The Journal of Membrane Biology

, Volume 78, Issue 3, pp 201–210 | Cite as

Incorporation of the human erythrocyte sialoglycoprotein into recombined membranes containing cholesterol

  • Philip L. Yeagle


Glycophorin, the major sialoglycoprotein from the human erythrocyte membrane, has been isolated and recombined with phosphatidylcholine and cholesterol. Sucrose density gradient analysis of the recombinants shows that it is possible not only to recombine this protein with phospholipid, but also with phospholipid-cholesterol mixtures. Surprisingly, by the same analysis, it was possible to make a recombinant with cholesterol and glycophorin, only, in the absence of added phospholipid. The accessibility of the protein to trypsin was tested in each of these recombinants. In all the recombinants which contained either phospholipid, or phospholipid and cholesterol, the protein was protected from extensive hydrolysis. This is consistent with closed vesicles and incorporation of the protein into the recombinant membrane. Extensive hydrolysis of the protein occurred in the cholesterol-glycophorin recombinant indicating some differences in structure. Freeze-fracture electron microscopy of the phospholipid and the phospholipid-cholesterol recombinants showed mostly unilamellar vesicles, 1000 to 5000 Å in diameter. Intramembranous particles were observed on both fracture faces, and the fracture planes were those expected for phospholipid bilayers. The glycophorin-cholesterol recombinants also showed fracture planes consistent with bilayers, and revealed intramembranous particles. Pieces of membrane-like structures as well as apparent vesicular structures were observed. Finally in the recombinants of glycophorin with phospholipid and cholesterol, cholesterol is shown to reduce the population of the motionally restricted phospholipid headgroup environment, in proportion to the mole percent cholesterol content.

Key Words

cholesterol glycophorin membrane protein erythrocyte membrane lipid-protein interactions membrane structure 



lithium diiodosalicylate


nuclear magnetic resonance


sodium dodecylsulfate


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  1. Allain, C.C., Poon, L.S., Chan, C.S.G., Richmond, W., Fu, P.C. 1974. Enzymatic determination of total serum cholesterol.Clin. Chem. (N.Y.) 20:470–477Google Scholar
  2. Bartlett, G.R. 1959. Phosphorus assay in column chromatography.J. Biol. Chem. 234:466–472PubMedGoogle Scholar
  3. Brulet, P., McConnell, H.M. 1976. Protein-lipid interactions: Glycophorin and DMPC.Biochem. Biophys. Res. Commun. 68:363–368CrossRefPubMedGoogle Scholar
  4. Dodge, J.T., Mitchell, C.D., Hanahan, D.J. 1963. The preparation and chemical characteristics of hemoglobin-free ghosts of human erythrocytes.Arch. Biochem. Biophys. 100:119–130PubMedGoogle Scholar
  5. Grefrath, S.P., Reynolds, J.A. 1974. The molecular weight of the major glycoprotein from the human erythrocyte membrane.Proc. Natl. Acad. Sci. USA 71:3193–3198Google Scholar
  6. Hong, K., Hubbell, W.L. 1972. Preparation and properties of phospholipid bilayers containing rhodopsin.Proc. Natl. Acad. Sci. USA 69:2617–2621PubMedGoogle Scholar
  7. Klappauf, E., Schubert, D. 1977. Band 3 from human erythrocyte membranes strongly interacts with cholesterol.FEBS Lett. 80:423–425CrossRefPubMedGoogle Scholar
  8. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature (London) 227:680–685Google Scholar
  9. Litman, B.J. 1973. Lipid model membrane characterization of mixed phospholipid vesicles.Biochemistry 13:2545–2550CrossRefGoogle Scholar
  10. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. 1951. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 193:265–275PubMedGoogle Scholar
  11. MacDonald, R.J., MacDonald, R.C. 1975. Assembly of phospholipid vesicles bearing sialoglycoprotein from erythrocyte membranes.J. Biol. Chem. 250:9206–9214Google Scholar
  12. Marchesi, V.T., Andrews, E.P. 1971. Glycophorin isolation from cell membranes with lithium diiodosalicylate.Science 174:1247–1248PubMedGoogle Scholar
  13. Mendelsohn, R., Dulby, R., Taraschi, T., Cameron, D.G., Mantsch, H.M. 1981. Raman and fourier transform infrared spectroscopic studies of the interaction between glycophorin and dimyristoyl phosphatidylcholine.Biochemistry 20:6699–6710CrossRefPubMedGoogle Scholar
  14. Mimms, L.T., Zamphighi, G., Nozaki, Y., Tanford, C., Reynolds, J.A. 1981. Phospholipid vesicle formation and transmembrane protein incorporation using octyl glucoside.Biochemistry 20:833–839PubMedGoogle Scholar
  15. Nunez, M.T., Glass, J. 1982. Reconstitution of the transferrin receptor in lipid vesicles. Effect of cholesterol on the binding of transferrin.Biochemistry 21:4139–4143CrossRefPubMedGoogle Scholar
  16. Ong, R.L., Marchesi, V.T., Prestegard, J.M. 1981. Small unilamellar vesicles containing glycophorin A.Biochemistry 20:4283–4289Google Scholar
  17. Rajan, S., Kang, S., Gutowsky, H.S., Oldfield, E. 1981. P-31 NMR study of membrane structure.J. Biol. Chem. 256:1160–1166PubMedGoogle Scholar
  18. Rogers, J., Lee, A.G., Wilton, D.C. 1977. The organization of cholesterol and ergosterol in lipid bilayers based on studies using non-perturbing fluorescent probes.Biochim. Biophys. Acta 552:23–27Google Scholar
  19. Romans, A.Y., Yeagle, P.L., O'Conner, S.E., Grisham, C.M. 1979. Interaction between glycophorin and phospholipids in recombined systems.J. Supramol. Struct. 10:241–251PubMedGoogle Scholar
  20. Seelig, J. 1978. P-31 NMR and the headgroup structure of phospholipids in membranes.Biochim. Biophys. Acta 515:105–140PubMedGoogle Scholar
  21. Smith, H.G., Jr., Stubbs, G.W., Litman, B.J. 1975. The isolation and purification of osmotically intact discs from retinal rod outer segments.Exp. Eye Res. 20:211–217PubMedGoogle Scholar
  22. Stewart, T.P., Hui, S.W., Portis, A., Papahadjopoulos, D. 1979. Complex phase mixing of PC and PS in multilamellar membrane vesicles.Biochim. Biophys. Acta 556:1–16PubMedGoogle Scholar
  23. Utsumi, H., Tunggal, B.D., Stoffel, W. 1980. C-13 NMR studies on the interactions of glycophorin with lecithin in reconstituted vesicles.Biochemistry 19:2385–2390CrossRefPubMedGoogle Scholar
  24. Yeagle, P.L., Romans, A.Y. 1981. The glycophorin-phospholipid interface in recombined systems.Biophys. J. 33:243–252PubMedGoogle Scholar
  25. Yeagle, P.L. 1982. P-31 NMR studies of the phospholipid-protein interface in cell membranes.Biophys. J. 37:227–239PubMedGoogle Scholar
  26. Yeagle, P.L. 1983. Cholesterol modulation of the Na+K+ ATPase ATP hydrolyzing activity in the human erythrocyte.Biochim. Biophys. Acta 727:39–44PubMedGoogle Scholar
  27. Zoelen, E.J.J. van, Dijck, P.M.W. van, Kruijff, B. de, Verkleij, A.J., Deenen, L.M.M. van 1978. Effect of glycophorin incorporation on the physico-chemical properties of phospholipid bilayers.Biochim. Biophys. Acta 514:9–24PubMedGoogle Scholar
  28. Zurcher, A., Heusser, H., Jeger, O., Geistich, P. 1954. Uber um wandlungen in den ringen B, C und D des ergosterins.Helv. Chim. Acta 37:1564–1571Google Scholar

Copyright information

© Springer-Verlag 1984

Authors and Affiliations

  • Philip L. Yeagle
    • 1
  1. 1.Department of BiochemistryState University of New York at Buffalo, School of MedicineBuffalo

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