Neurochemical Research

, Volume 16, Issue 12, pp 1303–1310 | Cite as

Characterization of an ATP diphosphohydrolase (EC in synaptosomes from cerebral cortex of adult rats

  • Ana M. O. Battastini
  • João B. T. da Rocha
  • Claudia K. Barcellos
  • Renato D. Dias
  • João J. F. Sarkis
Original Articles


Data from the literature have demonstrated that synaptosomal preparations from various sources can hydrolyze externally added ATP. Various authors characterized this activity as an ecto-ATPase. In the present report, we demonstrate that synaptosomal preparations obtained from the cerebral cortex of rats show ATPase activity that could not be dissociated from ADPase activity, suggesting that an ATP-diphosphohydrolase is involved in ATP and ADP hydrolysis. Furthermore, the ATP and ADP hydrolysis could not be attributed to associations of enzymes that could mimic an ATP-diphosphohydrolase because none of the following activities were detected in our assay conditions inorganic pyrophosphatase, adenylate kinase, or nonspecific phosphatases. A possible association between an ATPase and an ADPase was excluded on the basis of both the kinetics and much additional data on inhibitors, ion dependence, pH, etc. The present results demonstrate that in synaptosomal preparations from cerebral cortex an ATP-diphosphohydrolase is involved, at least in part, in ATP and ADP hydrolysis.

Key Words

ATP diphosphohydrolase apyrase ATPase ADPase neurotransmission 





ethylenediaminetetraacetic acid


N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid


inorganic phosphate



diphosphohydrolase, Apyrase (EC


ATP phosphohydrolase (EC 5′-nucleotidase (EC Hexokinase (EC Glucose-6-phosphate dehydrogenase (EC Adenylate kinase (EC Inorganic pyrophosphatase (EC


pyrophosphohydrolase (EC


lactate dehydrogenase (EC


succinate dehydrogenase (EC


acethylcholinesterase (EC


glucose-6-phosphatase (EC


cytoehrome c oxidoreductase (NCR) (EC


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  1. 1.
    Snyder, S. H., 1985. Adenosine as a modulator. Ann. Rev. Neurosc. 8:103–124.Google Scholar
  2. 2.
    Williams, M., 1987. Purine receptors in mammalian tissues: pharmacology and functional significance. Annu. Rev. Pharmacol. Toxicol. 27:315–345.PubMedGoogle Scholar
  3. 3.
    White, T. D. 1988. Role of adenine compounds in autonomic neurotransmission. Pharmacol. Ther. 38:129–168.PubMedGoogle Scholar
  4. 4.
    Richardson, P. J., and Brown, S. J. 1987. ATP release from affinity-purified rat cholinergic nerve terminals. J. Neurochem. 48:622–630.PubMedGoogle Scholar
  5. 5.
    Silinsky, E. M. 1975. On the association between transmitter secretion and the release of adenine nucleotides from mammalian motor nerve terminals. J. Physiol. (Lond.) 247:145–162.Google Scholar
  6. 6.
    Sorensen, R. G., and Mahler, H. R. 1982. Localization of endogenous ATPases at the nerve terminal. J. Bioenerg. Biomembr. 14:527–546.PubMedGoogle Scholar
  7. 7.
    Nagy, A., Shuster, T. A., and Rosenberg, M. D. 1983. Adenosine triphosphatase activity at the external surface of chicken brain synaptosomes. J. Neurochem. 40:226–234.PubMedGoogle Scholar
  8. 8.
    Keller, F., and Zimmerman, H. 1983. Ecto-adenosine triphosphatase activity at the cholinergic nerve endings of theTorpedo electric organ. Life Sci. 33:2635–2641.PubMedGoogle Scholar
  9. 9.
    Nagy, A., Shuster, T. A., and Delgado-Escueta, A. V. 1986. Ecto-ATPase of mammalian synaptosomes: identification and enzymic characterization. J. Neurochem. 47:976–986.PubMedGoogle Scholar
  10. 10.
    Grondal, E. M. J., and Zimmerman, H. 1986. Ectonucleotidase activities associated with cholinergic synaptossomes isolated fromTorpedo electric organ. J. Neurochem. 47:871–881.PubMedGoogle Scholar
  11. 11.
    Mellman, I., Fuchs, R., and Helenius, A. 1986. Acidification of the endocytic exocytic pathways. Annu. Rev. Biochem. 55:663–700.PubMedGoogle Scholar
  12. 12.
    Pullman, M. E., Penefsky, H. S., Datta, A., and Racker, E. 1960. Partial resolution of the enzymes catalyzing oxidative phosphorylation. J. Biol. Chem. 235:3322–3329.PubMedGoogle Scholar
  13. 13.
    Schadeck, R. J. G., Sarkis, J. J. F., Dias, R. D., Araujo, H. M. M., and Souza, D. O. G. 1989. Synaptosomal apyrase in the hypothalamus of adult rats. Brazilian J. Med. Biol. Res. 22:303–314.Google Scholar
  14. 14.
    Sarkis, J. J. F., and Saltó, C. 1991. Characterization of a synaptosomal ATP diphosphohydrolase from the electric organ ofTorpedo marmorata. Brain Res. Bull. “IN PRESS”Google Scholar
  15. 15.
    Richardson, P. J., Brown, S. J., Bayles, E. M., and Luzio, J. P. 1987. Ectoenzymes control adenosine modulation of immunoisolated cholinergic synapses. Nature. 327:232–234.PubMedGoogle Scholar
  16. 16.
    Brown, S. J. 1988. Adenosine and ATP: presynaptic effects at the cholinergic nerve terminal. Biochem. Soc. Trans. 16:442–443.PubMedGoogle Scholar
  17. 17.
    Nagy, A., and Delgado-Escueta, A. V. 1984. Rapid preparation of synaptosomes from mammalian brain using nontoxic isoosmotic gradient material (Percoll). J. Neurochem. 43:1114–1123.PubMedGoogle Scholar
  18. 18.
    Lanzetta, P. A., Alvarez, L. J., Reinach, P. S., and Candia, O. A. 1979. An improved assay for nanomole amounts of inorganic phosphate. Anal. Biochem. 100:95–97.PubMedGoogle Scholar
  19. 19.
    Whitaker, J. F. 1969. A general colorimetric procedure for the estimation of enzymes which are linked to the NADH-NAD+ system. Clin. Chim. Acta. 24:23–37.PubMedGoogle Scholar
  20. 20.
    Ellman, G. L., Courtney, K. D., Andres, V. Jr. and Featherstone, R. M. 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7:88–95.PubMedGoogle Scholar
  21. 21.
    Hubscher, G., and West, G. R. 1965. Specific assays of some phosphatases in subcellular fraction of small intestinal mucosa. Nature 205:99–780.PubMedGoogle Scholar
  22. 22.
    Booth, R. F. G., and Clark, J. B. 1978. A rapid method for the preparation of relatively pure metabolically competent synaptosomes from rat brain. Biochem. J. 176:365–370.PubMedGoogle Scholar
  23. 23.
    Bradford, M. M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248–254.PubMedGoogle Scholar
  24. 24.
    Lienhard, G. E., and Secemski, I. I. 1973. P1,p5-Di(adenosine-5′)pentaphosphate, a potent multisubstrate inhibitor of adenylate kinase. J. Biol. Chem. 248:1121–1123.PubMedGoogle Scholar
  25. 25.
    Russel, P. J., Horenstein, J. M., Goins, L., Jones, D., and Laver, M. 1974. Adenylate kinase in human tissues. J. Biol. Chem. 249:1874–1879.PubMedGoogle Scholar
  26. 26.
    Dixon, M., and Webb, E. C. 1979. Enzymes-3rd edn. Pages 73–74. Academic Press, New York.Google Scholar
  27. 27.
    Bowman, B. J., Mainzer, S. E., Allen, K. E., and Slayman, C. W. 1978. Effects of inhibitors on the plasma membrane and mitochondrial adenosine triphosphatases ofNeurospora crassa. Biochim. Biophys. Acta. 512:13–28.PubMedGoogle Scholar
  28. 28.
    Knowles, A. F., Isler, R. E., and Reece, J. F. 1983. The common accurrence of ATP diphosphohydrolase in mammalian plasma membranes. Biochim. Biophys. Acta 731:88–96.PubMedGoogle Scholar
  29. 29.
    Hosie, R. J. A. 1965. The localization of adenosine triphosphaases in morphologically characterized subcellular fractions of guinea pig brain. Biochem. J. 96:404–412.PubMedGoogle Scholar
  30. 30.
    Callaghan, O. H., and Weber, G. 1959. Kinetic studies on rabbitmuscle myokinase. Biochem. J. 73:473–485.PubMedGoogle Scholar
  31. 31.
    Markland, F. S., and Wadkins, C. L. 1966. Adenosine triphosphate-adenosine 5′-monophosphotransferase of bovine liver mitochondria. J. Biol. Chem. 241:4124–4135.PubMedGoogle Scholar
  32. 32.
    Kalckar, H. M. 1943. The role of myokinase in transphophorylations. II. The enzymatic action of myokinase on adenine nucleotides. J. Biol. Chem. 148:127–137.Google Scholar
  33. 33.
    Sarkis, J. J. F., Guimaraes, J. A., and Ribeiro, J. M. C. 1986. Salivary apyrase ofRhodnius prolixus. Biochem. J. 233:855–891.Google Scholar
  34. 34.
    Vara, F., and Serrano, R. 1981. Purification and characterization of a membrane-bound ATP diphosphohydrolase fromCicer arietinum (chick-pea) roots. Biochem. J. 197:637–646.PubMedGoogle Scholar
  35. 35.
    Tognoli, L., and Marre, E. 1981. Purification and characterization of a divalent cation-activated ATP-ADPase from pea stem microsomes. Biochim. Biophys. Acta 642:1–14.PubMedGoogle Scholar
  36. 36.
    Yagi, K., Arai, Y., Kato, N., Hirota, K., and Miura, Y. 1989. Purification of ATP diphosphohydrolase from bovine aorta microsomes. Eur. J. Biochem. 180:509–513.PubMedGoogle Scholar
  37. 37.
    LeBel, D., Poirier, G. C., Phaneuf, S., St-Jean, P., Laliberté, J. P., and Beaudoin, R. 1980. Characterization and purification of a calcium-sensitive ATP diphosphohydrolase from pig pancreas. J. Biol. Chem. 255:1227–1233.PubMedGoogle Scholar
  38. 38.
    Laliberte, J. F., and Beaudoin, A. R. 1983. Sequencial hydrolysis of the Y- and B- phosphates groups of ATP by the ATP diphosphohydrolase from pig pancreas. Biochim. Biophys. Acta 742:9–15.PubMedGoogle Scholar
  39. 39.
    Johnson, M. K., and Whittaker, V. P. 1963. Lactate dehydrogenase as a cytoplasmic marker in brain. Biochem. J. 88:404–409.PubMedGoogle Scholar
  40. 40.
    Harper, F., Lamy, F., and Calvert, R. 1978. Some properties of a Ca2+ and (or) Mg2+-requiring nucleoside di- and triphosphatase(s) associated with the membranes of rat pancreatic zymogen granules. Can. J. Biochem. 56:565–576.PubMedGoogle Scholar
  41. 41.
    Garcia-Alonso, J., Reglero, A., and Cabezas, J. A. 1990. Purification and properties of B-N-Acetylhexosaminidase A from pig brain. Int. J. Biochem. 22:645–651.PubMedGoogle Scholar
  42. 42.
    Nagy, A. K., Shuster, T. A., and Delgado-Escueta, A. V. 1989. Rat brain synaptosomal ATP:AMP-phosphotransferase activity. J. Neurochem. 53:1166–1172.PubMedGoogle Scholar

Copyright information

© Plenum Publishing Corporation 1991

Authors and Affiliations

  • Ana M. O. Battastini
    • 1
  • João B. T. da Rocha
    • 2
  • Claudia K. Barcellos
    • 3
  • Renato D. Dias
    • 3
  • João J. F. Sarkis
    • 3
  1. 1.Departamento de Ciências FisiológicasFundacão Universidade do Rio GrandeRio GrandeBrasil
  2. 2.Setor de Bioquímica-Centro de Ciências Naturais e ExatasUniversidade Federal de Santa MariaSanta MariaBrasil
  3. 3.Departamento de Bioquimica, Institute de BiociênciasUniversidade Federal do Rio Grande do SulPorto AlegreBrasil

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