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. 1992 Jun 15;284(Pt 3):621–624. doi: 10.1042/bj2840621

Soluble low-Km 5'-nucleotidase from electric-ray (Torpedo marmorata) electric organ and bovine cerebral cortex is derived from the glycosyl-phosphatidylinositol-anchored ectoenzyme by phospholipase C cleavage.

M Vogel 1, H Kowalewski 1, H Zimmermann 1, N M Hooper 1, A J Turner 1
PMCID: PMC1132579  PMID: 1535775

Abstract

Soluble and membrane-bound low-Km 5'-nucleotidase was isolated from high-speed supernatants and membrane fractions derived from the electric organ of the electric ray (Torpedo marmorata) or from bovine brain cerebral cortex. Purification of both enzymes included chromatography on concanavalin A-Sepharose and AMP-Sepharose. The contribution to the total of soluble enzyme activity was lower in electric organ (1.6%) than in bovine cerebral cortex (27.9%). Membrane-bound and soluble forms have very similar Km values for AMP and are inhibited by micromolar concentrations of ATP. Both forms cross-react with, and are inhibited by, an antibody against the membrane-bound surface-located (ecto-) 5'-nucleotidase from electric organ. The HNK-1 carbohydrate epitope is present on both forms of the Torpedo enzyme, but is entirely absent from bovine cerebral-cortex 5'-nucleotidase. An antibody specific for the inositol 1,2-(cyclic)monophosphate that is formed on phospholipase C cleavage of an intact glycosyl-phosphatidylinositol (GPI) anchor binds to the soluble, but not to the membrane-bound, form of the enzyme from both sources. Our results suggest that soluble low-Km 5'-nucleotidase in both electric organ and bovine brain is derived from the membrane-bound GPI-anchored form of the enzyme by the action of a phospholipase C and is not a soluble cytoplasmic enzyme.

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Selected References

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

  1. Davitz M. A., Hom J., Schenkman S. Purification of a glycosyl-phosphatidylinositol-specific phospholipase D from human plasma. J Biol Chem. 1989 Aug 15;264(23):13760–13764. [PubMed] [Google Scholar]
  2. Fini C., Coli M., Floridi A. Purification of 5'-nucleotidase from human seminal plasma. Biochim Biophys Acta. 1991 Sep 2;1075(1):20–27. doi: 10.1016/0304-4165(91)90069-s. [DOI] [PubMed] [Google Scholar]
  3. Fox J. A., Soliz N. M., Saltiel A. R. Purification of a phosphatidylinositol-glycan-specific phospholipase C from liver plasma membranes: a possible target of insulin action. Proc Natl Acad Sci U S A. 1987 May;84(9):2663–2667. doi: 10.1073/pnas.84.9.2663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Fritzson P., Haugen T. B., Tjernshaugen H. The presence and activity in normal and regenerating rat liver postmicrosomal supernatant fraction of an enzyme with properties similar to those of membrane-bound 5'-nucleotidase. Biochem J. 1986 Oct 1;239(1):185–190. doi: 10.1042/bj2390185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Grondal E. J., Zimmermann H. Purification, characterization and cellular localization of 5'-nucleotidase from Torpedo electric organ. Biochem J. 1987 Aug 1;245(3):805–810. doi: 10.1042/bj2450805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hoener M. C., Stieger S., Brodbeck U. Isolation and characterization of a phosphatidylinositol-glycan-anchor-specific phospholipase D from bovine brain. Eur J Biochem. 1990 Jul 5;190(3):593–601. doi: 10.1111/j.1432-1033.1990.tb15614.x. [DOI] [PubMed] [Google Scholar]
  7. Hooper N. M., Broomfield S. J., Turner A. J. Characterization of antibodies to the glycosyl-phosphatidylinositol membrane anchors of mammalian proteins. Biochem J. 1991 Jan 15;273(Pt 2):301–306. doi: 10.1042/bj2730301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hooper N. M., Turner A. J. Ectoenzymes of the kidney microvillar membrane. Isolation and characterization of the amphipathic form of renal dipeptidase and hydrolysis of its glycosyl-phosphatidylinositol anchor by an activity in plasma. Biochem J. 1989 Aug 1;261(3):811–818. doi: 10.1042/bj2610811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Huang K. S., Li S., Fung W. J., Hulmes J. D., Reik L., Pan Y. C., Low M. G. Purification and characterization of glycosyl-phosphatidylinositol-specific phospholipase D. J Biol Chem. 1990 Oct 15;265(29):17738–17745. [PubMed] [Google Scholar]
  10. Klemens M. R., Sherman W. R., Holmberg N. J., Ruedi J. M., Low M. G., Thompson L. F. Characterization of soluble vs membrane-bound human placental 5'-nucleotidase. Biochem Biophys Res Commun. 1990 Nov 15;172(3):1371–1377. doi: 10.1016/0006-291x(90)91601-n. [DOI] [PubMed] [Google Scholar]
  11. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  12. Lai K. M., Wong P. C. A comparison of the properties of 5'-nucleotidase purified from the cytosolic and synaptic plasma membrane fractions of rat forebrain. Int J Biochem. 1991;23(10):1123–1130. doi: 10.1016/0020-711x(91)90153-e. [DOI] [PubMed] [Google Scholar]
  13. Lanzetta P. A., Alvarez L. J., Reinach P. S., Candia O. A. An improved assay for nanomole amounts of inorganic phosphate. Anal Biochem. 1979 Nov 15;100(1):95–97. doi: 10.1016/0003-2697(79)90115-5. [DOI] [PubMed] [Google Scholar]
  14. Lisanti M. P., Darnell J. C., Chan B. L., Rodriguez-Boulan E., Saltiel A. R. The distribution of glycosyl-phosphatidylinositol anchored proteins is differentially regulated by serum and insulin. Biochem Biophys Res Commun. 1989 Oct 31;164(2):824–832. doi: 10.1016/0006-291x(89)91533-7. [DOI] [PubMed] [Google Scholar]
  15. Mallol J., Bozal J. Modification of 5'-nucleotidase activity by divalent cations and nucleotides. J Neurochem. 1983 May;40(5):1205–1211. doi: 10.1111/j.1471-4159.1983.tb13558.x. [DOI] [PubMed] [Google Scholar]
  16. Misumi Y., Ogata S., Ohkubo K., Hirose S., Ikehara Y. Primary structure of human placental 5'-nucleotidase and identification of the glycolipid anchor in the mature form. Eur J Biochem. 1990 Aug 17;191(3):563–569. doi: 10.1111/j.1432-1033.1990.tb19158.x. [DOI] [PubMed] [Google Scholar]
  17. Montero J. M., Fes J. B. Purification and characterization of bovine brain 5'-nucleotidase. J Neurochem. 1982 Oct;39(4):982–989. doi: 10.1111/j.1471-4159.1982.tb11486.x. [DOI] [PubMed] [Google Scholar]
  18. Ogata S., Hayashi Y., Misumi Y., Ikehara Y. Membrane-anchoring domain of rat liver 5'-nucleotidase: identification of the COOH-terminal serine-523 covalently attached with a glycolipid. Biochemistry. 1990 Aug 28;29(34):7923–7927. doi: 10.1021/bi00486a021. [DOI] [PubMed] [Google Scholar]
  19. Orford M., Mazurkiewicz D., Saggerson D. Soluble 5'-nucleotidase activities in rat brain. J Neurochem. 1991 Jan;56(1):141–146. doi: 10.1111/j.1471-4159.1991.tb02573.x. [DOI] [PubMed] [Google Scholar]
  20. Paul E., Leblond F. A., LeBel D. In resting conditions, the pancreatic granule membrane protein GP-2 is secreted by cleavage of its glycosylphosphatidylinositol anchor. Biochem J. 1991 Aug 1;277(Pt 3):879–881. doi: 10.1042/bj2770879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Peterson G. L. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem. 1977 Dec;83(2):346–356. doi: 10.1016/0003-2697(77)90043-4. [DOI] [PubMed] [Google Scholar]
  22. Piec G., Le Hir M. The soluble 'low-Km' 5'-nucleotidase of rat kidney represents solubilized ecto-5'-nucleotidase. Biochem J. 1991 Jan 15;273(Pt 2):409–413. doi: 10.1042/bj2730409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Stochaj U., Flocke K., Mathes W., Mannherz H. G. 5'-Nucleotidases of chicken gizzard and human pancreatic adenocarcinoma cells are anchored to the plasma membrane via a phosphatidylinositol-glycan. Biochem J. 1989 Aug 15;262(1):33–40. doi: 10.1042/bj2620033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Tanaka Y., Himeno M., Taguchi R., Ikezawa H., Kato K. 5'-Nucleotidase in rat liver lysosomal membranes is anchored via glycosyl-phosphatidylinositol. Cell Struct Funct. 1989 Oct;14(5):597–603. doi: 10.1247/csf.14.597. [DOI] [PubMed] [Google Scholar]
  25. Thomson L. F., Ruedi J. M., Glass A., Moldenhauer G., Moller P., Low M. G., Klemens M. R., Massaia M., Lucas A. H. Production and characterization of monoclonal antibodies to the glycosyl phosphatidylinositol-anchored lymphocyte differentiation antigen ecto-5'-nucleotidase (CD73). Tissue Antigens. 1990 Jan;35(1):9–19. doi: 10.1111/j.1399-0039.1990.tb01750.x. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Vogel M., Kowalewski H. J., Zimmermann H., Janetzko A., Margolis R. U., Wollny H. E. Association of the HNK-1 epitope with 5'-nucleotidase from Torpedo marmorata (electric ray) electric organ. Biochem J. 1991 Aug 15;278(Pt 1):199–202. doi: 10.1042/bj2780199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Volknandt W., Naito S., Ueda T., Zimmermann H. Synapsin I is associated with cholinergic nerve terminals in the electric organs of Torpedo, Electrophorus, and Malapterurus and copurifies with Torpedo synaptic vesicles. J Neurochem. 1987 Aug;49(2):342–347. doi: 10.1111/j.1471-4159.1987.tb02871.x. [DOI] [PubMed] [Google Scholar]
  30. Volknandt W., Vogel M., Pevsner J., Misumi Y., Ikehara Y., Zimmermann H. 5'-nucleotidase from the electric ray electric lobe. Primary structure and relation to mammalian and procaryotic enzymes. Eur J Biochem. 1991 Dec 18;202(3):855–861. doi: 10.1111/j.1432-1033.1991.tb16443.x. [DOI] [PubMed] [Google Scholar]
  31. Wortmann R. L., Veum J. A., Rachow J. W. Synovial fluid 5'-nucleotidase activity. Relationship to other purine catabolic enzymes and to arthropathies associated with calcium crystal deposition. Arthritis Rheum. 1991 Aug;34(8):1014–1020. doi: 10.1002/art.1780340811. [DOI] [PubMed] [Google Scholar]
  32. Zekri M., Harb J., Bernard S., Meflah K. Purification of bovine liver cytosolic 5'-nucleotidase. Kinetic and structural studies as compared to the membrane isoenzyme. Eur J Biochem. 1988 Feb 15;172(1):93–99. doi: 10.1111/j.1432-1033.1988.tb13860.x. [DOI] [PubMed] [Google Scholar]

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