DOCSLIB.ORG

The Mechanism of Action of 6-Mercaptopurine1'2 I. Biochemical Effects

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text

Load more

The Mechanism of Action of 6-Mercaptopurine1'2 I. Biochemical Effects JOSEPHINE SEE SALSER AND M. EARL BALlS (Divi8ion of Nucleoprolein Chemistry, Sloan-Kettering Institute for Cancer Research, Sloan-Kettering Divi8ion of Cornell University Medical College, New York, New York) SUMMARY The tumor Sarcoma 180 (5-180) was compared to host liver in an effort to evaluate the inhibitory action of 6-MP.3 The concentration of endogenous adenine nucleotides in S-180 was about 40 % of that found in liver, that of guanine nucleotides was about the same in both tissues, while that of inosinate was much lower in tumor. The liver of normal mice and 5-180-bearing mice exhibited the same capacity to convert IMP to AMP. A slightly greater conversion of 6-MP to 6-thio IMP in 5-180 was noted but it was not by itself of sufficient magnitude to explain the anti-tumor specificity of the compound. An 5-180 sensitive to 6-MP and one resistant to it each showed about one and a half times as much capacity to synthesize purines in vitro as host liver. The conversion of IMP to AMP by either 5-180 or liver was inhibited by 6-thio IMP ; the inhibition, though low, was greaten in the tumor. The actions of 6-thio IMP, reported by others in mammalian systems, are analogous to those previously found in bacterial and avian systems; i.e., the conversion of IMP to AMP and XMP is inhibited. It is suggested that the basis for the anti-tumor specificity may lie in differences in the enzymatic capacity of tumors and host tissues to carry out the con versions blocked by 6-thio IMP. Growth and isotope incorporation studies with bacterial tumor cells (1) have confirmed the observation that 6-thio systems (3—5,12, 13, 17, 18) have led to the hypothesis IMP is actually an effective competitive inhibitor of the that the active form of 6-MP is its ribonucleotide (thio conversion to xanthylate, the intermediate in the forma inosinate, 6-thio IMP), which inhibits the further conver tion of guanylate. sion by inosinate to other purine ribonucleotides (4, 5). Since the conversion of inosinate to other punine nibo This inhibition by thioinosinate of the formation of ade nucleotides occurs in both normal and tumor tissues, the nylate and guanylate has been demonstrated in cell-free selective susceptibility of tumors such as Sarcoma 180 preparations from Streptococcus faecalis and pigeon liver, (5-180) to the action of 6-MP must be considered in terms respectively (26). In L1210, studies on the incorporation of the total purine synthesis and interconversion mecha of labeled hypoxanthine into nucleic acid punines are con nisms. This paper presents some aspects of purine me sistent with the inhibition at the level of hypoxanthine, tabolism in 5-180 and host liver pertinent to nucleotide but only the conversion to adenylate seems to be seriously synthesis and conversion. The synthesis in vivo of 6-thio affected by 6-MP (10). More recent studies with partially IMP, the synthesis of purines de novo in in vitro systems, purified inosine-5'-phosphate-NAD-oxidoreductase (IMP and the inhibition of adenylate formation by 6-thio IMP dehydrogenase) from bacteria (15) and Ehrlich ascites in S-180 and liver have been studied. 1 This investigation was supported in part by funds from the National Cancer Institute, NIH, USPHS (CA-03190-08), and MATERIALS AND METHODS (5-K3-CA-16, 673-02). IMP@8@HC was obtained from Schwarz BioResearch, 2 A preliminary report has been presented (25). Inc. ; 6-MP-8-'4C and 4-amino-5-imidazole-carboxamide4- 3 The abbreviations used are : 6-MP, 6-mercaptopurine; 6-thio IMP, 6-thioinosinate; IMP, inosine monophosphate; AMP, aden ‘@C(AICA),from Southern Research Institute; glycine-1- osine monophosphate ; XMP, xanthosine monophosphate ; NAD, ‘4C,fromTracerlab. ATP (disodium salt) ; IMP (sodium nicotinamide adenine dinucleotide; AICA, 4-amino.5-imidazole salt); NAD (DPN); fructose-i ,6-diphosphate (sodium carboxamide; ATP, adenosine triphosphate; DPN, diphospho salt) ; a-ketoglutarate; L-glutamine ; nibose-5-phosphate pynidine nucleotide; TCA, trichloroacetic acid; GMP, guanosine monophosphate. (sodium salt); and tetrahydnofolic acid, were obtained Receivedfor publication September10,1964;revisedDecember from Sigma Chemical Company. 6-Thioinosinate (6-thio 23, 1964. IMP) was kindly supplied by Dr. A. Hampton. 539 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1965 American Association for Cancer Research. 540 Cancer Research Vol.25,May 1965 HA/ICR Swiss mice (approximately 20 gm each) with With labeled AlGA (specific activity = 4.11 X 10@cpm/ a 5- or a 7-day-old subcutaneous implant of 5-180 were smole) as precursor, the incubation medium was that de kindly supplied by Dr. H. Schwartz and Dr. C. Reilly of scribed by Schulman and Buchanan (27). In all the incu Sloan-Kettering Institute. Mice with 11-day-old sub bations, there was 250 mg wet weight of tissue/mi. cutaneous implants of a resistant variant of 5-180 were After a 15-min equilibration period, the labeled pre supplied by Dr. D. Clarke. Necrotic tumors were dis cursor was added and the incubation carried out for 2 hr at carded in all instances. 37.5°Cin a Dubnoff metabolic shaker. The reaction was Acetone powders of the tissues were prepared by stopped by immersion of the vessels in an acetone-Dry Ice homogenizing the liver or tumor with 10 volumes of cold bath. Carrier IMP was added to give a final concentra acetone (—25°C)in a Waring Blendor, followed by filter tion of 5 @moIes/ml. All subsequent steps were carried ing and rehomogenizing the resuspended cake in fresh cold out at 4°Cunless otherwise specified. acetone. The last 2 steps were repeated twice. The yield Sufficient cold 60 % TCA was then added to give a final of acetone powder from different batches of liver was about TCA concentration of 10 %. The precipitated protein 25 % of the wet weight while that from 5-180 was 12—15%. was removed by centnifugation and washed in the manner 6-MP incorporation in vivo.—Mice with 5-day-old im described above. After ether extraction, the supernatant plants of S-l80 were given i.v. (caudal vein) injections of solutions were lyophilized to dryness. Since the incuba 6-MP-8-'4C (50 mg/kg body weight, specific activity = tions were carried out in duplicate, one set of samples was 2.3 X 10@cpm/,@mo1e). The animals were sacrificed at dissolved in a minimal volume of 0.01 N HC1 and the nu 2, 6, and 24 hr after a single injection and at 48 hr after 3 cleotides were isolated as barium salts (24). To minimize successive daily injections of the labeled compound. The the adsorption of traces of labeled precursor, unlabeled liver and tumor were removed and chilled in chopped ice. AICA or glycine was added during the precipitation and All subsequent steps, unless otherwise specified, were 2 reprecipitations. The radioactivity was determined carried out at 4°C. The chilled tissues were homogenized and was shown by paper chromatography (i-butyric briefly in a Potter-Elvehjem homogenizer with 1 volume acid-NH3) to be associated with inosinate. of cold distilled water. Sufficient cold 20 % TCA was The duplicate set of samples was dissolved in water, ad added to give a final concentration of 10 % TCA and ho justed to pH 11 with NH4OH and put on Dowex-1 (chlo mogenization was continued for another 3 min. The pre ride form, X10, 200-400 mesh) columns. These were cipitated protein was removed by centrifugation, resus washed with at least 100 volumes of water and 0.003 N HC1 pended and rehomogenized 3 more times with cold 5% until the eluates were free of radioactivity. Inosinate TCA. The combined supernatant solutions were adjusted was subsequently eiuted with 0.005 N HC1, lyophilized to to 0.01 N with respect to HBr and extracted 8—10times dryness, and finally dissolved in water for radioactivity de with ether to remove the TCA. termination. The identity was further confirmed spec The components of this acid-soluble fraction (adjusted trally and by paper chromatography. to PH 9 with concentrated NH4OH) were separated on Conversion of inosinate to adenylate.—Cell-free extracts Dowex-1 (bromide form, XlO, 200—400mesh) colunms were prepared by homogenizing acetone powder or frozen into 3 fractions : (a) feed and wash water, containing tissue in a medium containing 0.16 M KC1 and 0.05 M Tnis nucleosides, nicotinamide, amino acids, and flavins, (b) buffer, pH 7.4, (21), (4 gm wet weight or the equivalent 0.006 N HBr, containing free purines, kNIP, and IMP, and amount of acetone powder/b ml) in a Virtis “45―ho (c) 0.18 N HBr, containing 6-thio I@1P with traces of GMP mogenizer for 3 min. To partially fractionate the active and ATP. These fractions were concentrated to dryness system(s) involved in this conversion, aliquots of the and taken up in a minimal measured volume of water. homogenate were centrifuged at 15,000 X g for 30 min and The radioactivities of aliquots of the fractions were de at 34,800 X g for 60 min5. All the operations were carried termined on infinitely thin films on aluminum planchets out at 4°C. in a Geiger-Muller internal flow counter. Paper chroma Each 10 ml of incubation mixture contained 2 gm wet tography of the 3rd fraction in various solvent systems weight or the equivalent amount of acetone powder; 2.5 (16) showed that all the radioactivity was associated with mmoles ATP ; 2.5 mmoles L-aspartate ; 5 mmoles fructose 6-thio IMP.
Recommended publications
  • 35 Disorders of Purine and Pyrimidine Metabolism

    35 Disorders of Purine and Pyrimidine Metabolism

    35 Disorders of Purine and Pyrimidine Metabolism Georges van den Berghe, M.- Françoise Vincent, Sandrine Marie 35.1 Inborn Errors of Purine Metabolism – 435 35.1.1 Phosphoribosyl Pyrophosphate Synthetase Superactivity – 435 35.1.2 Adenylosuccinase Deficiency – 436 35.1.3 AICA-Ribosiduria – 437 35.1.4 Muscle AMP Deaminase Deficiency – 437 35.1.5 Adenosine Deaminase Deficiency – 438 35.1.6 Adenosine Deaminase Superactivity – 439 35.1.7 Purine Nucleoside Phosphorylase Deficiency – 440 35.1.8 Xanthine Oxidase Deficiency – 440 35.1.9 Hypoxanthine-Guanine Phosphoribosyltransferase Deficiency – 441 35.1.10 Adenine Phosphoribosyltransferase Deficiency – 442 35.1.11 Deoxyguanosine Kinase Deficiency – 442 35.2 Inborn Errors of Pyrimidine Metabolism – 445 35.2.1 UMP Synthase Deficiency (Hereditary Orotic Aciduria) – 445 35.2.2 Dihydropyrimidine Dehydrogenase Deficiency – 445 35.2.3 Dihydropyrimidinase Deficiency – 446 35.2.4 Ureidopropionase Deficiency – 446 35.2.5 Pyrimidine 5’-Nucleotidase Deficiency – 446 35.2.6 Cytosolic 5’-Nucleotidase Superactivity – 447 35.2.7 Thymidine Phosphorylase Deficiency – 447 35.2.8 Thymidine Kinase Deficiency – 447 References – 447 434 Chapter 35 · Disorders of Purine and Pyrimidine Metabolism Purine Metabolism Purine nucleotides are essential cellular constituents 4 The catabolic pathway starts from GMP, IMP and which intervene in energy transfer, metabolic regula- AMP, and produces uric acid, a poorly soluble tion, and synthesis of DNA and RNA. Purine metabo- compound, which tends to crystallize once its lism can be divided into three pathways: plasma concentration surpasses 6.5–7 mg/dl (0.38– 4 The biosynthetic pathway, often termed de novo, 0.47 mmol/l). starts with the formation of phosphoribosyl pyro- 4 The salvage pathway utilizes the purine bases, gua- phosphate (PRPP) and leads to the synthesis of nine, hypoxanthine and adenine, which are pro- inosine monophosphate (IMP).
  • Utilisation Des Cellules Souches Pluripotentes Pour Le Criblage À Haut Débit De Molécules Thérapeutiques

    Utilisation Des Cellules Souches Pluripotentes Pour Le Criblage À Haut Débit De Molécules Thérapeutiques

    Utilisation des cellules souches pluripotentes pour le criblage à haut débit de molécules thérapeutiques dans la maladie de Lesch-Nyhan : 2019SACLE011 NNT Thèse de doctorat de l'Université Paris-Saclay, préparée à l’Université d’Evry Val-d’Essonne École doctorale n°569 Innovation thérapeutique : du fondamental à l’appliqué (ITFA) Spécialité de doctorat: Immunologie Thèse présentée et soutenue à Corbeil-Essonnes, le 01 juillet 2019, par Valentin Ruillier Composition du Jury : Dr. Olivier Goureau Institut de la Vision, Université Sorbonne, Paris Président Pr. Odile Boespflug-Tanguy Université Paris Diderot, Hôpital Robert Debré, Paris Rapporteur Dr. Amélie Piton IGBMC, Hôpitaux Universitaires de Strasbourg Rapporteur Dr. Terence Beghyn APTEEUS, Lille Examinateur Pr. Christelle Monville UEVE/INSERM U861, I-STEM, Corbeil-Essonnes Directeur de thèse Dr. Alexandra Benchoua CECS, I-STEM, Corbeil-Essonnes Co-Encadrant Résumé - Abstract Mots clés : Maladie de Lesch-Nyhan, cellules souches pluripotentes, iPSC, criblage à haut débit, HGPRT, purines Résumé : Les mutations affectant la fonction identifier, par une approche de criblage à haut d’enzymes impliquées dans le cycle des purines débit, de nouvelles molécules chimiques capables sont responsables d’une multitude de syndromes de corriger ces défauts. Plus de 3000 molécules pédiatriques, caractérisés par des atteintes ont été testées et 6 composés, tous dérivés de neurologiques et comportementales. A ce jour, l’adénosine, ont pu être identifiés comme aucune stratégie thérapeutique n’a été compensant le métabolisme par un mécanisme réellement efficace pour contrôler ces d’action indépendant de l’HGPRT. De manière symptômes. La maladie de Lesch-Nyhan (MLN), intéressante, un des composés, la S- associée à la perte de fonction de l’enzyme de adenosylmethionine (SAM) a par le passé déjà recyclage HGPRT, constitue un bon modèle démontré des effets bénéfiques sur les d’étude.
  • Forms of Hypoxanthine Or, Through Interconversions, Guanine Or Adenine

    Forms of Hypoxanthine Or, Through Interconversions, Guanine Or Adenine

    826 GENETICS: GOTS AND GOLLUB PROC. N. A. S. Summary.-The ribonucleic acids of isolated thymus nuclei can be separated into two distinct fractions, one of which probably represents ribonucleic acid of the nu- cleolus. Studies of the incorporation of orotic acid-6-C"4 and adenosine-8-C'4 into these RNA fractions in vitro show great differences in their metabolic activity and different susceptibilities to an inhibitor of RNA synthesis, the "nucleolar" RNA being by far the more active. It is a pleasure to acknowledge our indebtedness to Mr. Rudolf Meudt for his careful and expert technical assistance. * This research was supported in part by a grant (RG-4919 M&G) from the United States Public Health Service. I V. G. Allfrey, A. E. Mirsky, and S. Osawa, Nature, 176, 1042, 1955. 2 V. G. Allfrey, A. E. Mirsky, and S. Osawa, J. Gen. Physiol., 40, 451, 1957. 3 R. Logan and J. N. Davidson, Biochim. et Biophys. Acta, 24, 196, 1957. 4 S. Osawa, K. Takata, and Y. Hotta (in press). 6 J. M. Webb, J. Biol. Chem., 221, 635, 1956. 6 M. Bessis, in Traite de cytologie sanguine (Paris: Masson & Cie, 1954), p. 83. J. N. Davidson and R. M. S. Smellie. Biochem. J.. 52, 594, 1952. SEQUENTIAL BLOCKADE IN ADENINE BIOSYNTHESIS BY GENETIC LOSS OF AN APPARENT BIFUNCTIONAL DEACYLASE* By JOSEPH S. GOTS AND EDITH G. GOLLUB DEPARTMENT OF MICROBIOLOGY, SCHOOL OF MEDICINE, UNIVERSITY OF PENNSYLVANIA, PHILADELPHIA, PENNSYLVANIA Communicated by D. Wright Wilson, July 3, 1957 Along the biosynthetic pathway to the purines of nucleic acids, inosinic acid occurs as a pivotal point in a bifurcation which leads to adenylic acid along one branch and to guanylic acid along the other: (B) r- Adenylic acid (AMP) (A) Inosinic acid (IMP) (C) L|_ * Guanylic acid (GMP) Bacterial mutations may result in genetic impairments at three locations with respect to the pivotal inosinic acid, thus yielding auxotrophs with three broad classes of nutritional response.
  • Reconstituted IMPDH Polymers Accommodate Both Catalytically Active and Inactive Conformations

    Reconstituted IMPDH Polymers Accommodate Both Catalytically Active and Inactive Conformations

    M BoC | BRIEF REPORT Reconstituted IMPDH polymers accommodate both catalytically active and inactive conformations Sajitha A. Anthonya,†, Anika L. Burrellb,†, Matthew C. Johnsonb, Krisna C. Duong-Lya, Yin-Ming Kuoa, Jacqueline C. Simoneta, Peter Michenerc, Andrew Andrewsa, Justin M. Kollmanb,†,*, and Jeffrey R. Petersona,†,* aCancer Biology Program, Fox Chase Cancer Center, Philadelphia, PA 19111; bDepartment of Biochemistry, University of Washington, Seattle, WA 98195; cDepartment of Biochemistry & Molecular Biology, Drexel University College of Medicine, Philadelphia, PA 19102 ABSTRACT Several metabolic enzymes undergo reversible polymerization into macromo- Monitoring Editor lecular assemblies. The function of these assemblies is often unclear, but in some cases they Diane Barber regulate enzyme activity and metabolic homeostasis. The guanine nucleotide biosynthetic University of California, San Francisco enzyme inosine monophosphate dehydrogenase (IMPDH) forms octamers that polymerize into helical chains. In mammalian cells, IMPDH filaments can associate into micron-length Received: Apr 25, 2017 assemblies. Polymerization and enzyme activity are regulated in part by binding of purine Revised: Aug 2, 2017 nucleotides to an allosteric regulatory domain. ATP promotes octamer polymerization, Accepted: Aug 4, 2017 whereas guanosine triphosphate (GTP) promotes a compact, inactive conformation whose ability to polymerize is unknown. Also unclear is whether polymerization directly alters IMPDH catalytic activity. To address this, we identified point mutants of human IMPDH2 that either prevent or promote polymerization. Unexpectedly, we found that polymerized and nonassembled forms of recombinant IMPDH have comparable catalytic activity, substrate affinity, and GTP sensitivity and validated this finding in cells. Electron microscopy revealed that substrates and allosteric nucleotides shift the equilibrium between active and inactive conformations in both the octamer and the filament.
  • Mechanisms of Synthesis of Purine Nucleotides in Heart Muscle Extracts

    Mechanisms of Synthesis of Purine Nucleotides in Heart Muscle Extracts

    Mechanisms of Synthesis of Purine Nucleotides in Heart Muscle Extracts David A. Goldthwait J Clin Invest. 1957;36(11):1572-1578. https://doi.org/10.1172/JCI103555. Research Article Find the latest version: https://jci.me/103555/pdf MECHANISMS OF SYNTHESIS OF PURINE NUCLEOTIDES IN HEART MUSCLE EXTRACTS1 BY DAVID A. GOLDTHWAIT2 (From the Departments of Biochemistry and Medicine, Western Reserve University, Cleveland, Ohio) (Submitted for publication February 18, 1957; accepted July 18, 1957) The key role of ATP, a purine nucleotide, in 4. Adenine or Hypoxanthine + PRPP -> AMP the conversion of chemical energy into mechanical or Inosinic Acid (IMP) + P-P. work by myocardial tissue is well established (1, The third mechanism of synthesis is through the 2). The requirement for purine nucleotides has phosphorylation of a purine nucleoside (8, 9): also been demonstrated in the multiple synthetic 5. Adenosine + ATP -, AMP + ADP. reactions which maintain all animal cells in the Several enzymatic mechanisms are known which steady state. Since the question immediately arises result in the degradation of purine nucleotides and whether the purine nucleotides are themselves in nucleosides. The deamination of adenylic acid is a steady state, in which their rates of synthesis well known (10): equal their rates of degradation, it seems reason- 6. AMP -* IMP + NH8. able to investigate first what mechanisms of syn- Non-specific phosphatases (11) as well as spe- thesis and degradation may be operative. cific 5'-nucleotidases (12) have been described At present, there are three known pathways for which result in dephosphorylation: the synthesis of purine nucleotides. The first is 7.
  • Calcium 5'-Ribonucleotides

    Calcium 5'-Ribonucleotides

    CALCIUM 5'-RIBONUCLEOTIDES Prepared at the 18th JECFA (1974), published in NMRS 54B (1975) and in FNP 52 (1992). Metals and arsenic specifications revised at the 57th JECFA (2001). An ADI ‘not specified’ was established at the 18th JECFA (1974). SYNONYMS Calcium ribonucleotides, INS No. 634 DEFINITION Chemical names (Mixture of) calcium inosine-5'-monophosphate and calcium guanosine-5'- monophosphate Chemical formula C10H11CaN4O8P · x H2O and C10H12CaN5O8P · x H2O Structural formula Calcium 5’-guanylate Calcium 5’-inosinate Assay Not less than 97% and not more than the equivalent of 102% of C10H11CaN4O8P and C10H12CaN5O8P, calculated on the anhydrous basis. The proportion of C10H11CaN4O8P or C10H12CaN5O8P to the sum of them is between 47% and 53%. DESCRIPTION Odourless, white or off-white crystals or powder FUNCTIONAL USES Flavour enhancer CHARACTERISTICS IDENTIFICATION Solubility (Vol. 4) Sparingly soluble in water Test for ribose (Vol. 4) Passes test Test for organic phosphate Passes test (Vol. 4) Test 5 ml of a 1 in 2,000 solution Test for inosinic acid To 2 ml of a 1 in 2,000 solution add 2 ml of 10% hydrochloric acid and 0.1 g of zinc powder, heat in a water bath for 10 min, and filter. Cool the filtrate in ice water, add 1 ml of a 3 in 1,000 sodium nitrite solution, shake well, and allow to stand for 10 min. Add 1 ml of a 1 in 200 ammonium sulfamate solution, shake well, and allow to stand for 5 min. Add 1 ml of a 1 in 500 N-(1- naphthyl)-ethylenediamine dihydrochloride solution.
  • Small Elevations of Glucose Concentration Redirect and Amplify the Synthesis of Guanosine 5'-Triphosphate in Rat Islets

    Small Elevations of Glucose Concentration Redirect and Amplify the Synthesis of Guanosine 5'-Triphosphate in Rat Islets

    Small elevations of glucose concentration redirect and amplify the synthesis of guanosine 5'-triphosphate in rat islets. S A Metz, … , M E Rabaglia, A Kowluru J Clin Invest. 1993;92(2):872-882. https://doi.org/10.1172/JCI116662. Research Article Recent studies suggest a permissive requirement for guanosine 5'-triphosphate (GTP) in insulin release, based on the use of GTP synthesis inhibitors (such as myocophenolic acid) acting at inosine monophosphate (IMP) dehydrogenase; herein, we examine the glucose dependency of GTP synthesis. Mycophenolic acid inhibited insulin secretion equally well after islet culture at 7.8 or 11.1 mM glucose (51% inhibition) but its effect was dramatically attenuated when provided at < or = 6.4 mM glucose (13% inhibition; P < 0.001). These observations were explicable by a stimulation of islet GTP synthesis derived from IMP since, at high glucose: (a) total GTP content was augmented; (b) a greater decrement in GTP (1.75 vs. 1.05 pmol/islet) was induced by mycophenolic acid; and (c) a smaller "pool" of residual GTP persisted after drug treatment. Glucose also accelerated GTP synthesis from exogenous guanine ("salvage" pathway) and increased content of a pyrimidine, uridine 5'-triphosphate (UTP), suggesting that glucose augments production of a common regulatory intermediate (probably 5-phosphoribosyl-1-pyrophosphate). Pathway-specific radiolabeling studies confirmed that glucose tripled both salvage and de novo synthesis of nucleotides. We conclude that steep changes in the biosynthesis of cytosolic pools of GTP occur at modest changes in glucose concentrations, a finding which may have relevance to the adaptive (patho) physiologic responses of islets to changes in ambient glucose levels.
  • Effects of Allopurinol and Oxipurinol on Purine Synthesis in Cultured Human Cells

    Effects of Allopurinol and Oxipurinol on Purine Synthesis in Cultured Human Cells

    Effects of allopurinol and oxipurinol on purine synthesis in cultured human cells William N. Kelley, James B. Wyngaarden J Clin Invest. 1970;49(3):602-609. https://doi.org/10.1172/JCI106271. Research Article In the present study we have examined the effects of allopurinol and oxipurinol on thed e novo synthesis of purines in cultured human fibroblasts. Allopurinol inhibits de novo purine synthesis in the absence of xanthine oxidase. Inhibition at lower concentrations of the drug requires the presence of hypoxanthine-guanine phosphoribosyltransferase as it does in vivo. Although this suggests that the inhibitory effect of allopurinol at least at the lower concentrations tested is a consequence of its conversion to the ribonucleotide form in human cells, the nucleotide derivative could not be demonstrated. Several possible indirect consequences of such a conversion were also sought. There was no evidence that allopurinol was further utilized in the synthesis of nucleic acids in these cultured human cells and no effect of either allopurinol or oxipurinol on the long-term survival of human cells in vitro could be demonstrated. At higher concentrations, both allopurinol and oxipurinol inhibit the early steps ofd e novo purine synthesis in the absence of either xanthine oxidase or hypoxanthine-guanine phosphoribosyltransferase. This indicates that at higher drug concentrations, inhibition is occurring by some mechanism other than those previously postulated. Find the latest version: https://jci.me/106271/pdf Effects of Allopurinol and Oxipurinol on Purine Synthesis in Cultured Human Cells WILLIAM N. KELLEY and JAMES B. WYNGAARDEN From the Division of Metabolic and Genetic Diseases, Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina 27706 A B S TR A C T In the present study we have examined the de novo synthesis of purines in many patients.
  • Nucleotide Metabolism Pathway: the Achilles' Heel for Bacterial Pathogens

    Nucleotide Metabolism Pathway: the Achilles' Heel for Bacterial Pathogens

    REVIEW ARTICLES Nucleotide metabolism pathway: the achilles’ heel for bacterial pathogens Sujata Kumari1,2,* and Prajna Tripathi1,3 1National Institute of Immunology, New Delhi 110 067, India 2Present address: Department of Zoology, Magadh Mahila College, Patna University, Patna 800 001, India 3Present address: Institute of Molecular Medicine, Jamia Hamdard, New Delhi 110 062, India de novo pathway, the nucleotides are synthesized from Pathogens exploit their host to extract nutrients for their survival. They occupy a diverse range of host simple precursor molecules. In the salvage pathway, the niches during infection which offer variable nutrients preformed nucleobases or nucleosides which are present accessibility. To cause a successful infection a patho- in the cell or transported from external environmental gen must be able to acquire these nutrients from the milieu to the cell are utilized to form nucleotides. host as well as be able to synthesize the nutrients on its own, if required. Nucleotides are the essential me- tabolite for a pathogen and also affect the pathophysi- Purine biosynthesis pathway ology of infection. This article focuses on the role of nucleotide metabolism of pathogens during infection The purine biosynthesis pathway is universally conserved in a host. Nucleotide metabolism and disease pathoge- in living organisms (Figure 1). As an example, we here nesis are closely related in various pathogens. Nucleo- present the pathway derived from well-studied Gram- tides, purines and pyrimidines, are biosynthesized by positive bacteria Lactococcus lactis. In the de novo the de novo and salvage pathways. Whether the patho- pathway the purine nucleotides are synthesized from sim- gen will employ the de novo or salvage pathway dur- ple molecules such as phosphoribosyl pyrophosphate ing infection is dependent on various factors, like (PRPP), amino acids, CO2 and NH3 by a series of enzy- availability of nucleotides, energy condition and pres- matic reactions.
  • Alternative Pathways of Glucose Metabolism II. Nucleotides from The

    Alternative Pathways of Glucose Metabolism II. Nucleotides from The

    Alternative Pathways of Glucose Metabolism II . Nucleotides from the Acid-soluble Fraction of Normal and Tumor Tissues and Studies on Nucleic Acid Synthesis in Tumors*t HANNS SCHMITZ4 VAN R. POTTER, ROBERT B. HTJRLBERT,@ AND DWAIN M. WHITE (McArdie Memovial Laboratory, the Medical School, University of Wisconain, Madison, WI..) The first paper (18) in this series on the alterna MATERIALS AND METHODS tive pathways of glucose metabolism established The present study has involved the measure the fact that radioactivity from glucose-i-C'4 was ment of the specific activities of the free 5' mono-, readily incorporated into the pentose moiety of the di-, and triphosphates of adenosine, guanosine, ribonucleic and desoxyribonucleic acids of Flexner cytidine, and uridine from the acid-soluble extract Jobling tumors in rats, and described the over-all of tumor tissue in relation to the specific activities distribution of radioactivity in the acid-soluble of the corresponding nucleotides that were oh and acid-insoluble fractions of tumor and liver tis tamed by chemical or enzymatic hydrolysis of the sue at various time periods. The acid-soluble frac nucleic acids from the same tissue samples, at tion of tissue contains an appreciable amount of specified time intervals after the injection of glu free nucleotides which are possible intermediary cose-1-C'4. The experimental plan corresponds compounds in the synthesis of the nucleic acids exactly to that described in the preceding paper; (6, 7, 8, 9, 13, 16). It was noted (18) that as the C'4 many of
  • Mechanism of Excessive Purine Biosynthesis in Hypoxanthine- Guanine Phosphoribosyltransferase Deficiency

    Mechanism of Excessive Purine Biosynthesis in Hypoxanthine- Guanine Phosphoribosyltransferase Deficiency

    Mechanism of excessive purine biosynthesis in hypoxanthine- guanine phosphoribosyltransferase deficiency Leif B. Sorensen J Clin Invest. 1970;49(5):968-978. https://doi.org/10.1172/JCI106316. Research Article Certain gouty subjects with excessive de novo purine synthesis are deficient in hypoxanthineguanine phosphoribosyltransferase (HG-PRTase [EC 2.4.2.8]). The mechanism of accelerated uric acid formation in these patients was explored by measuring the incorporation of glycine-14C into various urinary purine bases of normal and enzyme-deficient subjects during treatment with the xanthine oxidase inhibitor, allopurinol. In the presence of normal HG-PRTase activity, allopurinol reduced purine biosynthesis as demonstrated by diminished excretion of total urinary purine or by reduction of glycine-14C incorporation into hypoxanthine, xanthine, and uric acid to less than one-half of control values. A boy with the Lesch-Nyhan syndrome was resistant to this effect of allopurinol while a patient with 12.5% of normal enzyme activity had an equivocal response. Three patients with normal HG-PRTase activity had a mean molar ratio of hypoxanthine to xanthine in the urine of 0.28, whereas two subjects who were deficient in HG-PRTase had reversal of this ratio (1.01 and 1.04). The patterns of 14C-labeling observed in HG-PRTase deficiency reflected the role of hypoxanthine as precursor of xanthine. The data indicate that excessive uric acid in HG-PRTase deficiency is derived from hypoxanthine which is insufficiently reutilized and, as a consequence thereof, catabolized inordinately to uric acid. The data provide evidence for cyclic interconversion of adenine and hypoxanthine derivatives. Cleavage of inosinic acid to hypoxanthine via inosine does […] Find the latest version: https://jci.me/106316/pdf Mechanism of Excessive Purine Biosynthesis in Hypoxanthine-Guanine Phosphoribosyltransferase Deficiency LEIF B.
  • 1 T CELL ACTIVATION TRIGGERS REVERSIBLE INOSINE-5'-MONOPHOSPHATE DEHYDROGENASE ASSEMBLY Krisna C. Duong-Ly 1, Yin-Ming Kuo 2

    1 T CELL ACTIVATION TRIGGERS REVERSIBLE INOSINE-5'-MONOPHOSPHATE DEHYDROGENASE ASSEMBLY Krisna C. Duong-Ly 1, Yin-Ming Kuo 2

    bioRxiv preprint doi: https://doi.org/10.1101/315929; this version posted May 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. T CELL ACTIVATION TRIGGERS REVERSIBLE INOSINE-5’-MONOPHOSPHATE DEHYDROGENASE ASSEMBLY Krisna C. Duong-Ly1, Yin-Ming Kuo2, Matthew C. Johnson3, Justin M. Kollman3, Jonathan Soboloff4, Glenn F. Rall5, Andrew J. Andrews2, and Jeffrey R. Peterson1 1Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, PA 2Cancer Epigenetics Program, Fox Chase Cancer Center, Philadelphia, PA 3Department of Biochemistry, University of Washington, Seattle, WA 4Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA 5Blood Cell Development and Function Program, Fox Chase Cancer Center, Philadelphia, PA Correspondence to Jeffrey R. Peterson: [email protected], Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, ORCID: 0000-0002-0604-718X; Yin-Ming Kuo’s present address is Department of Radiology, University of Pennsylvania School of Medicine, Philadelphia, PA Short running title: IMPDH FILAMENT ASSEMBLY IN T CELLS Abbreviations used: IMP: inosine-5’-monophosphate IMPDH: inosine-5’-monophosphate dehydrogenase 1 bioRxiv preprint doi: https://doi.org/10.1101/315929; this version posted May 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.