DOCSLIB.ORG

Triphosphate- and Deoxyadenosine 5'- Triphosphate-Activated Nucleotidase from Human Malignant Lymphocytes1

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Triphosphate- and Deoxyadenosine 5'- Triphosphate-Activated Nucleotidase from Human Malignant Lymphocytes1 [CANCER RESEARCH 42, 4321-4324, November 1982] 0008-5472/82/0042-OOOOS02.00 Characterization of an Adenosine S'-Triphosphate- and Deoxyadenosine 5'- Triphosphate-activated Nucleotidase from Human Malignant Lymphocytes1 Dennis A. Carson and D. Bruce Wasson Department of Clinical Research, Scripps Clinic and Research Foundation, La Jolla, California 92037 ABSTRACT dATP (5, 14, 19, 23). Subsequently, ATP levels, and indeed the total intracellular pools of adenine nucleotides, slowly de The kinetic properties of a soluble, magnesium-dependent cline (1,5,19). The individual enzymes potentially catalyzing 5'-nucleotidase from human malignant lymphocytes have been nucleotide degradation in lymphocytes with elevated dATP determined. The partially purified enzyme is distinct from levels have not been characterized. Recently, an ATP- and plasma membrane-associated 5'-nucleotidase and is free of dATP-stimulated IMP-dephosphorylating activity was de nonspecific phosphatase activity. Among purine ribonucleo- scribed in crude extracts of a human T-lymphoblastoid cell line tides, it reacted efficiently with inosine 5'-monophosphate and guanosine 5'-monophosphate and to a lesser degree with (1). deoxyguanosine 5'-monophosphate. Adenosine 5'-monophos- In the present investigations, we have partially purified from phate and deoxyadenosine 5'-monophosphate were 30-fold malignant human lymphocytes a soluble nucleotidase with properties analogous to the rat and chicken liver enzymes. The less efficient substrates. Increasing concentrations of adeno- sine S'-triphosphate and deoxyadenosine 5'-triphosphate from kinetics of the enzyme has been determined, with special 0 to 3 rriM enhanced 5'-nucleotidase activity up to 7-fold. reference to the effects of adenine deoxynucleotides as sub Guanosine 5'-triphosphate and deoxyguanosine 5'-triphos- strates and activators. phate were much less effective enzyme activators, while uridine S'-triphosphate was without effect. Inorganic phosphate in MATERIALS AND METHODS hibited dephosphorylating activity in both adenosine 5'-triphos- Enzyme Purification. Splenic tissue, largely replaced by a well- phate-supplemented and unsupplemented buffer. The activa differentiated lymphocytic lymphoma, was removed during surgery and tion of this 5'-nucleotidase by deoxyadenosine 5'-triphosphate, then frozen at -70° for 3 weeks. 5'-Nucleotidase was purified from combined with the relative inability of the enzyme to dephos- the rapidly thawed specimen, following the method developed by Itoh phorylate deoxyadenosine 5'-monophosphate, conceivably (11), except that the second phosphocellulose column was eluted with may contribute to the adenine nucleotide degradation induced a linear gradient of 200 to 800 mw NaCI instead of with ATP and the by deoxyadenosine in normal and malignant lymphocytes. low-ionic strength precipitation step was omitted. When stored as described by Itoh (11), the enzyme was stable at -70° for at least 1 month. INTRODUCTION 5'-Nucleotidase Assay. 5'-Nucleotidase activity was determined radiochemically using [8-14C]IMP as substrate and by measuring the Intracellular nucleotide degradation in human cells is highly release of inorganic phosphate from various nucleoside 5'-monophos- regulated (10). However, the exact enzymes catalyzing the phates, exactly as described earlier (4). Unless stated otherwise, the dephosphorylation of purine 5'-monophosphates have not buffer was 100 mw imidazole-HCI (pH 6.5), 50 rnw MgCI2, 500 mw been well characterized. The most abundant human 5'-nucle- NaCI, 0.1% bovine serum albumin, containing varying concentrations otidase [5'-ribonucleotide phosphohydrolase (EC 3.1.3.5)] is of nucleoside 5'-monophosphate, and effectors as indicated. The re on the external surface of the plasma membrane and probably actions were initiated by the addition of 1 to 5 HQenzyme protein and, after 15 to 45 min at 37°, were terminated by heating to 100° for 1 plays no role in intracellular nucleotide degradation (2, 5, 18, 19). min. For the radiochemical assay, inosine was separated from nucleo Recently, several investigators have described a novel 5'- tides by chromatography on polyethyleneimine-cellulose jn methanol: water (1:1) in the presence of appropriate standards (4). The nucleo- nucleotidase in the cytoplasm of rat liver and chicken liver that is distinct from the plasma membrane enzyme (11 -13, 24-26). sides were visualized under UV, cut out, and counted in a liquid scintillation spectrometer. Enzyme activities are expressed as nmol The activity of the cytosolic enzyme was enhanced by ATP at product per min per mg protein and were linear with protein concentra concentrations that inhibited plasma membrane 5'-nucleotid- tion and with time for the data reported. Protein content was determined ase. Several lines of evidence suggested that in rat liver the by the method of Lowry ef al. (18), using bovine serum albumin as a ATP-stimulated cytoplasmic nucleotidase was a predominant standard. enzyme catalyzing the dephosphorylation of purine nucleotides Materials. All common nucleosides and nucleotides were purchased (24-26). from Sigma Chemical Co. (St. Louis, Mo.). The [8-14C]IMP (50 mCi/ In the presence of an inhibitor of adenosine deaminase, mmol) came from Amersham/Searle Corp. (Arlington Heights, III.) and was purified by high-performance liquid chromatography. ATP and nondividing human lymphocytes exposed to /¿Mconcentrations GTP were purified immediately before use by DEAE-cellulose chro of deoxyadenosine in vitro or in vivo progressively accumulate matography, using a 100 to 500 ITIMNaCI gradient in 5 mw potassium phosphate, pH 5.0, and contained less than 2 nmol orthophosphate ' Supported by Grants GM 23200 and CA 31497 from the USPHS. per /imol nucleotide. Other materials were of the highest grade com Received April 26. 1982; accepted July 21, 1982. mercially available. NOVEMBER 1982 4321 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1982 American Association for Cancer Research. D. A. Carson and D. B. Wasson RESULTS Enzyme Purification. Because the IMP-dephosphorylating activity in the crude lymphocyte extract (5.0 nmol/min/mg protein) represented a mixture of nucleotidases and phospha- tases, it was not possible to determine the exact purification dGMP factor or yield of the 5'-nucleotidase. However, the estimated cytosolic nucleotidase activity in lymphocytes ranged from 0.4 to 1.0 nmol/min/mg protein. The final enzyme preparation was free of detectable ß-glycerophosphate- and ATP-dephosphor- ylating activities (<0.5 nmol dephosphorylated/min/mg pro AMP tein at a substrate concentration of 40 ITIM)(4). It also lacked measurable adenosine deaminase or purine nucleoside phos- phorylase (3). At an IMP concentration of 3 mM in the standard 4 5 buffer lacking ATP, the specific activity of the partially purified Substrate(mM) enzyme was 1.4 /imol/min/mg protein. The total yield from Chart 2. Relation of substrate concentration to enzyme velocity. The reaction 14.2 starting material was 2.7 mg protein, containing 3.78 conditions were the same as in Chart 1 except that varying concentrations of jumol IMP-dephosphorylating activity per min. IMP, dIMP, QMP, dGMP, and AMP were used. Effect of pH and MgCI2. When assayed in imidazole-HCI buffer with IMP as substrate, the pH optimum of the enzyme was 6.4 (Chart 1). At neutral pH, 5'-nucleotidase activity was approximately 50% maximal. The enzyme activity was not inhibited by 10 mM tartrate or 5 mM jS-glycerophosphate. Ten mM fluoride inhibited enzyme activity by 65%. In the absence of added MgCI2, no IMP- dephosphorylating activity was measurable. Il Substrate Specificity. The lymphocyte 5'-nucleotidase de- phosphorylated all 8 purine and pyrimidine 5'-monophosphates studied (Table 1). Over the range of concentrations tested, 0.4 10 20 30 40 50 Substrate (mM) Chart 3. Relation of substrate concentration to enzyme velocity and effect of ATP. The reaction conditions were as described in Chart 1 except that varying concentrations of AMP and dAMP were used, insert, effect of 3 mM ATP upon 0.1 the rate of the reactions. substrate-velocity plots were hyperbolic with the preferred 5.5 6 6.5 7.5 substrates IMP, dIMP, GMP, dGMP (Chart 2), and CMP (not pH shown). Linear regression analysis of Eadie-Hofstee plots of Chart 1. Effect of pH on 5'-nucleotidase activity. Enzyme activity was assayed the data by the method of least squares yielded r* values in 100 mM imidazole-HCI containing 50 IÕIMMgCI.. 0.1% bovine serum albumin, 2 mM IMP, and 1.5 «gprotein in a total volume of 100 »IProduct formation was >0.90 in each case. determined after 45 min at 37" by the inorganic phosphate method. With the inefficient substrates AMP and dAMP (Chart 3), Table 1 substrate-velocity plots were approximately linear at concen Substrate specificity of the ¿'-nucleotidase trations up to 12 mM. Maximal enzyme activity was approached for each nucleotide, product formation was determined by the release of only at nucleotide concentrations of 30 to 50 mM, far above inorganic phosphate at substrate concentrations from 0.1 to 100 mM. The any achievable physiological range. With the latter 2 sub reaction buffer lacked ATP or inorganic phosphate. The V,,,,. values for the respective nucleotides are shown relative to IMP, for which the specific activity strates, the nucleotide concentration at which dephosphorylat- was 1.4 ;imol/min/ mg protein. The S •¿isthe substrate concentration at which ing activity was half-maximal (S0 5) O 5) was used to
Load more

Recommended publications
  • 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).
    [Show full text]
  • Deoxyguanosine Cytotoxicity by a Novel Inhibitor of Furine Nucleoside Phosphorylase, 8-Amino-9-Benzylguanine1
    [CANCER RESEARCH 46, 519-523, February 1986] Potentiation of 2'-Deoxyguanosine Cytotoxicity by a Novel Inhibitor of Furine Nucleoside Phosphorylase, 8-Amino-9-benzylguanine1 Donna S. Shewach,2 Ji-Wang Chern, Katherine E. Pillóte,Leroy B. Townsend, and Peter E. Daddona3 Departments of Internal Medicine [D.S.S., P.E.D.], Biological Chemistry [P.E.D.], and Medicinal Chemistry [J-W.C., K.E.P., L.B.T.], University ol Michigan, Ann Arbor, Michigan 48109 ABSTRACT to the ADA-deficient disease state (2). PNP is an essential enzyme of the purine salvage pathway, We have synthesized and evaluated a series of 9-substituted catalyzing the phosphorolysis of guanosine, inosine, and their analogues of 8-aminoguanine, a known inhibitor of human purine 2'-deoxyribonucleoside derivatives to the respective purine nucleoside phosphorylase (PNP) activity. The ability of these bases. To date, several inhibitors of PNP have been identified, agents to inhibit PNP has been investigated. All compounds were and most of these compounds resemble purine bases or nucleo found to act as competitive (with inosine) inhibitors of PNP, with sides. The most potent inhibitors exhibit apparent K¡values in K¡values ranging from 0.2 to 290 /¿M.Themost potent of these the range of 10~6to 10~7 M (9-12). Using partially purified human analogues, 8-amino-9-benzylguanine, exhibited a K, value that erythrocyte PNP, the diphosphate derivative of acyclovir dis was 4-fold lower than that determined for the parent base, 8- played K¡values of 5.1 x 10~7 to 8.7 x 10~9 M, depending on aminoguanine.
    [Show full text]
  • 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]
  • Metabolism of Purines and Pyrimidines in Health and Disease
    39th Meeting of the Polish Biochemical Society Gdañsk 16–20 September 2003 SESSION 6 Metabolism of purines and pyrimidines in health and disease Organized by A. C. Sk³adanowski, A. Guranowski 182 Session 6. Metabolism of purines and pyrimidines in health and disease 2003 323 Lecture The role of DNA methylation in cytotoxicity mechanism of adenosine analogues in treatment of leukemia Krystyna Fabianowska-Majewska Zak³ad Chemii Medycznej IFiB, Uniwersytet Medyczny, ul. Mazowiecka 6/8, 92 215 £ódŸ Changes in DNA methylation have been recognized tory effects of cladribine and fludarabine on DNA as one of the most common molecular alterations in hu- methylation, after 48 hr growth of K562 cells with the man neoplastic diseases and hypermethylation of drugs, are non-random and affect mainly CpG rich is- gene-promoter regions is one of the most frequent lands or CCGG sequences but do not affect sepa- mechanisms of the loss of gene functions. For this rea- rately-located CpG sequences. The analysis showed son, DNA methylation may be a tool for detection of that cladribine (0.1 mM) reduced the methylated early cell transformations as well as predisposition to cytosines in CpG islands and CCGG sequences to a sim- metastasis process. Moreover, DNA methylation seems ilar degree. The inhibition of cytosine methylation by to be a promissing target for new preventive and thera- fludarabine (3 mM) was observed mainly in CCGG se- peutic strategies. quences, sensitive to HpaII, but the decline in the meth- Our studies on DNA methylation and cytotoxicity ylated cytosine, located in CpG island was 2-fold lower mechanism of antileukemic drugs, cladribine and than that with cladribine.
    [Show full text]
  • Determining HDAC8 Substrate Specificity by Noah Ariel Wolfson A
    Determining HDAC8 substrate specificity by Noah Ariel Wolfson A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Biological Chemistry) in the University of Michigan 2014 Doctoral Committee: Professor Carol A. Fierke, Chair Professor Robert S. Fuller Professor Anna K. Mapp Associate Professor Patrick J. O’Brien Associate Professor Raymond C. Trievel Dedication My thesis is dedicated to all my family, mentors, and friends who made getting to this point possible. ii Table of Contents Dedication ....................................................................................................................................... ii List of Figures .............................................................................................................................. viii List of Tables .................................................................................................................................. x List of Appendices ......................................................................................................................... xi Abstract ......................................................................................................................................... xii Chapter 1 HDAC8 substrates: Histones and beyond ...................................................................... 1 Overview ..................................................................................................................................... 1 HDAC introduction
    [Show full text]
  • Inhibitory Effects of Cordycepin on Platelet Activation Via Regulation of Cyclic Adenosine Monophosphate-Downstream Pathway
    Biomedical Science Letters 2017, 23(3): 251~260 Original Article https://doi.org/10.15616/BSL.2017.23.3.251 eISSN : 2288-7415 Inhibitory Effects of Cordycepin on Platelet Activation via Regulation of Cyclic Adenosine Monophosphate-downstream Pathway Dong-Ha Lee† Department of Biomedical Laboratory Science, Korea Nazarene University, Cheonan 31172, Korea Platelet activation is essential at the sites of vascular injury, which leads to hemostasis through adhesion, aggregation, and secretion process. However, potent and continuous platelet activation may be an important reason of circulatory disorders. Therefore, proper regulation of platelet activation may be an effective treatment for vascular diseases. In this research, inhibitory effects of cordycepin (3'-deoxyadenosine) on platelet activation were determined. As the results, cordycepin increased cAMP and cGMP, which are intracellular Ca2+-antagonists. In addition, cordycepin reduced collagen- 2+ elevated [Ca ]i mobilization, which was increased by a cAMP-dependent protein kinase (PKA) inhibitor (Rp-8-Br- cAMPS), but not a cGMP-protein kinase (PKG) inhibitor (Rp-8-Br-cGMPS). Furthermore, cordycepin increased IP3RI 1756 2+ (Ser ) phosphorylation, indicating inhibition of IP3-mediated Ca release from internal store via the IP3RI, which was strongly inhibited by Rp-8-Br-cAMPS, but was not so much inhibited by Rp-8-Br-cGMPS. These results suggest that the 2+ 1756 reduction of [Ca ]i mobilization is caused by the cAMP/A-kinase-dependent IP3RI (Ser ) phosphorylation. In addition, cordycepin increased the phosphorylation of VASP (Ser157) known as PKA substrate, but not VASP (Ser239) known as PKG substrate. Cordycepin-induced VASP (Ser157) phosphorylation was inhibited by Rp-8-Br-cAMPS, but was not inhibited by Rp-8-Br-cGMPS, and cordycepin inhibited collagen-induced fibrinogen binding to αIIb/β3, which was increased by Rp-8-Br-cAMPS, but was not inhibited by Rp-8-Br-cGMPS.
    [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]
  • Cancer Science Standard Abbreviations List
    Cancer Science Standard Abbreviations List Common abbreviations, acronyms and short names are listed below. These shortened forms can be used without definition in articles published in Cancer Science. The same form is used in the plural. 7-AAD 7-amino-actinomycin D (stain) ES cell embryonic stem cell Ab antibody EST expressed sequence tag ADP adenosine 5′-diphosphate FACS fluorescence-activated cell sorter dADP 2′-deoxyadenosine 5′-diphosphate FBS fetal bovine serum AIDS acquired immunodeficiency syndrome FCS fetal calf serum Akt protein kinase B FDA Food and Drug Administration AML acute myelogenous leukemia FISH fluorescence in situ hybridization AMP adenosine 5′-monophosphate FITC fluorescein isothiocyanate dAMP 2′-deoxyadenosine 5′-monophosphate FPLC fast protein liquid chromatography ANOVA analysis of variance FRET fluorescence resonance energy transfer ATCC American Type Culture Collection GAPDH glyceraldehyde-3-phosphate dehydrogenase ATP adenosine 5′-triphosphate GDP guanosine 5′-diphosphate dATP 2′-deoxyadenosine 5′-triphosphate dGDP 2′-deoxyguanosine 5′-diphosphate beta-Gal, β-Gal beta-galactosidase GFP green fluorescent protein bp base pair(s) GMP guanosine 5′-monophosphate BrdU 5-bromodeoxyuridine dGMP 2′-deoxyguanosine 5′-monophosphate BSA bovine serum albumin GST glutathione S-transferase CCK-8 Cell Counting Kit-8 (tradename) GTP guanosine 5′-triphosphate CDP cytidine 5′-diphosphate dGTP 2′-deoxyguanosine 5′-triphosphate cCDP 2′-deoxycytidine 5′-diphosphate HA hemagglutinin CHAPS 3-[(3-cholamidopropyl)dimethylamino]-1-
    [Show full text]
  • Plasma Deoxyadenosine, Adenosine, and Erythrocyte Deoxyatp Are Elevated at Birth in an Adenosine Deaminase-Deficient Child
    Plasma deoxyadenosine, adenosine, and erythrocyte deoxyATP are elevated at birth in an adenosine deaminase-deficient child. R Hirschhorn, … , A Rubinstein, P Papageorgiou J Clin Invest. 1980;65(3):768-771. https://doi.org/10.1172/JCI109725. Research Article We have determined concentrations of adenosine, deoxyadenosine, and deoxyATP (dATP) in cord blood from an infant prenatally diagnosed as ADA deficient. Plasma deoxyadenosine and adenosine were already elevated in cord blood (0.7 and 0.5 microM vs. normal of less than 0.07 microM). Elevation of plasma deoxyadenosine has not previously been documented in these children. Erythrocyte dATP content was also elevated at birth (215 nmol/ml packed erythrocytes vs. normal of 2.9). These elevated concentrations of adenosine, deoxyadenosine, and dATP are similar to those we observed in another older adenosine deaminase-deficient patient and may explain the impaired immune function and lymphopenia seen at birth. Find the latest version: https://jci.me/109725/pdf RAPID PUBLICATIONS Plasma Deoiyadenosine, Adenosine, and Erythrocyte deoxyATP are Elevated at Birth in an Adenosine Deaminase-deficient Child ROCHELLE HIRSCHHORN and VIVIAN ROEGNER, Department of Medicine, New York University School of Medicine, New York 10016 ARYE RUBINSTEIN, Department of Pediatrics, Albert Einstein College of Medicine, New York 10461 PHOTINI PAPAGEORGIOU, Department of Pediatrics, Rutgers University Medical School, New Brunswick, Netv Jersey 08854 A B S T RA C T We have determined concentrations of amounts of deoxyadenosine, another substrate of ADA, adenosine, deoxyadenosine, and deoxyATP (dATP) in in their urine (4-11). Additionally, deoxyATP (dATP), a cord blood from an infant prenatally diagnosed as ADA metabolite of deoxyadenosine, is markedly increased deficient.
    [Show full text]
  • Cyclic Adenosine 3':5'-Monophc;Phate and Cyclic Guanosine 3':5'
    1 CYCLIC ADENOSINE 3':5'-MONOPHC;PHATE AND CYCLIC GUANOSINE 3':5'- MONOPHOSPHATE METABOLISM DURING THE MITOTIC CYCLE OF THE ACELLULAR SLIME MOULD PHYSABUM POLYCEPHALUM ( SCHWEINITZ ) by James Richard Lovely B.Sc. A.R.C.S. Submitted in part fulfilment for the degree of Doctor of Philosophy of the University of London. September 1977 Department of Botany, Imperial College, London SW7 2AZ. ABSTRACT. Several methods have been assessed and the most effective used for the extraction, purification, separation and assay of cyclic AMP and cyclic GMP from the acellular slime mould Physarum polycephalum. Both cyclic nucleotides have been measured at half hourly intervals in synchronous macroplasmodia. Cyclic AMP was maximal in the last quarter of G2 while cyclic GMP showed two maxima, one occurring during S phase and the other late in G2. Enzymes synthesising and degrading these cyclic nucleotides and their sensitivity to certain inhibitors and cations have been assayed by new radiometric methods in which the tritium labelled substrate and product are separated by thin layer chromatography on cellulose. Synchronous surface plasmodia have been homogenised and sepa- rated into three fractions by differential centrifugation. Phosphodiesterase activity of each fraction has been measured using tritium labelled cyclic AMP and cyclic GMP as substrate. During the 8 hour mitotic cycle, no significant change of either enzyme in any fraction was detected. Adenylate and guanylate cyclase activity has been measured using the tritium labelled imidophosphate analogues as substrate. Adenylate cyclase activity in a low speed particulate fraction increased by about 50% in late G2. No significant changes were detected at any time in the high speed particulate or soluble fractions.
    [Show full text]
  • TITLE Adenylate Kinase 2 Deficiency Causes NAD+ Depletion and Impaired Purine Metabolism During Myelopoiesis
    bioRxiv preprint doi: https://doi.org/10.1101/2021.07.05.450633; this version posted July 6, 2021. 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. TITLE Adenylate Kinase 2 deficiency causes NAD+ depletion and impaired purine metabolism during myelopoiesis AUTHORS Wenqing Wang1, Andew DeVilbiss2, Martin Arreola1, Thomas Mathews2, Misty Martin-Sandoval2, Zhiyu Zhao2, Avni Awani1, Daniel Dever1, Waleed Al-Herz3, Luigi Noratangelo4, Matthew H. Porteus1, Sean J. Morrison2, Katja G. Weinacht1, * 1. Department of Pediatrics, Stanford University School of Medicine, Stanford, California 94305 USA 2. Children’s Research Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75390 USA 3. Department of Pediatrics, Faculty of Medicine, Kuwait University, Safat, 13110 Kuwait 4. Laboratory of Clinical Immunology and Microbiology, National Institute of Health, BETHESDA MD 20814 USA * Corresponding author ABSTRACT Reticular Dysgenesis is a particularly grave from of severe combined immunodeficiency (SCID) that presents with severe congenital neutropenia and a maturation arrest of most cells of the lymphoid lineage. The disease is caused by biallelic loss of function mutations in the mitochondrial enzyme Adenylate Kinase 2 (AK2). AK2 mediates the phosphorylation of adenosine monophosphate (AMP) to adenosine diphosphate (ADP) as substrate for adenosine triphosphate (ATP) synthesis in the mitochondria. Accordingly, it has long been hypothesized that a decline in OXPHOS metabolism is the driver of the disease. The mechanistic basis for Reticular Dysgenesis, however, remained incompletely understood, largely due to lack of appropriate model systems to phenocopy the human disease.
    [Show full text]
  • Biochemical Basis for Differential Deoxyadenosine Toxicity To
    Proc. Natl. Acad. Sci. USA Vol. 76, No. 5, pp. 2434-2437, May 1979 Medical Sciences Biochemical basis for differential deoxyadenosine toxicity to T and B lymphoblasts: Role for 5'-nucleotidase (deoxyadenosine kinase/deoxyadenylate kinase/immunodeficiency) ROBERT L. WORTMANN, BEVERLY S. MITCHELL, N. LAWRENCE EDWARDS, AND IRVING H. Fox Human Purine Research Center, Departments of Internal Medicine and Biological Chemistry, Clinical Research Center, University of Michigan Medical Center, Ann Arbor, Michigan 48109 Communicated by James B. Wyngaarden, March 7, 1979 ABSTRACT Deoxyadenosine metabolism was investigated tained from Calbiochem. Erythro-9-[3-(2-hydroxynonyl)]- in cultured human cells to elucidate the biochemical basis for adenine (EHNA) was a gift from G. B. Elion of Burroughs the sensitivity of T lymphoblasts and the resistance of B lym- Wellcome (Research Triangle Park, NC). Horse serum was phoblasts to deoxyadenosine toxicity. T lymphoblasts have a 20- to 45-fold greater capacity to synthesize deoxyadenosine nu- obtained from Flow Laboratories (Rockville, MD), and Eagle's cleotides than B lymphoblasts at deoxyadenosine concentrations minimal essential medium was purchased from GIBCO. From of 50-300 ,uM. During the synthesis of dATP, T lymphoblasts Amersham/Searle, [U-14C]deoxyadenosine (505 mCi/mmol), accumulate large quantities of dADP, whereas B lymphoblasts [8-14C]hypoxanthine (52.5 mCi/mmol), and [U-14C]deoxy- do not accumu ate dADP. Enzymes affecting deoxyadenosine adenosine monophosphate (574 mCi/mmol) were purchased; nucleotide synthesis were assayed in these cells. No substantial and, from ICN, [8-14C]adenosine monophosphate (34.4 mCi/ differences were evident in activities of deoxyadenosine kinase (ATP: deoxyadenosine 5'-phosphotransferase, EC 2.7.1.76) or mmol) was purchased (1 Ci = 3.7 X 1010 becquerels).
    [Show full text]