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Effect ofClostridium perfringens phospholipase C (alpha-toxin) on the human diploid fibroblast membrane

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Summary

Human diploid embryonic lung fibroblasts were cultivated in Eagle's Minimum Essential Medium and labeled with3H-uridine. The release of soluble radioactive substances into the medium was used as an indicator of damage to the cell membrane. The assay method described is simple, sensitive and rapid and allows quantitative estimation of changes in membrane permeability before any morphological damage is observed microscopically. Crude commercial preparations of phospholipase C (E.C. 3.1.4.3.) (40 μg/ml) were highly active on the cell membrane but most of the membrane damaging activity was found to be due to contaminating theta-toxin. However, also highly purified phospholipase C caused a membrane damage as measured by release of isotope through the plasma membrane. The release could be increased by including an optimal concentration of calcium ions in the incubation buffer, by treating the cells in a hypotonic medium and by simultaneous treatment with sublytic concentrations of Triton X-100. To our knowledge this is the first report of membrane damage on a live, intact, metabolizing human diploid cell caused by a highly purified phospholipase C. The results are in agreement with a dynamic membrane structure with the polar groups of a part of the phospholipids accessible at the membrane surface.

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References

  1. Banerjee, S. P., Dwosh, I. L., Khanna, V. K., Sen, A. K. 1970. Solubilization of guinea pig kidney (Na++K+)-ATPase with Lubrol W and Triton X-100.Biochim. Biophys. Acta 211:345

    Google Scholar 

  2. Bligh, E. G., Dyer, W. J. 1959. A rapid method of total lipid extraction and purification.Canad. J. Biochem. Physiol. 37:911

    Google Scholar 

  3. Burnstein, C., Loyter, A., Racker, E. 1971. Effect of phospholipases on the structure and function of mitochondria.J. Biol. Chem. 246:4075

    Google Scholar 

  4. Coleman, R., Finean, J. B., Knutton, S., Limbrick, A. R. 1970. A structural study of the modification of erythrocyte ghosts by phospholipase C.Biochim. Biophys. Acta 219:81

    Google Scholar 

  5. Condrea, E., de Vries, A. 1964. Hemolysis and splitting of human erythrocyte phospholipids by snake venoms.Biochim. Biophys. Acta 84:60

    Google Scholar 

  6. Cuatrecasas, P. 1971. Perturbation of the insulin receptor of isolated fat cells with proteolytic enzymes.J. Biol. Chem. 246:6522

    Google Scholar 

  7. Dunnick, J. K., Marinetti, G. V., Greenland, P. 1972. Effects of detergents and sodium fluoride on the enzyme activities of the rat liver plasma membrane.Biochim. Biophys. Acta 266:684

    Google Scholar 

  8. Eagle, H. 1959. Amino acid metabolism in mammalian cell cultures.Science 130:432

    Google Scholar 

  9. Finean, J. B., Coleman, R. 1970. Integration of structural and biochemical approaches in the study of cell membranes.In: Membranes Structure and Function (FEBS symposium). J. R. Villanueva and F. Ponz, editors. Vol 20, p. 9. Academic Press Inc., New York-London

    Google Scholar 

  10. Finean, J. B., Martonosi, A. 1965. The action of phospholipase C on muscle microsomes. A correlation of electron microscope and biochemical data.Biochim. Biophys. Acta 98:547

    Google Scholar 

  11. Glaser, M., Simpkins, H., Singer, S. J., Sheetz, M., Chan, S. I. 1970. On the interactions of lipids and proteins in the red blood cell membrane.Proc. Nat. Acad. Sci. 65:721

    Google Scholar 

  12. Helenius, A., Simons, K. 1972. The binding of detergents to lipophilic and hydrophilic proteins.J. Biol. Chem. 247:3656

    Google Scholar 

  13. Johnson, C. E., Bonventre, P. F. 1967. Lethal toxins ofBacillus cereus. I. Relationships and nature of toxin, hemolysin and phospholipase.J. Bacteriol. 94:306

    Google Scholar 

  14. Kaplan, S. S., Finch, S. C., Basford, R. E. 1972. Polymorphonuclear leukocyte activation: Effects of phospholipase C.Proc. Soc. Exp. Biol. N.Y. 140:540

    Google Scholar 

  15. Klibansky, C., London, Y., Frenkel, A., de Vries, A. 1968. Enhancing action of synthetic and natural basic polypeptides on erythrocyte, ghost phospholipid hydrolysis by phospholipase A.Biochim. Biophys. Acta 150:15

    Google Scholar 

  16. Lankisch, P. G., Vogt, W. 1972. Direct haemolytic activity of phospholipase A.Biochim. Biophys. Acta 270:241

    Google Scholar 

  17. Lenard, J., Singer, S. J. 1968. Structure of membranes: Reaction of red blood cell membranes with phospholipase C.Science 159:738

    Google Scholar 

  18. Litwin, J., Thelestam, M. 1972. The lysis of human diploid fibroblasts with borate buffer.Acta Path. Microbiol. Scand. 80B:115

    Google Scholar 

  19. 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

    Google Scholar 

  20. MacFarlane, M. G., Knight, B. C. J. G. 1941. The biochemistry of bacterial toxins. I. Lecithinase activity ofCl. welchii toxins.Biochem. J. 35:884

    Google Scholar 

  21. Martonosi, A. 1968. Sarcoplasmic reticulum. IV. Solubilization of microsomal adenosine triphosphatase.J. Biol. Chem. 243:71

    Google Scholar 

  22. McIlwain, D. L., Rapport, M. M. 1971. The effects of phospholipase C (Clostridium perfringens) on purified myelin.Biochim. Biophys. Acta 239:71

    Google Scholar 

  23. Miller, D. M. 1970. Total solubilization of erythrocyte membranes by nonionic detergents.Biochem. Biophys. Res. Commun. 40:716

    Google Scholar 

  24. Mitzutani, H., Mitzutani, H. 1964. Action of phospholipase on influenza virus.Nature 204:718

    Google Scholar 

  25. Möllby, R., Nord, C.-E., Wadström, T. 1973. Biological activities contaminating preparations of phospholipase C (alpha-toxin) fromClostridium perfringens.Toxicon 11:139

    Google Scholar 

  26. Möllby, R., Wadström, T. 1973. Purification of phospholipase C (alpha-toxin) fromClostridium perfringens.Biochim. Biophys. Acta 321:569

    Google Scholar 

  27. Op den Kamp, J. A. F., Kauerz, M. T., van Deenen, L. L. M. 1972. Action of phospholipase A2 and phospholipase C onBacillus subtilis protoplasts.J. Bacteriol. 112:1090

    Google Scholar 

  28. Ottolenghi, A. C., Bowman, M. H. 1970. Membrane structure: Morphological and chemical alterations in phospholipase-C-treated mitochondria and red cell ghosts.J. Membrane Biol. 2:180

    Google Scholar 

  29. Patriarca, P., Zatti, M., Cramer, R., Rossi, F. 1970. Stimulation of the respiration of polymorphonuclear leukocytes by phospholipase C.Life Sci. 9:841

    Google Scholar 

  30. Razin, S., Barash, V. 1969. Solubilization of mycoplasma membranes by the nonionic detergent Triton X-100.FEBS Lett. 3:217

    Google Scholar 

  31. Robertson, J. D. 1964. Unit membranes: A review with recent new studies of experimental alterations and a new subunit structure in synaptic membranes.In: Cellular Membranes in Development. M. Locke, editor. p. 1. Academic Press Inc., New York-London

    Google Scholar 

  32. Roelofsen, B., Zwaal, R. F. A., Comfurius, P., Woodward, C. B., van Deenen, L. L. M. 1971. Action of pure phospholipase A2 and phospholipase c on human erythrocytes and ghosts.Biochim. Biophys. Acta 241:925

    Google Scholar 

  33. Roth, F. B., Pillemer, L. 1953. The separation of alpha-toxin (lecithinase) from filtrates ofClostridium welchii.J. Immunol. 70:533

    Google Scholar 

  34. Simons, K., Helenius, A., Garoff, H. 1973. Solubilization of the membrane proteins from Semliki Forest virus with Triton X-100.J. Mol. Biol. 80:119

    Google Scholar 

  35. Singer, S. J. 1971. Molecular organisation of biological membranes.In: Structure and Function of Biological Membranes. L. J. Rothfield, editor. p. 192. Academic Press Inc., New York-London

    Google Scholar 

  36. Singer, S. J., Nicolson, G. L. 1972. The fluid mosaic model of the structure of cell membranes.Science 175:720

    Google Scholar 

  37. Sugahara, T., Ohsaka, A. 1970. Two molecular forms ofClostridium perfringens alpha-toxin associated with lethal, hemolytic and enzymatic activities.Jap. J. Med. Sci. Biol. 23:61

    Google Scholar 

  38. Takeda, Y., Hizukuri, S. 1972. Effect of Triton X-100 on sweet potato beta-amylase.Biochim. Biophys. Acta 268:175

    Google Scholar 

  39. Thelestam, M., Möllby, R., Wadström, T. 1973. Effects of staphylococcal alpha-, beta-, delta-, and gamma-hemolysins on human diploid fibroblasts and HeLa cells: Evaluation of a new quantitative assay for measuring cell damage.Infect. Immunity 8:938

    Google Scholar 

  40. Wadström, T., Möllby, R. 1971. Studies on extracellular proteins fromStaphylococcus aureaus. VI. Production and purification of beta-haemolysin in large scale.Biochim. Biophys. Acta 242:288

    Google Scholar 

  41. Wadström, T., Möllby, R. 1971. Studies on extracellular proteins fromStaphylococcus aureus. VII. Studies on beta-haemolysin.Biochim. Biophys. Acta 242:308

    Google Scholar 

  42. Wadström, T., Möllby, R. 1972. Some biological properties of purified staphylococcal haemolysins.Toxicon 10:511

    Google Scholar 

  43. van Heyningen, W. E. 1941. The biochemistry of gas gangrene toxins. 2. Partial purification of the toxins ofCl. welchii type A.Biochem. J. 35:1257

    Google Scholar 

  44. Woodward, C. B., Zwaal, R. F. A. 1972. The lytic behavior of pure phospholipases A2 and C towards osmotically swollen erythrocytes and resealed ghosts.Biochim. Biophys. Acta 274:272

    Google Scholar 

  45. Zwaal, R. F. A., Roelofsen, B., Comfurius, P., van Deenen, L. L. M. 1971. Complete purification and some properties of phospholipase C fromBacillus cereus.Biochim. Biophys. Acta 233:474

    Google Scholar 

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Authors and Affiliations

  1. Departments of Bacteriology and Applied Microbiology, Karolinska Institutet, S-10401, Stockholm 60

    Roland Möllby, Monica Thelestam & Torkel Wadström

  2. Department of Bacteriology, Statens Bakteriologiska Laboratorium, S-10521, Stockholm, Sweden

    Roland Möllby, Monica Thelestam & Torkel Wadström

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  1. Roland Möllby
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  2. Monica Thelestam
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  3. Torkel Wadström
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Möllby, R., Thelestam, M. & Wadström, T. Effect ofClostridium perfringens phospholipase C (alpha-toxin) on the human diploid fibroblast membrane. J. Membrain Biol. 16, 313–330 (1974). https://doi.org/10.1007/BF01872421

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  • DOI: https://doi.org/10.1007/BF01872421

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