Skip to main content
NCBI home page
Infection and Immunity logoLink to Infection and Immunity
. 1987 Mar;55(3):579–586. doi: 10.1128/iai.55.3.579-586.1987

Purification of Pseudomonas aeruginosa exoenzyme S.

D E Woods, J U Que
PMCID: PMC260377  PMID: 3102377

Abstract

Pseudomonas aeruginosa produces two distinct ADP-ribosyl transferases, exotoxin A and exoenzyme S, which differ in a number of properties including substrate specificity. Exoenzyme S was purified from culture supernatants of P. aeruginosa DG1. The procedure for purification consists of four major steps: ammonium sulfate precipitation, anion-exchange chromatography on DEAE-Sephacel, acetone precipitation in the presence of 1 M NaCl, and G-100 Superfine gel filtration chromatography. Exoenzyme S was monitored during purification by an assay for ADP-ribosyl transferase activity, mouse toxicity, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified material exhibited ADP-ribosyl transferase activity, reacted with monoclonal antibodies to exoenzyme S, and was toxic to mice and a variety of tissue culture cell lines.

Full text

PDF
579

Images in this article

583Image
on p.583
584Image
on p.584
584Image
on p.584

Selected References

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

  1. Andrews P. Estimation of the molecular weights of proteins by Sephadex gel-filtration. Biochem J. 1964 May;91(2):222–233. doi: 10.1042/bj0910222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Avrameas S., Guilbert B. A method for quantitative determination of cellular immunoglobulins by enzyme-labeled antibodies. Eur J Immunol. 1971 Nov;1(5):394–396. doi: 10.1002/eji.1830010518. [DOI] [PubMed] [Google Scholar]
  3. BAUMSTARK J. S., LAFFIN R. J., BARDAWIL W. A. A PREPARATIVE METHOD FOR THE SEPARATION OF 7S GAMMA GLOBULIN FROM HUMAN SERUM. Arch Biochem Biophys. 1964 Dec;108:514–522. doi: 10.1016/0003-9861(64)90436-9. [DOI] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  5. Bryan L. E., Kureishi A., Rabin H. R. Detection of antibodies to Pseudomonas aeruginosa alginate extracellular polysaccharide in animals and cystic fibrosis patients by enzyme-linked immunosorbent assay. J Clin Microbiol. 1983 Aug;18(2):276–282. doi: 10.1128/jcm.18.2.276-282.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chung D. W., Collier R. J. Enzymatically active peptide from the adenosine diphosphate-ribosylating toxin of Pseudomonas aeruginosa. Infect Immun. 1977 Jun;16(3):832–841. doi: 10.1128/iai.16.3.832-841.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gill D. M., Pappenheimer A. M., Jr Structure-activity relationships in diphtheria toxin. J Biol Chem. 1971 Mar 10;246(5):1492–1495. [PubMed] [Google Scholar]
  8. Hissey P. H., Thompson K. J., Bawden L. Single-step monoclonal antibody affinity purification of human urogastrone from urine. J Immunol Methods. 1985 Apr 22;78(2):211–216. doi: 10.1016/0022-1759(85)90078-x. [DOI] [PubMed] [Google Scholar]
  9. Iglewski B. H., Kabat D. NAD-dependent inhibition of protein synthesis by Pseudomonas aeruginosa toxin,. Proc Natl Acad Sci U S A. 1975 Jun;72(6):2284–2288. doi: 10.1073/pnas.72.6.2284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Iglewski B. H., Sadoff J. C. Toxin inhibitors of protein synthesis: production, purification, and assay of Pseudomonas aeruginosa toxin A. Methods Enzymol. 1979;60:780–793. doi: 10.1016/s0076-6879(79)60071-x. [DOI] [PubMed] [Google Scholar]
  11. Iglewski B. H., Sadoff J., Bjorn M. J., Maxwell E. S. Pseudomonas aeruginosa exoenzyme S: an adenosine diphosphate ribosyltransferase distinct from toxin A. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3211–3215. doi: 10.1073/pnas.75.7.3211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Köhler G., Howe S. C., Milstein C. Fusion between immunoglobulin-secreting and nonsecreting myeloma cell lines. Eur J Immunol. 1976 Apr;6(4):292–295. doi: 10.1002/eji.1830060411. [DOI] [PubMed] [Google Scholar]
  13. Köhler G., Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975 Aug 7;256(5517):495–497. doi: 10.1038/256495a0. [DOI] [PubMed] [Google Scholar]
  14. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  15. Liu P. V. Extracellular toxins of Pseudomonas aeruginosa. J Infect Dis. 1974 Nov;130 (Suppl)(0):S94–S99. doi: 10.1093/infdis/130.supplement.s94. [DOI] [PubMed] [Google Scholar]
  16. Middlebrook J. L., Dorland R. B. Bacterial toxins: cellular mechanisms of action. Microbiol Rev. 1984 Sep;48(3):199–221. doi: 10.1128/mr.48.3.199-221.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Nicas T. I., Frank D. W., Stenzel P., Lile J. D., Iglewski B. H. Role of exoenzyme S in chronic Pseudomonas aeruginosa lung infections. Eur J Clin Microbiol. 1985 Apr;4(2):175–179. doi: 10.1007/BF02013593. [DOI] [PubMed] [Google Scholar]
  18. Nicas T. I., Iglewski B. H. Contribution of exoenzyme S to the virulence of Pseudomonas aeruginosa. Antibiot Chemother (1971) 1985;36:40–48. doi: 10.1159/000410470. [DOI] [PubMed] [Google Scholar]
  19. Nicas T. I., Iglewski B. H. Isolation and characterization of transposon-induced mutants of Pseudomonas aeruginosa deficient in production of exoenzyme S. Infect Immun. 1984 Aug;45(2):470–474. doi: 10.1128/iai.45.2.470-474.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Oakley B. R., Kirsch D. R., Morris N. R. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem. 1980 Jul 1;105(2):361–363. doi: 10.1016/0003-2697(80)90470-4. [DOI] [PubMed] [Google Scholar]
  21. Stanker L. H., Vanderlaan M., Juarez-Salinas H. One-step purification of mouse monoclonal antibodies from ascites fluid by hydroxylapatite chromatography. J Immunol Methods. 1985 Jan 21;76(1):157–169. doi: 10.1016/0022-1759(85)90488-0. [DOI] [PubMed] [Google Scholar]
  22. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Woods D. E., Iglewski B. H. Toxins of Pseudomonas aeruginosa: new perspectives. Rev Infect Dis. 1983 Sep-Oct;5 (Suppl 4):S715–S722. doi: 10.1093/clinids/5.supplement_4.s715. [DOI] [PubMed] [Google Scholar]
  24. Woods D. E., Sokol P. A. Use of transposon mutants to assess the role of exoenzyme S in chronic pulmonary disease due to Pseudomonas aeruginosa. Eur J Clin Microbiol. 1985 Apr;4(2):163–169. doi: 10.1007/BF02013591. [DOI] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES