1887

Abstract

Phospholipase C from the Dallas 1E strain of serogroup 5 was purified from buffered yeast extract culture supernate by ion-exchange chromatography followed by fractionation by manganous chloride and ammonium sulphate precipitation steps. Enzyme activity was assayed by hydrolysis of -nitrophenylphosphorylcholine and confirmed by release of radioactivity from tritiated L-α-dipalmitoylphosphatidylcholine labelled in the methyl groups of choline. After SDS-PAGE, the purified preparation yielded a single band upon Coomassie-blue staining. This protein migrated with an apparent of 50000–54000. Phospholipase C activity was maximal at pH ≥ 8.4 and was enhanced in the presence of sorbitol and of several nonionic detergents but was eliminated by SDS. EDTA, Cu, Fe and Zn inhibited enzyme activity, whereas Ba, Ca, Co, Mg and Mn restored activity to EDTA-treated material. No haemolytic activity was demonstrated with the purified enzyme.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-134-2-489
1988-02-01
2021-05-12
Loading full text...

Full text loading...

/deliver/fulltext/micro/134/2/mic-134-2-489.html?itemId=/content/journal/micro/10.1099/00221287-134-2-489&mimeType=html&fmt=ahah

References

  1. Alberts B., Bray D., Lewis J., Raff M., Roberts K., Watson J.D. 1983 Molecular Biology of the Cell pp. 255–263 New York: Garland Publishing;
    [Google Scholar]
  2. Baine W.B. 1985; Cytolytic and phospholipase C activity in Legionella species. Journal of General Microbiology 131:1383–1391
    [Google Scholar]
  3. Baine W.B., Rasheed J.K. 1979; Aromatic substrate specificity of browning by cultures of the Legionnaires’ disease bacterium. Annals of Internal Medicine 90:619–620
    [Google Scholar]
  4. Baine W.B., Rasheed J.K., Feeley J.C., Gorman G.W., Casida L.E. Jr 1978; Effect of supplemental l-tyrosine on pigment production in cultures of the Legionnaires’ disease bacterium. Current Microbiology 1:93–94
    [Google Scholar]
  5. Baine W.B., Rasheed J.K., Mackel D.C., Bopp C.A., Wells J.G., Kaufmann A.F. 1979; Exotoxin activity associated with the Legionnaires’ disease bacterium. Journal of Clinical Microbiology 9:453–456
    [Google Scholar]
  6. Berka R.M., Vasil M.L. 1982; Phospholipase C (heat-labile hemolysin) of Pseudomonas aeruginosa:purification and preliminary characterization. Journal of Bacteriology 152:239–245
    [Google Scholar]
  7. Dennis E.A. 1983; Phospholipases. In The Enzymes, 3rd edn. 16 pp. 307–353 Boyer P. D. Edited by New York: Academic Press;
    [Google Scholar]
  8. De Vries T. 1961; Potentials of electrochemical reactions at 25 °C. In Handbook of Chemistry and Physics, 43rd edn.. pp. 1743–1745 Edited by Hodgman C. D., Weast R. C., Shankland R. S., Selby S. M. Cleveland: The Chemical Rubber Publishing Co.;
    [Google Scholar]
  9. El-Sayed M.Y., Roberts M.F. 1985; Charged detergents enhance the activity of phospholipase C (Bacillus cereus) towards micellar short-chain phosphatidylcholine. Biochimica et biophysica acta 831:133–141
    [Google Scholar]
  10. El-Sayed M.Y., De Bose C.D., Coury L.A., Roberts M.F. 1985; Sensitivity of phospholipase C (Bacillus cereus) activity to phosphatidylcholine structural modifications. Biochimica et biophysica acta 837:325–335
    [Google Scholar]
  11. England A.C. III McKinney R.M., Skaliy P., Gorman G.W. 1980; A fifth serogroup of Legionella pneumophila. . Annals of Internal Medicine 93:58–59
    [Google Scholar]
  12. Feeley J.C., Gibson R.J., Gorman G.W., Langford N.C., Rasheed J.K., Mackel D.C., Baine W.B. 1979; Charcoal-yeast extract agar: primary isolation medium for Legionella pneumophila. . Journal of Clinical Microbiology 10:437–441
    [Google Scholar]
  13. Green A.A., Hughes W.L. 1955; Protein fractionation on the basis of solubility in aqueous solutions of salts and organic solvents. Methods in Enzymology 1:67–90
    [Google Scholar]
  14. Grossman S., Oestreicher G., Hogue P.K., Cobley J.G., Singer T.P. 1974; Microanalytical determination of the activities of phospholipases A, C, and D and of their mixtures. Analytical Biochemistry 58:301–309
    [Google Scholar]
  15. Gulig P.A., Hansen E.J. 1985; Coprecipitation of lipopolysaccharide and the 39000-molecular-weight major outer membrane protein of Haemophilus influenzae type b by lipopolysaccharide-directed monoclonal antibody. Infection and Immunity 49:819–827
    [Google Scholar]
  16. Hames B.D. 1981; An introduction to polyacrylamide gel electrophoresis. In Gel Electrophoresis of Proteins pp. 1–91 Hames B.D., Rickwood D. Edited by Oxford: IRL Press;
    [Google Scholar]
  17. Heppel L. 1955; Separation of proteins from nucleic acids. Methods in Enzymology 1:137–138
    [Google Scholar]
  18. Himmelhoch S.R. 1971; Chromatography of proteins on ion-exchange adsorbents. Methods in Enzymology 22:273–286
    [Google Scholar]
  19. Hindahl M.S., Iglewski B.H. 1986; Outer membrane proteins from Legionella pneumophila serogroups and other Legionella species. Infection and Immunity 51:94–101
    [Google Scholar]
  20. Hoffman P. 1984; Bacterial physiology. In Legionella, Proceedings of the 2nd International Symposium, pp. 61–67 Thomsberry C., Balows A., Feeley J. C., Jakubowski W. Edited by Washington, DC: American Society for Microbiology;
    [Google Scholar]
  21. Horwitz M.A. 1983; The Legionnaires’ disease bacterium (Legionella pneumophila) inhibits phagosome-lysosome fusion in human monocytes. Journal of Experimental Medicine 158:2108–2126
    [Google Scholar]
  22. Horwitz M.A., Maxfield F.R. 1984; Legionella pneumophila inhibits acidification of its phagosome in human monocytes. Journal of Cell Biology 99:1936–1943
    [Google Scholar]
  23. Klaus M.H., Clements J.A., Havel R.J. 1961; Composition of surface-active material isolated from beef lung. Proceedings of the National Academy of Sciences of the United States of America 47:1858–1859
    [Google Scholar]
  24. Krug E..L., Kent C. 1981; Assay for phospholipase C. Methods in Enzymology 72D:347–351
    [Google Scholar]
  25. Kurioka S., Matsuda M. 1976; Phospholipase C assay using p-nitrophenylphosphorylcholine together with sorbitol and its application to studying the metal and detergent requirement of the enzyme. Analytical Biochemistry 75:281–289
    [Google Scholar]
  26. Malmqvist T., Thelestam M., Möllby R. 1984; Hydrophobicity of cultured mammalian cells and some effects of bacterial phospholipases C. Acta pathologica, microbiologica et immunologica scandinavica B92:127–133
    [Google Scholar]
  27. Möllby R. 1978; Bacterial phospholipases. In Bacterial Toxins and Cell Membranes, pp. 367–424 Jeljaszewicz J., Wadström T. Edited by New York: Academic Press;
    [Google Scholar]
  28. Nishizuka Y. 1986; Studies and perspectives of protein kinase C. Science 233:305–312
    [Google Scholar]
  29. Otani S., Matsui I., Kuramoto A., Morisawa S. 1984; Induction of ornithine decarboxylase in guinea-pig lymphocytes and its relation to phospholipid metabolism. Biochimica et biophysica acta 800:96–101
    [Google Scholar]
  30. Pasculle A.W., Feeley J.C., Gibson R.J., Cordes L.G., Myerowitz R.L., Patton C.M., Gorman G.W., Carmack C.L., Ezzell J.W., Dowling J.N. 1980; Pittsburgh pneumonia agent: direct isolation from human lung tissue. Journal of Infectious Diseases 141:727–732
    [Google Scholar]
  31. Pine L., George J.R., Reeves M.W., Harrell W.K. 1979; Development of a chemically defined liquid medium for growth of Legionella pneumophila. . Journal of Clinical Microbiology 9:615–626
    [Google Scholar]
  32. Segel I.H. 1976 Biochemical Calculations pp. 233–246 New York: John Wiley;
    [Google Scholar]
  33. Selander R.K., Mckinney R.M., Whittam T.S., Bibb W.F., Brenner D.J., Nolte F.S., Pattison P.E. 1985; Genetic structure of populations of Legionella pneumophila . Journal of Bacteriology 163:1021–1037
    [Google Scholar]
  34. Turner J.C., Anderson H.M., Gandal C.P. 1958; Species differences in red blood cell phospha-tides separated by column and paper chromatography. Biochimica et biophysica acta 30:130–134
    [Google Scholar]
  35. Waite M. 1985; Approaches to the study of mammalian cellular phospholipases. Journal of Lipid Research 26:1379–1388
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-134-2-489
Loading
/content/journal/micro/10.1099/00221287-134-2-489
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error