Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1984 Sep;45(3):667–673. doi: 10.1128/iai.45.3.667-673.1984

Identification and extraction of Pasteurella haemolytica membrane proteins.

P G Squire, D W Smiley, R B Croskell
PMCID: PMC263347  PMID: 6205995

Abstract

The inner and outer membranes of Pasteurella haemolytica were separated by sucrose density gradient centrifugation after plasmolysis of the cells in 20% sucrose and fragmentation in a French pressure cell. Assays of the two membrane fractions for 2-keto-3-deoxyoctonate, succinate dehydrogenase, and NADH dehydrogenase and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that each of the two membrane fractions was purified fivefold relative to the other. The outer membrane fraction contained two major proteins of molecular weights 30,000 and 42,000 (30K and 42K proteins), and the inner membrane fraction contained five proteins in approximately equal amounts. Intact bacteria as well as membrane fractions were extracted by procedures used by others for vaccine preparation to determine whether the outer membrane proteins were released. Extraction of the isolated membranes with 0.5 M potassium thiocyanate in 0.425 M NaCl with or without EDTA or with M sodium salicylate failed to release more than traces of the outer membrane proteins. Sodium dodecyl sulfate extracted essentially all of the proteins of both membranes, but the products of this procedure were of low solubility and presumably denatured. The inner membrane proteins were extracted with 0.5% Sarkosyl in 0.02 M sodium phosphate buffer (pH 7.5). The 42K outer membrane protein, most of the lipopolysaccharide, and some of the 30K outer membrane protein were extracted with 1% Zwittergent 3-16 in 0.25 M NaCl (pH 8), and the remaining 30K outer membrane protein was extracted with 1% deoxycholate in 0.25% NaCl (pH 8). Extraction of membranes in this sequence yielded partially purified membrane proteins that were soluble in dilute buffers.

Full text

PDF
672

Images in this article

669Image
on p.669
669Image
on p.669

Selected References

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

  1. Baluyut C. S., Simonson R. R., Bemrick W. J., Maheswaran S. K. Interaction of Pasteurella haemolytica with bovine neutrophils: identification and partial characterization of a cytotoxin. Am J Vet Res. 1981 Nov;42(11):1920–1926. [PubMed] [Google Scholar]
  2. Benson M. L., Thomson R. G., Valli V. E. The bovine alveolar macrophage. II. In vitro studies with Pasteurella haemolytica. Can J Comp Med. 1978 Jul;42(3):368–369. [PMC free article] [PubMed] [Google Scholar]
  3. Chopra I., Shales S. W. Comparison of the polypeptide composition of Escherichia coli outer membranes prepared by two methods. J Bacteriol. 1980 Oct;144(1):425–427. doi: 10.1128/jb.144.1.425-427.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Darveau R. P., Charnetzky W. T., Hurlbert R. E. Outer membrane protein composition of Yersinia pestis at different growth stages and incubation temperatures. J Bacteriol. 1980 Aug;143(2):942–949. doi: 10.1128/jb.143.2.942-949.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ellar D. J., Muñoz E., Salton M. R. The effect of low concentrations of glutaraldehyde on Micrococcus lysodeikticus membranes: changes in the release of membrane-associated enzymes and membrane structure. Biochim Biophys Acta. 1971 Jan 5;225(1):140–150. doi: 10.1016/0005-2736(71)90292-6. [DOI] [PubMed] [Google Scholar]
  6. Filip C., Fletcher G., Wulff J. L., Earhart C. F. Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodium-lauryl sarcosinate. J Bacteriol. 1973 Sep;115(3):717–722. doi: 10.1128/jb.115.3.717-722.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Friend S. C., Wilkie B. N., Thomson R. G., Barnum D. A. Bovine pneumonic pasteurellosis: experimental induction in vaccinated and nonvaccinated calves. Can J Comp Med. 1977 Jan;41(1):77–83. [PMC free article] [PubMed] [Google Scholar]
  8. Gaunt G., Moffat R., Mukkur T. K. Fowl cholera: immunization of chickens with potassium thiocyanate (KSCN) extract of Pasteurella multocida serotype 3. Avian Dis. 1977 Oct-Dec;21(4):543–548. [PubMed] [Google Scholar]
  9. Gentry M. J., Corstvet R. E., Panciera R. J. Extraction of capsular material from Pasteurella haemolytica. Am J Vet Res. 1982 Nov;43(11):2070–2073. [PubMed] [Google Scholar]
  10. Gilmour N. J., Angus K. W., Donachie W., Fraser J. Vaccination against experimental pneumonic pasteurellosis. Vet Rec. 1982 May 8;110(19):450–450. doi: 10.1136/vr.110.19.450-a. [DOI] [PubMed] [Google Scholar]
  11. Gilmour N. J. Pasteurellosis in sheep. Vet Rec. 1978 Feb 4;102(5):100–102. doi: 10.1136/vr.102.5.100. [DOI] [PubMed] [Google Scholar]
  12. Hedstrom R. C., Pavlovskis O. R., Galloway D. R. Antibody response of infected mice to outer membrane proteins of Pseudomonas aeruginosa. Infect Immun. 1984 Jan;43(1):49–53. doi: 10.1128/iai.43.1.49-53.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Himmel M. E., Yates M. D., Lauerman L. H., Squire P. G. Purification and partial characterization of a macrophage cytotoxin from Pasteurella haemolytica. Am J Vet Res. 1982 May;43(5):764–767. [PubMed] [Google Scholar]
  14. Jermyn M. A. Increasing the sensitivity of the anthrone method for carbohydrate. Anal Biochem. 1975 Sep;68(1):332–335. doi: 10.1016/0003-2697(75)90713-7. [DOI] [PubMed] [Google Scholar]
  15. Kaehler K. L., Markham R. J., Muscoplat C. C., Johnson D. W. Evidence of species specificity in the cytocidal effects of Pasteurella haemolytica. Infect Immun. 1980 Nov;30(2):615–616. doi: 10.1128/iai.30.2.615-616.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kelley J. T., Parker C. D. Identification and preliminary characterization of Vibrio cholerae outer membrane proteins. J Bacteriol. 1981 Feb;145(2):1018–1024. doi: 10.1128/jb.145.2.1018-1024.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kodama H., Matsumoto M., Snow L. M. Immunogenicity of capsular antigens of Pasteurella multocida in turkeys. Am J Vet Res. 1981 Oct;42(10):1838–1841. [PubMed] [Google Scholar]
  18. Koplow J., Goldfine H. Alterations in the outer membrane of the cell envelope of heptose-deficient mutants of Escherichia coli. J Bacteriol. 1974 Feb;117(2):527–543. doi: 10.1128/jb.117.2.527-543.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kuusi N., Nurminen M., Saxen H., Valtonen M., Mäkelä P. H. Immunization with major outer membrane proteins in experimental salmonellosis of mice. Infect Immun. 1979 Sep;25(3):857–862. doi: 10.1128/iai.25.3.857-862.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Loeb M. R., Zachary A. L., Smith D. H. Isolation and partial characterization of outer and inner membranes from encapsulated Haemophilus influenzae type b. J Bacteriol. 1981 Jan;145(1):596–604. doi: 10.1128/jb.145.1.596-604.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. MASTER R. W. POSSIBLE SYNTHESIS OF POLYRIBONUCLEOTIDES OF KNOWN BASE-TRIPLET SEQUENCES. Nature. 1965 Apr 3;206:93–93. doi: 10.1038/206093b0. [DOI] [PubMed] [Google Scholar]
  23. Markwell M. A., Haas S. M., Bieber L. L., Tolbert N. E. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem. 1978 Jun 15;87(1):206–210. doi: 10.1016/0003-2697(78)90586-9. [DOI] [PubMed] [Google Scholar]
  24. Moriyon I., Berman D. T. Effects of nonionic, ionic, and dipolar ionic detergents and EDTA on the Brucella cell envelope. J Bacteriol. 1982 Nov;152(2):822–828. doi: 10.1128/jb.152.2.822-828.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mukkur T. K. Immunologic and physiologic responses of calves inoculated with potassium thiocyanate extract of Pasteurella multocida type A. Am J Vet Res. 1978 Aug;39(8):1269–1273. [PubMed] [Google Scholar]
  26. Nagy L. K., Penn C. W. Protection of cattle against experimental haemorrhagic septicaemia by the capsular antigens of Pasteurella multocida, types B and E. Res Vet Sci. 1976 May;20(3):249–253. [PubMed] [Google Scholar]
  27. Osborn M. J., Gander J. E., Parisi E., Carson J. Mechanism of assembly of the outer membrane of Salmonella typhimurium. Isolation and characterization of cytoplasmic and outer membrane. J Biol Chem. 1972 Jun 25;247(12):3962–3972. [PubMed] [Google Scholar]
  28. Osborn M. J., Wu H. C. Proteins of the outer membrane of gram-negative bacteria. Annu Rev Microbiol. 1980;34:369–422. doi: 10.1146/annurev.mi.34.100180.002101. [DOI] [PubMed] [Google Scholar]
  29. Penn C. W., Nagy L. K. Isolation of a protective, non-toxic capsular antigen from Pasteurella multocida, types B and E. Res Vet Sci. 1976 Jan;20(1):90–96. [PubMed] [Google Scholar]
  30. Schnaitman C. A. Protein composition of the cell wall and cytoplasmic membrane of Escherichia coli. J Bacteriol. 1970 Nov;104(2):890–901. doi: 10.1128/jb.104.2.890-901.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Schnaitman C. A. Solubilization of the cytoplasmic membrane of Escherichia coli by Triton X-100. J Bacteriol. 1971 Oct;108(1):545–552. doi: 10.1128/jb.108.1.545-552.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Tabatabai L. B., Deyoe B. L., Ritchie A. E. Isolation and characterization of toxic fractions from Brucella abortus. Infect Immun. 1979 Nov;26(2):668–679. doi: 10.1128/iai.26.2.668-679.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Verstreate D. R., Creasy M. T., Caveney N. T., Baldwin C. L., Blab M. W., Winter A. J. Outer membrane proteins of Brucella abortus: isolation and characterization. Infect Immun. 1982 Mar;35(3):979–989. doi: 10.1128/iai.35.3.979-989.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wilkie B. N., Markham R. J., Shewen P. E. Response of calves to lung challenge exposure with Pasteurella haemolytica after parenteral or pulmonary immunization. Am J Vet Res. 1980 Nov;41(11):1773–1778. [PubMed] [Google Scholar]
  35. Winter A. J., Verstreate D. R., Hall C. E., Jacobson R. H., Castleman W. L., Meredith M. P., McLaughlin C. A. Immune response to porin in cattle immunized with whole cell, outer membrane, and outer membrane protein antigens of Brucella abortus combined with trehalose dimycolate and muramyl dipeptide adjuvants. Infect Immun. 1983 Dec;42(3):1159–1167. doi: 10.1128/iai.42.3.1159-1167.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Yates W. D. A review of infectious bovine rhinotracheitis, shipping fever pneumonia and viral-bacterial synergism in respiratory disease of cattle. Can J Comp Med. 1982 Jul;46(3):225–263. [PMC free article] [PubMed] [Google Scholar]
  37. Zollinger W. D., Mandrell R. E., Griffiss J. M., Altieri P., Berman S. Complex of meningococcal group B polysaccharide and type 2 outer membrane protein immunogenic in man. J Clin Invest. 1979 May;63(5):836–848. doi: 10.1172/JCI109383. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES