Endotoxin pp 233-245 | Cite as

Fluorescent Detection of Lipopolysaccharide Interactions with Model Membranes

  • D. M. Jacobs
  • H. Yeh
  • R. M. Price
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 256)


The critical importance of the lipid A moiety of LPS in resistance and pathogenesis in gram negative infections has led to the assumption that LPS interaction with target cells is due to hydrophobic interaction with plasma membranes. However, work from several laboratories, including our own, is consistent with the presence of a cell membrane structure with characterstics of a “receptor”. We have proposed a two-step model for LPS-membrane interaction which resolves the two views, and have developed a model system to control the first step (binding to membrane protein) and study the second step (intercalation into lipid bilayer). We examined the interaction of LPS with small unilamellar phosphatidylcholine vesicles labeled in the hydrophobic portion of the bilayer with the fluorescent probe diphenylhexatrine (DPH) and detected changes in the physical properties of the bilayer by measuring DPH fluorescence anisotropy (Δr). We have found that purified, phenol-extracted S. typhimurium LPS interacts with the bilayer as measured by an increase in Δr and conclude that the LPS aggregate coalesced with the lipid bilayer. The greatest change in Δr was achieved with lipid A, Ra-Re glycolipids and diphosphoryl lipid A. Monophosphoryl lipid A and lipid X were less effective. Preparations of wild-type LPS fractionated according to the length of the O-antigen side chain and unfractionated LPS had least effect on Δr. Thus other factors such as serum components or membrane proteins may be necessary to enhance the interaction of LPS with target cells.


Lipid Bilayer Acyl Chain Malachite Green Membrane Lipid Bilayer Fluorescence Anisotropy 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Benedetto, D. A., Shands, J. W., Jr. and Shah, D.O., 1973, The interaction of bacterial lipopolysaccharide with phospholipid bilayers and monolayers. Biochim. Biophys. Acta. 298: 145.PubMedCrossRefGoogle Scholar
  2. 2.
    Bona, C., Juy, D., Truffa-Bachi, P., and Kaplan, G. J., 1976, Binding, capping and internalization of lipopolysaccharide in thymic and non-thymic lymphocytes of the mouse. Biological and autoradiographic study. J. Microscopie Biol. Cell. 25: 47.Google Scholar
  3. 3.
    Carter, S. G., and Karl, D. W., 1982, Inorganic phosphate assay with malachite green: An improvement and evaluation. J. Biochem. Biophys. Methods 7: 7.PubMedCrossRefGoogle Scholar
  4. 4.
    Cynkin, M. A., and Ashwell, G., 1960, Estimation of 3-deoxy sugars by means of the malonaldehydethiobarbituric acid reaction. Nature 186: 155.PubMedCrossRefGoogle Scholar
  5. 5.
    Emmerling, G., Henning, U., and Gulik-Kryzwicki, T., 1977, Order disorder conformational transition of hydrocarbon chains in lipopolysaccharide from Escherichia coli, Eur. J. Biochem. 78: 503.PubMedCrossRefGoogle Scholar
  6. 6.
    Fried, V. A., and Rothfield, L. I., 1978, Interactions between lipopolysaccharide and phosphatidylethanolamine in molecular monolayers. Biochim. Biphys. Acta. 514: 69.CrossRefGoogle Scholar
  7. 7.
    Goldman, R. C., and Leive, L., 1980, Heterogeneity of antigenic-sidechain length in lipolysaccharide from Escherichia coli 0111 and Salmonella typhimurium LT2. Eur. J. Biochem. 107: 145.PubMedCrossRefGoogle Scholar
  8. 8.
    Gregory, S. H., Zimmerman, D. H., and Kern, M., 1980, The Lipid A moiety of lipolysaccharide is specificaly bound to B cell subpopulations of responder and nonresponder animals. J. Immunol. 125: 102.PubMedGoogle Scholar
  9. 9.
    Jacobs, D. M., 1984, Structural features of binding of lipopolysaccharide to murine lymphocytes. Rev. Infect. Dis. 6: 501.PubMedCrossRefGoogle Scholar
  10. 10.
    Jacobs, D. M., and Eldridge, J. H., 1984, Surface phenotype of LPS-binding murine lymphocytes. Proc. Soc. Exp. Biol. Med. 175: 458.PubMedCrossRefGoogle Scholar
  11. 11.
    Jacobs, D. M. and Price, R. M., 1987, A model for lipopolysaccharidemembrane interaction, in: “Recent Advances in Mucosal Immunology, Part A,” J. Mestecky, J. R. McGhee, J. Bienestock, P. L. Ogre, eds. Plenum Publishing Corp.Google Scholar
  12. 12.
    Jann, B., Reske, K., and Jann, K., 1975, Heterogeneity of lipopolysaccharides. Analysis of polysaccharide chain lengths by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Eur. J. Biochem. 60: 239.PubMedCrossRefGoogle Scholar
  13. 13.
    Kabis, S., and Rosenstreich, D., 1977, Binding of bacterial endotoxin to murine spleen lymphocytes. Infect. Immun. 15: 156.Google Scholar
  14. 14.
    Lakowicz, J. R., 1983. in: “Principles of Fluorescence Spectroscopy,” Plenum Press, New York pp. 111–153 and 258–296.Google Scholar
  15. 15.
    Lakowicz, J. R., Prendergast, F. G., and Hogen. D., 1979, Fluorescence anisotropy measurements under oxygen quenching conditions as a method to quantify the depolarizing rotations of fluorophores. Application to diphenylhexatrine in istoropic solvents and in lipid bilayers. Biochem. 18: 520.CrossRefGoogle Scholar
  16. 16.
    Larsen, N. E., Enelow, R. I., Simmons, E. R., and Sullivan, R., 1985, Effect of bacterial endotoxin on the transmembrane electrical potential and plasma membrane fluidity of human monocytes. Biochim. Biophys. Acta. 815: 1.PubMedCrossRefGoogle Scholar
  17. 17.
    Liu, M. S., Onji, T., and Snelgrove, N. E., 1982, Changes in the phase transition temperature of phospholipids induced by endotoxin. Biochim. Biophys. Acta. 710: 248.PubMedCrossRefGoogle Scholar
  18. 18.
    MacKay, A. L., Nichol, C. P., Weeks, G., and Davis, J. H., 1984, A proton and deuterium nuclear magnetic resonance study of orientational order in aqueous dispersons of lipopolysaccharide and lipopolysaccharide/dipalmitoylphosphatidylcholine mixtures. Biochim. Biophys. Acta. 774: 181.CrossRefGoogle Scholar
  19. 19.
    Moller, G., Anderson, J., Pohlit, H., and Sjoberg, O., 1973. Quantitation of the number of mitogen molecules activating DNA synthesis in T and B lymphocytes. Clin. Exp. Immunol. 13: 89.PubMedGoogle Scholar
  20. 20.
    Nikaido, H., Takeuchi, Y., Ohnishi, S. T., and Nakae, T., 1977. Outer membrane of Salmonella typhimurium. Electron spin resonance studies. Biochim. Biophys. Acta. 465: 152.PubMedCrossRefGoogle Scholar
  21. 21.
    Onji, T., and Liu, M. S., 1979. Changes in the surface charge density of liposomes induced by Escherichia coli endotoxin. Biochim. Biophys. Acta. 558: 320.PubMedCrossRefGoogle Scholar
  22. 22.
    Palva, E. T., and Makela, P. H., 1980, Lipopolysaccharide heterogeneity in Salmonella typhimurium analyzed by sodium dodecylsulfate/polyacrylamide gel electrophoresis. Eur. J. Biochem. 107: 137.PubMedCrossRefGoogle Scholar
  23. 23.
    Peterson, A. A. and McGoarty, E. J., 1985, High-molecular-weight components in lipopolysaccharides of Salmonella typhimurium. Salmonella minne-sots. and Escherichia coli. J. Bacteriol. 162: 738.PubMedGoogle Scholar
  24. 24.
    Price, R. M. and Jacobs, D. M., 1986. Fluorescent detection of lipopolysaccharide interactions with model membranes. Biochem. Biophys. Acta. 859: 26.PubMedCrossRefGoogle Scholar
  25. 25.
    Price, R. M., Gersten, D. M. and Ramwell, P. W., 1985, Macromolecules mediate prostacyclin release from human umbilical artery. Biochim. Biophys. Acta. 836: 246.PubMedCrossRefGoogle Scholar
  26. 26.
    Quinn, P. J., 1981, The fluidity of cell membranes and its regulation. Prog. Biophys. Molec. Biol. 38: 1.CrossRefGoogle Scholar
  27. 27.
    Rothfield, L., and Horne, R. W., 1967, Reassociation of purified lipopolysaccharide and phospholipid of the bacterial cell envelope: Electron microscopic and monolayer studies. J. Bacteriol. 93: 1705.PubMedGoogle Scholar
  28. 28.
    Rottem, S., 1978, The effect of Lipid A on the fluidity and permeability properties of phospholipid dispersons. FEBS Letter 95: 121.CrossRefGoogle Scholar
  29. 29.
    Salesse, R., and Garnier, J., 1984, Adenylate cyclase and membrane fluidity. Molec. Cell Biochim. 60: 17.Google Scholar
  30. 30.
    Swartzwelder, F. and Jacobs, D. M., 1984, Lipopolysaccharide capping on murine lymphocytes. Rev. Infect. Dis. 6: 578.Google Scholar
  31. 31.
    Symond, D. B. A., and Clarkson, C. A., 1979, The binding of LPS to the lymphocyte surface. Immunology 38: 503.Google Scholar
  32. 32.
    Szoka, F., and Papahadjopoulos, D., 1980, Comparative properties and methods of preparation of lipid vesicles (liposomes). Ann. Rev. Biophys. Bioeng. 9: 467.CrossRefGoogle Scholar
  33. 33.
    Takeuchi, Y. and Nikaido, H., 1981, Persistence of segregated phospholipid domains in phospholipid-lipopolysaccharide mixed bilayers: Studies with spin-labeled phospholipids. Biochemistry 20: 523.PubMedCrossRefGoogle Scholar
  34. 34.
    Van Alphen, L., Verkleij, A., Burnell, E., and Lugtenberg, B., 1980, 31p nuclear magnetic resonance and freeze-fracture electron microscopy studies on Escherichia coli. II. Lipopolysaccharide and lipopolysaccharide-phospholipid complexes. Biochim. Biophys. Acta. 597: 502.PubMedCrossRefGoogle Scholar
  35. 35.
    Zimmerman, D. H., Gregory, S., and Kern, M., 1977, Differentiation of lymphoid cells: The preferential binding of the Lipid A moiety of lipopolysaccharide to B lymphocyte populations. J. Immunol 119: 1018.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1990

Authors and Affiliations

  • D. M. Jacobs
    • 1
  • H. Yeh
    • 1
  • R. M. Price
    • 1
  1. 1.Department of Microbiology, School of Medicine and Biomedical SciencesState University of New York at BuffaloBuffaloUSA

Personalised recommendations