of detection and lower limits of quantification were sufficient to detect the presence of each nucleotide in biological specimens. The HILIC method, optimised for this Masters project, separated successfully twelve nucleotides in a standard and identified endogenous nucleotides in biological specimens (RBCX, WBCX, adult and neonatal saliva). iv hplc analysis of nucleotides The results indicated that the differences in nucleotide concentrations between adult and neonatal saliva are worthy of further study, as this phenomenon may have evolved as a selective mechanism to shape the oral and gut microbiota. This novel HILIC method overcomes limitations of previous nucleotide analyses. The citrate ion is theoretically MS-compatible, providing the basis for future studies of MS-based HPLC nucleotide analysis. hplc analysis of nucleotides v Table of Contents Keywords ................................................................................................................................. ii Abstract ................................................................................................................................... iii Table of Contents .................................................................................................................... vi List of Figures ....................................................................................................................... viii List of Tables ............................................................................................................................x List of Abbreviations .............................................................................................................. xi Statement of Original Authorship ......................................................................................... xiii Acknowledgements ............................................................................................................... xiv Chapter 1: Introduction ...................................................................................... 1 1.1 Nucleotides .....................................................................................................................2 1.2 Nucleotide Cellular Functions ........................................................................................3 1.3 Dietary and Saliva Nucleotides ......................................................................................5 1.4 Intracellular and Extracellular Nucleotides ....................................................................6 1.5 Gastrointestinal Tract Microbiota and Human Health ....................................................7 1.6 Neonatal gastrointestinal tract and oral microbiota in response to delivery method and feeding ............................................................................................................................9 1.7 The Role of Saliva Nucleosides and Bases on Regulating Microbiota ........................10 1.8 Nucleotide Analysis by High Performance Liquid Chromatography (HPLC) .............11 1.8.1 Reversed-phase (RP) HPLC ...............................................................................15 1.8.2 Normal-phase HPLC ..........................................................................................15 1.8.3 Ion-exchange HPLC ...........................................................................................16 1.8.4 Ion-pairing HPLC ...............................................................................................17 1.8.5 HILIC HPLC ......................................................................................................17 1.9 Hypothesis and Aims ....................................................................................................19 1.9.1 Hypothesis ..........................................................................................................19 1.9.2 Aims ...................................................................................................................19 1.9.3 Objectives ...........................................................................................................20 Chapter 2: Materials and Methods .................................................................. 21 2.1 Ethics ............................................................................................................................22 2.2 Materials .......................................................................................................................22 2.2.1 Chemicals ...........................................................................................................22 2.2.2 Columns..............................................................................................................23 2.2.3 Nucleotides .........................................................................................................23
Load more

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. 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.
    [Show full text]
  • 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.
    [Show full text]
  • 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.
    [Show full text]
  • 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.
    [Show full text]
  • 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].
    [Show full text]
  • 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.
    [Show full text]
  • 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.
    [Show full text]
  • 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.
    [Show full text]
  • 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.
    [Show full text]
  • 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.
    [Show full text]
  • 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. ..............................................................................
    [Show full text]
  • Concentration and Synthesis of Phosphoribosylpyrophosphate in Erythrocytes from Normal, Hyperuricemic, and Gouty Subjects
    Concentration and Synthesis of Phosphoribosylpyrophosphate in Erythrocytes From Normal, Hyperuricemic, and Gouty Subjects By FRANK L. MEYSKENS AND HIBBARD E. WILLIAMS Phosphoribosylpyrophosphate (PRPP) ADP, GDP, and 2,3-DPG was intact. The synthetase activity and the intracellular concentration of PRPP in erythrocytes concentration of PRPP were assayed in was higher in normal females than in erythrocytes from patients with primary normal males, higher in normal subjects hyperuricemia and primary metabolic than in gouty patients, and lower in hy- gout. Sensitivity of the enzyme to feed- peruricemic patients taking allopurinol back inhibition by adenosine diphosphate than in those hyperuricemic patients not (ADP), guanosine diphosphate (GDP), taking this drug. The difference in intra- and 2,3-diphosphoglycerate (2,3-DPG) cellular levels of PRPP in erythrocytes in was determined. All patients with gout gout versus hyperuricemic patients was and four of ten patients with hyperuric- not significant. The significance of these emia were taking ailopurinol during the findings is discussed in relation to the study. Mean PRPP synthetase activity in regulation of PRPP synthetase and in the erythrocytes from hyperuricemic and important regulatory role of PRPP in gouty patients was similar to that in nor- purine metabolism. mal subjects, and feedback inhibition by HE INTRACELLULAR CONCENTRATION of 5-phosphoribosyl-1- T pyrophosphate (PRPP) appears to be important in the regulation of purine metabolism. 1-4 Altered intracellular levels of PRPP have
    [Show full text]