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. 1989 Aug 15;262(1):33–40. doi: 10.1042/bj2620033

5'-Nucleotidases of chicken gizzard and human pancreatic adenocarcinoma cells are anchored to the plasma membrane via a phosphatidylinositol-glycan.

U Stochaj 1, K Flocke 1, W Mathes 1, H G Mannherz 1
PMCID: PMC1133225  PMID: 2554891

Abstract

We have analysed the membrane anchorage of plasma-membrane 5'-nucleotidase, an ectoenzyme which can mediate binding to components of the extracellular matrix. We demonstrated that the purified enzyme obtained from chicken gizzard and a human pancreatic adenocarcinoma cell line were both completely transformed into a hydrophilic form by treatment with phospholipases C and D, cleaving glycosylphosphatidylinositol (GPI). These data indicate the presence of a glycolipid linker employed for membrane anchoring of the 5'-nucleotidase obtained from both sources. Incubation of plasma membranes under identical conditions revealed that about half of the AMPase activity was resistant to GPI-hydrolysing phospholipases. Investigation of the enzymic properties of purified chicken gizzard 5'-nucleotidase revealed only minor changes after removal of the phosphatidylinositol linker. However, cleavage of the membrane anchor resulted in an increased sensitivity towards inhibition by concanavalin A. After tissue fractionation, chicken gizzard 5'-nucleotidase could be obtained as either a membrane-bound or a soluble protein; the latter is suspected to be released from the plasma membrane by endogenous phospholipases. Higher-molecular-mass proteins immuno-cross-reactive with the purified chicken gizzard 5'-nucleotidase were detected as both soluble and membrane-bound forms.

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Selected References

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

  1. Baron M. D., Pope B., Luzio J. P. The membrane topography of ecto-5'-nucleotidase in rat hepatocytes. Biochem J. 1986 Jun 1;236(2):495–502. doi: 10.1042/bj2360495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Davitz M. A., Hereld D., Shak S., Krakow J., Englund P. T., Nussenzweig V. A glycan-phosphatidylinositol-specific phospholipase D in human serum. Science. 1987 Oct 2;238(4823):81–84. doi: 10.1126/science.2443973. [DOI] [PubMed] [Google Scholar]
  3. Davitz M. A., Low M. G., Nussenzweig V. Release of decay-accelerating factor (DAF) from the cell membrane by phosphatidylinositol-specific phospholipase C (PIPLC). Selective modification of a complement regulatory protein. J Exp Med. 1986 May 1;163(5):1150–1161. doi: 10.1084/jem.163.5.1150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dickson G., Gower H. J., Barton C. H., Prentice H. M., Elsom V. L., Moore S. E., Cox R. D., Quinn C., Putt W., Walsh F. S. Human muscle neural cell adhesion molecule (N-CAM): identification of a muscle-specific sequence in the extracellular domain. Cell. 1987 Sep 25;50(7):1119–1130. doi: 10.1016/0092-8674(87)90178-4. [DOI] [PubMed] [Google Scholar]
  5. Dieckhoff J., Knebel H., Heidemann M., Mannherz H. G. An improved procedure for purifying 5'-nucleotidase from various sources. Evidence for tissue and species differences in their molecular mass and affinity for F-actin. Eur J Biochem. 1985 Sep 2;151(2):377–383. doi: 10.1111/j.1432-1033.1985.tb09112.x. [DOI] [PubMed] [Google Scholar]
  6. Dieckhoff J., Lietzke R., Mannherz H. G. Monoclonal antibodies against 5'-nucleotidase from chicken gizzard. Evidence for species and tissue specific differences of 5'-nucleotidase. J Biol Chem. 1986 Jan 15;261(2):922–927. [PubMed] [Google Scholar]
  7. Dieckhoff J., Niggemeyer B., Lietzke R., Mannherz H. G. Reconstitution of purified chicken gizzard 5'-nucleotidase in phospholipid vesicles. Evidence for its transmembraneous character and the existence of functional domains on both sides of the phospholipid bilayer. Eur J Biochem. 1987 Jan 15;162(2):451–459. doi: 10.1111/j.1432-1033.1987.tb10623.x. [DOI] [PubMed] [Google Scholar]
  8. Dustin M. L., Selvaraj P., Mattaliano R. J., Springer T. A. Anchoring mechanisms for LFA-3 cell adhesion glycoprotein at membrane surface. 1987 Oct 29-Nov 4Nature. 329(6142):846–848. doi: 10.1038/329846a0. [DOI] [PubMed] [Google Scholar]
  9. Ferguson M. A., Homans S. W., Dwek R. A., Rademacher T. W. Glycosyl-phosphatidylinositol moiety that anchors Trypanosoma brucei variant surface glycoprotein to the membrane. Science. 1988 Feb 12;239(4841 Pt 1):753–759. doi: 10.1126/science.3340856. [DOI] [PubMed] [Google Scholar]
  10. Gower H. J., Barton C. H., Elsom V. L., Thompson J., Moore S. E., Dickson G., Walsh F. S. Alternative splicing generates a secreted form of N-CAM in muscle and brain. Cell. 1988 Dec 23;55(6):955–964. doi: 10.1016/0092-8674(88)90241-3. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. He H. T., Finne J., Goridis C. Biosynthesis, membrane association, and release of N-CAM-120, a phosphatidylinositol-linked form of the neural cell adhesion molecule. J Cell Biol. 1987 Dec;105(6 Pt 1):2489–2500. doi: 10.1083/jcb.105.6.2489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Huizinga T. W., van der Schoot C. E., Jost C., Klaassen R., Kleijer M., von dem Borne A. E., Roos D., Tetteroo P. A. The PI-linked receptor FcRIII is released on stimulation of neutrophils. Nature. 1988 Jun 16;333(6174):667–669. doi: 10.1038/333667a0. [DOI] [PubMed] [Google Scholar]
  14. Ishihara M., Fedarko N. S., Conrad H. E. Involvement of phosphatidylinositol and insulin in the coordinate regulation of proteoheparan sulfate metabolism and hepatocyte growth. J Biol Chem. 1987 Apr 5;262(10):4708–4716. [PubMed] [Google Scholar]
  15. Jemmerson R., Low M. G. Phosphatidylinositol anchor of HeLa cell alkaline phosphatase. Biochemistry. 1987 Sep 8;26(18):5703–5709. doi: 10.1021/bi00392a019. [DOI] [PubMed] [Google Scholar]
  16. Kern H. F., Bosslet K., Sedlacek H. H., Schorlemmer H. U. Monocyte-related functions expressed in cell lines established from human pancreatic adenocarcinoma. II. Inhibition of stimulated activity by monoclonal antibodies reacting with surface antigens on tumor cells. Pancreas. 1988;3(1):2–10. doi: 10.1097/00006676-198802000-00002. [DOI] [PubMed] [Google Scholar]
  17. Lin P. H., Selinfreund R., Wakshull E., Wharton W. Rapid and efficient purification of plasma membrane from cultured cells: characterization of epidermal growth factor binding. Biochemistry. 1987 Feb 10;26(3):731–736. doi: 10.1021/bi00377a012. [DOI] [PubMed] [Google Scholar]
  18. Low M. G., Finean J. B. Specific release of plasma membrane enzymes by a phosphatidylinositol-specific phospholipase C. Biochim Biophys Acta. 1978 Apr 20;508(3):565–570. doi: 10.1016/0005-2736(78)90100-1. [DOI] [PubMed] [Google Scholar]
  19. Low M. G., Prasad A. R. A phospholipase D specific for the phosphatidylinositol anchor of cell-surface proteins is abundant in plasma. Proc Natl Acad Sci U S A. 1988 Feb;85(4):980–984. doi: 10.1073/pnas.85.4.980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Menon A. K., Mayor S., Ferguson M. A., Duszenko M., Cross G. A. Candidate glycophospholipid precursor for the glycosylphosphatidylinositol membrane anchor of Trypanosoma brucei variant surface glycoproteins. J Biol Chem. 1988 Feb 5;263(4):1970–1977. [PubMed] [Google Scholar]
  21. Mikol D. D., Stefansson K. A phosphatidylinositol-linked peanut agglutinin-binding glycoprotein in central nervous system myelin and on oligodendrocytes. J Cell Biol. 1988 Apr;106(4):1273–1279. doi: 10.1083/jcb.106.4.1273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Newby A. C., Luzio J. P., Hales C. N. The properties and extracellular location of 5'-nucleotidase of the rat fat-cell plasma membrane. Biochem J. 1975 Mar;146(3):625–633. doi: 10.1042/bj1460625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Newby A. C. The pigeon heart 5'-nucleotidase responsible for ischaemia-induced adenosine formation. Biochem J. 1988 Jul 1;253(1):123–130. doi: 10.1042/bj2530123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ogata S., Hayashi Y., Takami N., Ikehara Y. Chemical characterization of the membrane-anchoring domain of human placental alkaline phosphatase. J Biol Chem. 1988 Jul 25;263(21):10489–10494. [PubMed] [Google Scholar]
  25. Panagia V., Heyliger C. E., Choy P. C., Dhalla N. S. Solubilization of rat heart sarcolemma 5'-nucleotidase by phosphatidylinositol-specific phospholipase C. Biochim Biophys Acta. 1981 Feb 6;640(3):802–806. doi: 10.1016/0005-2736(81)90110-3. [DOI] [PubMed] [Google Scholar]
  26. Stroynowski I., Soloski M., Low M. G., Hood L. A single gene encodes soluble and membrane-bound forms of the major histocompatibility Qa-2 antigen: anchoring of the product by a phospholipid tail. Cell. 1987 Aug 28;50(5):759–768. doi: 10.1016/0092-8674(87)90334-5. [DOI] [PubMed] [Google Scholar]
  27. Thompson L. F., Ruedi J. M., Low M. G. Purification of 5'-nucleotidase from human placenta after release from plasma membranes by phosphatidylinositol-specific phospholipase C. Biochem Biophys Res Commun. 1987 May 29;145(1):118–125. doi: 10.1016/0006-291x(87)91295-2. [DOI] [PubMed] [Google Scholar]
  28. Truong V. L., Collinson A. R., Lowenstein J. M. 5'-Nucleotidases in rat heart. Evidence for the occurrence of two soluble enzymes with different substrate specificities. Biochem J. 1988 Jul 1;253(1):117–121. doi: 10.1042/bj2530117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Worku Y., Luzio J. P., Newby A. C. Identification of histidyl and cysteinyl residues essential for catalysis by 5'-nucleotidase. FEBS Lett. 1984 Feb 27;167(2):235–240. doi: 10.1016/0014-5793(84)80133-7. [DOI] [PubMed] [Google Scholar]
  30. Yeh E. T., Reiser H., Bamezai A., Rock K. L. TAP transcription and phosphatidylinositol linkage mutants are defective in activation through the T cell receptor. Cell. 1988 Mar 11;52(5):665–674. doi: 10.1016/0092-8674(88)90404-7. [DOI] [PubMed] [Google Scholar]
  31. Zachowski A., Evans W. H., Paraf A. Immunological evidence that plasma-membrane 5'-nucleotidase is a transmembrane protein. Biochim Biophys Acta. 1981 Jun 9;644(1):121–126. doi: 10.1016/0005-2736(81)90066-3. [DOI] [PubMed] [Google Scholar]
  32. de Almeida M. L., Turner M. J., Stambuk B. B., Schenkman S. Identification of an acid-lipase in human serum which is capable of solubilizing glycophosphatidylinositol-anchored proteins. Biochem Biophys Res Commun. 1988 Jan 15;150(1):476–482. doi: 10.1016/0006-291x(88)90545-1. [DOI] [PubMed] [Google Scholar]

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