Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1997 May 1;323(Pt 3):677–684. doi: 10.1042/bj3230677

Diadenosine polyphosphate hydrolase from presynaptic plasma membranes of Torpedo electric organ.

J Mateo 1, P Rotllan 1, E Marti 1, I Gomez De Aranda 1, C Solsona 1, M T Miras-Portugal 1
PMCID: PMC1218370  PMID: 9169600

Abstract

The diadenosine polyphosphate hydrolase present in presynaptic plasma membranes from the Torpedo electric organ has been characterized using fluorogenic substrates of the form di-(1, N6-ethenoadenosine) 5',5'''-P1,Pn-polyphosphate. The enzyme hydrolyses diadenosine polyphosphates (ApnA, where n=3-5), producing AMP and the corresponding adenosine (n-1) 5'-phosphate, Ap(n-1). The Km values of the enzyme were 0.543+/-0.015, 0.478+/-0.043 and 0. 520+/-0.026 microM, and the Vmax values were 633+/-4, 592+/-18 and 576+/-45 pmol/min per mg of protein, for the etheno derivatives of Ap3A (adenosine 5',5'''-P1,P3-triphosphate), Ap4A (adenosine 5',5"'-P1,P4-tetraphosphate) and Ap5A (adenosine 5',5'''-P1,P5-pentaphosphate) respectively. Ca2+, Mg2+ and Mn2+ are enzyme activators, with EC50 values of 0.86+/-0.11, 1.35+/-0.24 and 0.58+/-0.10 mM respectively. The fluoride ion is an inhibitor with an IC50 value of 1.38+/-0.19 mM. The ATP analogues adenosine 5'-tetraphosphate and adenosine 5'-[gamma-thio]triphosphate are potent competitive inhibitors and adenosine 5'-[alpha,beta-methylene]diphosphate is a less potent competitive inhibitor, the Ki values being 0.29+/-0.03, 0.43+/-0.05 and 7.18+/-0.8 microM respectively. The P2-receptor antagonist pyridoxal phosphate 6-azophenyl-2',4'-disulphonic acid behaves as a non-competitive inhibitor with a Ki value of 29.7+/-3.1 microM, and also exhibits a significant inhibitory effect on Torpedo apyrase activity. The effect of pH on the Km and Vmax values, together with inhibition by diethyl pyrocarbonate, strongly suggests the presence of functional histidine residues in Torpedo diadenosine polyphosphate hydrolase. The enzyme from Torpedo shows similarities with that of neural origin from neurochromaffin cells, and significant differences compared with that from endothelial vascular cells.

Full Text

The Full Text of this article is available as a PDF (477.6 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bailey S. J., Hourani S. M. Effects of suramin on contractions of the guinea-pig vas deferens induced by analogues of adenosine 5'-triphosphate. Br J Pharmacol. 1995 Mar;114(6):1125–1132. doi: 10.1111/j.1476-5381.1995.tb13324.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beukers M. W., Kerkhof C. J., van Rhee M. A., Ardanuy U., Gurgel C., Widjaja H., Nickel P., IJzerman A. P., Soudijn W. Suramin analogs, divalent cations and ATP gamma S as inhibitors of ecto-ATPase. Naunyn Schmiedebergs Arch Pharmacol. 1995 May;351(5):523–528. doi: 10.1007/BF00171044. [DOI] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Fredholm B. B., Abbracchio M. P., Burnstock G., Daly J. W., Harden T. K., Jacobson K. A., Leff P., Williams M. Nomenclature and classification of purinoceptors. Pharmacol Rev. 1994 Jun;46(2):143–156. [PMC free article] [PubMed] [Google Scholar]
  5. Goldman S. J., Gordon E. L., Slakey L. L. Hydrolysis of diadenosine 5',5''-P',P''-triphosphate (Ap3A) by porcine aortic endothelial cells. Circ Res. 1986 Sep;59(3):362–366. doi: 10.1161/01.res.59.3.362. [DOI] [PubMed] [Google Scholar]
  6. Grondal E. J., Zimmermann H. Ectonucleotidase activities associated with cholinergic synaptosomes isolated from Torpedo electric organ. J Neurochem. 1986 Sep;47(3):871–881. doi: 10.1111/j.1471-4159.1986.tb00692.x. [DOI] [PubMed] [Google Scholar]
  7. Grummt F., Waltl G., Jantzen H. M., Hamprecht K., Huebscher U., Kuenzle C. C. Diadenosine 5',5'''-P1,P4-tetraphosphate, a ligand of the 57-kilodalton subunit of DNA polymerase alpha. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6081–6085. doi: 10.1073/pnas.76.12.6081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gualix J., Abal M., Pintor J., Garcia-Carmona F., Miras-Portugal M. T. Nucleotide vesicular transporter of bovine chromaffin granules. Evidence for a mnemonic regulation. J Biol Chem. 1996 Jan 26;271(4):1957–1965. doi: 10.1074/jbc.271.4.1957. [DOI] [PubMed] [Google Scholar]
  9. Guranowski A. Fluoride is a strong and specific inhibitor of (asymmetrical) Ap4A hydrolases. FEBS Lett. 1990 Mar 26;262(2):205–208. doi: 10.1016/0014-5793(90)80190-t. [DOI] [PubMed] [Google Scholar]
  10. Harden T. K., Boyer J. L., Nicholas R. A. P2-purinergic receptors: subtype-associated signaling responses and structure. Annu Rev Pharmacol Toxicol. 1995;35:541–579. doi: 10.1146/annurev.pa.35.040195.002545. [DOI] [PubMed] [Google Scholar]
  11. Heidenreich S., Tepel M., Schlüter H., Harrach B., Zidek W. Regulation of rat mesangial cell growth by diadenosine phosphates. J Clin Invest. 1995 Jun;95(6):2862–2867. doi: 10.1172/JCI117992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Komoszyński M., Wojtczak A. Apyrases (ATP diphosphohydrolases, EC 3.6.1.5): function and relationship to ATPases. Biochim Biophys Acta. 1996 Feb 2;1310(2):233–241. doi: 10.1016/0167-4889(95)00135-2. [DOI] [PubMed] [Google Scholar]
  13. Leff P., Wood B. E., O'Connor S. E. Suramin is a slowly-equilibrating but competitive antagonist at P2x-receptors in the rabbit isolated ear artery. Br J Pharmacol. 1990 Nov;101(3):645–649. doi: 10.1111/j.1476-5381.1990.tb14134.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lienhard G. E., Secemski I. I. P 1 ,P 5 -Di(adenosine-5')pentaphosphate, a potent multisubstrate inhibitor of adenylate kinase. J Biol Chem. 1973 Feb 10;248(3):1121–1123. [PubMed] [Google Scholar]
  15. Martí E., Cantí C., Gómez de Aranda I., Miralles F., Solsona C. Action of suramin upon ecto-apyrase activity and synaptic depression of Torpedo electric organ. Br J Pharmacol. 1996 Jul;118(5):1232–1236. doi: 10.1111/j.1476-5381.1996.tb15528.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Morel N., Marsal J., Manaranche R., Lazereg S., Mazie J. C., Israel M. Large-scale purification of presynaptic plasma membranes from Torpedo marmorata electric organ. J Cell Biol. 1985 Nov;101(5 Pt 1):1757–1762. doi: 10.1083/jcb.101.5.1757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ogilvie A., Lüthje J., Pohl U., Busse R. Identification and partial characterization of an adenosine(5')tetraphospho(5')adenosine hydrolase on intact bovine aortic endothelial cells. Biochem J. 1989 Apr 1;259(1):97–103. doi: 10.1042/bj2590097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Pearson J. D., Coade S. B., Cusack N. J. Characterization of ectonucleotidases on vascular smooth-muscle cells. Biochem J. 1985 Sep 1;230(2):503–507. doi: 10.1042/bj2300503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Pintor J., Porras A., Mora F., Miras-Portugal M. T. Dopamine receptor blockade inhibits the amphetamine-induced release of diadenosine polyphosphates, diadenosine tetraphosphate and diadenosine pentaphosphate, from neostriatum of the conscious rat. J Neurochem. 1995 Feb;64(2):670–676. doi: 10.1046/j.1471-4159.1995.64020670.x. [DOI] [PubMed] [Google Scholar]
  20. Pintor J., Rotllán P., Torres M., Miras-Portugal M. T. Characterization and quantification of diadenosine hexaphosphate in chromaffin cells: granular storage and secretagogue-induced release. Anal Biochem. 1992 Feb 1;200(2):296–300. doi: 10.1016/0003-2697(92)90469-n. [DOI] [PubMed] [Google Scholar]
  21. Pintor J., Torres M., Miras-Portugal M. T. Carbachol induced release of diadenosine polyphosphates--Ap4A and Ap5A--from perfused bovine adrenal medulla and isolated chromaffin cells. Life Sci. 1991;48(24):2317–2324. doi: 10.1016/0024-3205(91)90268-g. [DOI] [PubMed] [Google Scholar]
  22. Plesner L. Ecto-ATPases: identities and functions. Int Rev Cytol. 1995;158:141–214. doi: 10.1016/s0074-7696(08)62487-0. [DOI] [PubMed] [Google Scholar]
  23. Ramos A., Pintor J., Miras-Portugal M. T., Rotllán P. Use of fluorogenic substrates for detection and investigation of ectoenzymatic hydrolysis of diadenosine polyphosphates: a fluorometric study on chromaffin cells. Anal Biochem. 1995 Jun 10;228(1):74–82. doi: 10.1006/abio.1995.1317. [DOI] [PubMed] [Google Scholar]
  24. Ramos A., Rotllán P. Specific dinucleoside polyphosphate cleaving enzymes from chromaffin cells: a fluorimetric study. Biochim Biophys Acta. 1995 Nov 15;1253(1):103–111. doi: 10.1016/0167-4838(95)00154-m. [DOI] [PubMed] [Google Scholar]
  25. Rodriguez del Castillo A., Torres M., Delicado E. G., Miras-Portugal M. T. Subcellular distribution studies of diadenosine polyphosphates--Ap4A and Ap5A--in bovine adrenal medulla: presence in chromaffin granules. J Neurochem. 1988 Dec;51(6):1696–1703. doi: 10.1111/j.1471-4159.1988.tb01147.x. [DOI] [PubMed] [Google Scholar]
  26. Rodriguez-Pascual F., Torres M., Rotllán P., Miras-Portugal M. T. Extracellular hydrolysis of diadenosine polyphosphates, ApnA, by bovine chromaffin cells in culture. Arch Biochem Biophys. 1992 Aug 15;297(1):176–183. doi: 10.1016/0003-9861(92)90657-i. [DOI] [PubMed] [Google Scholar]
  27. Rotllan P., Miras Portugal M. T. Adenosine kinase from bovine adrenal medulla. Eur J Biochem. 1985 Sep 2;151(2):365–371. doi: 10.1111/j.1432-1033.1985.tb09110.x. [DOI] [PubMed] [Google Scholar]
  28. Schulze-Lohoff E., Zanner S., Ogilvie A., Sterzel R. B. Vasoactive diadenosine polyphosphates promote growth of cultured renal mesangial cells. Hypertension. 1995 Dec;26(6 Pt 1):899–904. doi: 10.1161/01.hyp.26.6.899. [DOI] [PubMed] [Google Scholar]
  29. Torres M., Pintor J., Miras-Portugal M. T. Presence of ectonucleotidases in cultured chromaffin cells: hydrolysis of extracellular adenine nucleotides. Arch Biochem Biophys. 1990 May 15;279(1):37–44. doi: 10.1016/0003-9861(90)90460-g. [DOI] [PubMed] [Google Scholar]
  30. Ziganshin A. U., Ziganshina L. E., King B. F., Pintor J., Burnstock G. Effects of P2-purinoceptor antagonists on degradation of adenine nucleotides by ecto-nucleotidases in folliculated oocytes of Xenopus laevis. Biochem Pharmacol. 1996 Apr 12;51(7):897–901. doi: 10.1016/0006-2952(95)02252-x. [DOI] [PubMed] [Google Scholar]
  31. Zimmermann H. 5'-Nucleotidase: molecular structure and functional aspects. Biochem J. 1992 Jul 15;285(Pt 2):345–365. doi: 10.1042/bj2850345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Zimmermann H. Signalling via ATP in the nervous system. Trends Neurosci. 1994 Oct;17(10):420–426. doi: 10.1016/0166-2236(94)90016-7. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES