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. 1987 Dec;55(12):3131–3136. doi: 10.1128/iai.55.12.3131-3136.1987

Isolation of a membrane-associated Bacteroides gingivalis glycylprolyl protease.

D Grenier 1, B C McBride 1
PMCID: PMC260038  PMID: 3316034

Abstract

A low-molecular-weight proteolytic enzyme was purified 47-fold from outer membranes of Bacteroides gingivalis ATCC 33277 by preparative polyacrylamide gel electrophoresis. The enzyme was present in all B. gingivalis strains tested but was not found in other species of black-pigmented Bacteroides. The molecular weight, determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, was 19,500 when the enzyme was heated to 100 degrees C in SDS before electrophoresis and 29,000 when it was mixed with SDS but not heated. The optimum pH, with azocasein as the substrate, was between 6.0 and 6.5. The activity was inhibited by phenylmethylsulfonyl fluoride, N-alpha-p-tosyl-L-lysine chloromethyl ketone, Hg2+, and various reducing agents. The enzyme was active against azocasein, azocoll, proline-rich protein from saliva, and the synthetic peptide glycyl-L-proline-p-nitroanilide. The enzyme did not degrade acid-soluble collagen nor did it hydrolyze various arginine- and lysine-containing synthetic substrates.

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Selected References

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

  1. Abiko Y., Hayakawa M., Murai S., Takiguchi H. Glycylprolyl dipeptidylaminopeptidase from Bacteroides gingivalis. J Dent Res. 1985 Feb;64(2):106–111. doi: 10.1177/00220345850640020201. [DOI] [PubMed] [Google Scholar]
  2. Berdal B. P., Olsvik O. Legionella extracellular protease activity on chromogenic peptides: a simplified procedure for biochemical enzyme identification. Acta Pathol Microbiol Immunol Scand B. 1983 Feb;91(1):89–91. doi: 10.1111/j.1699-0463.1983.tb00014.x. [DOI] [PubMed] [Google Scholar]
  3. Boyd J., McBride B. C. Fractionation of hemagglutinating and bacterial binding adhesins of Bacteroides gingivalis. Infect Immun. 1984 Aug;45(2):403–409. doi: 10.1128/iai.45.2.403-409.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Carlsson J., Herrmann B. F., Höfling J. F., Sundqvist G. K. Degradation of the human proteinase inhibitors alpha-1-antitrypsin and alpha-2-macroglobulin by Bacteroides gingivalis. Infect Immun. 1984 Feb;43(2):644–648. doi: 10.1128/iai.43.2.644-648.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carlsson J., Höfling J. F., Sundqvist G. K. Degradation of albumin, haemopexin, haptoglobin and transferrin, by black-pigmented Bacteroides species. J Med Microbiol. 1984 Aug;18(1):39–46. doi: 10.1099/00222615-18-1-39. [DOI] [PubMed] [Google Scholar]
  6. Cimasoni G., Song M., McBride B. C. Effect of crevicular fluid and lysosomal enzymes on the adherence of streptococci and bacteroides to hydroxyapatite. Infect Immun. 1987 Jun;55(6):1484–1489. doi: 10.1128/iai.55.6.1484-1489.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Foltmann B., Szecsi P. B., Tarasova N. I. Detection of proteases by clotting of casein after gel electrophoresis. Anal Biochem. 1985 May 1;146(2):353–360. doi: 10.1016/0003-2697(85)90551-2. [DOI] [PubMed] [Google Scholar]
  8. Fujimura S., Nakamura T. Isolation and characterization of a protease from Bacteroides gingivalis. Infect Immun. 1987 Mar;55(3):716–720. doi: 10.1128/iai.55.3.716-720.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fujimura S., Nakamura T. Isolation and characterization of proteases from Bacteroides melaninogenicus. Infect Immun. 1981 Sep;33(3):738–742. doi: 10.1128/iai.33.3.738-742.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. GIBBONS R. J., MACDONALD J. B. Degradation of collagenous substrates by Bacteroides melaninogenicus. J Bacteriol. 1961 Apr;81:614–621. doi: 10.1128/jb.81.4.614-621.1961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Grenier D., Mayrand D. Functional characterization of extracellular vesicles produced by Bacteroides gingivalis. Infect Immun. 1987 Jan;55(1):111–117. doi: 10.1128/iai.55.1.111-117.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Grenier D., Mayrand D. Selected characteristics of pathogenic and nonpathogenic strains of Bacteroides gingivalis. J Clin Microbiol. 1987 Apr;25(4):738–740. doi: 10.1128/jcm.25.4.738-740.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hancock R. E., Poole K., Benz R. Outer membrane protein P of Pseudomonas aeruginosa: regulation by phosphate deficiency and formation of small anion-specific channels in lipid bilayer membranes. J Bacteriol. 1982 May;150(2):730–738. doi: 10.1128/jb.150.2.730-738.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kaufman E. J., Mashimo P. A., Hausmann E., Hanks C. T., Ellison S. A. Fusobacterial infection: enhancement by cell free extracts of Bacteroides melaninogenicus possessing collagenolytic activity. Arch Oral Biol. 1972 Mar;17(3):577–580. doi: 10.1016/0003-9969(72)90073-8. [DOI] [PubMed] [Google Scholar]
  15. Kelleher P. J., Juliano R. L. Detection of proteases in polyacrylamide gels containing covalently bound substrates. Anal Biochem. 1984 Feb;136(2):470–475. doi: 10.1016/0003-2697(84)90246-x. [DOI] [PubMed] [Google Scholar]
  16. Kilian M. Degradation of immunoglobulins A2, A2, and G by suspected principal periodontal pathogens. Infect Immun. 1981 Dec;34(3):757–765. doi: 10.1128/iai.34.3.757-765.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kortt A. A., Burns J. E., Stewart D. J. Detection of the extracellular proteases of Bacteroides nodosus in polyacrylamide gels: a rapid method of distinguishing virulent and benign ovine isolates. Res Vet Sci. 1983 Sep;35(2):171–174. [PubMed] [Google Scholar]
  18. 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]
  19. Mayrand D., McBride B. C. Exological relationships of bacteria involved in a simple, mixed anaerobic infection. Infect Immun. 1980 Jan;27(1):44–50. doi: 10.1128/iai.27.1.44-50.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Moore W. E., Holdeman L. V., Smibert R. M., Hash D. E., Burmeister J. A., Ranney R. R. Bacteriology of severe periodontitis in young adult humans. Infect Immun. 1982 Dec;38(3):1137–1148. doi: 10.1128/iai.38.3.1137-1148.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Muñoz M. L., Rojkind M., Calderón J., Tanimoto M., Arias-Negrete S., Martínez-Palomo A. Entamoeba histolytica: collagenolytic activity and virulence. J Protozool. 1984 Aug;31(3):468–470. doi: 10.1111/j.1550-7408.1984.tb02995.x. [DOI] [PubMed] [Google Scholar]
  22. Oppenheim F. G., Hay D. I., Franzblau C. Proline-rich proteins from human parotid saliva. I. Isolation and partial characterization. Biochemistry. 1971 Nov;10(23):4233–4238. doi: 10.1021/bi00799a013. [DOI] [PubMed] [Google Scholar]
  23. Sakai D. K. Loss of virulence in a protease-deficient mutant of Aeromonas salmonicida. Infect Immun. 1985 Apr;48(1):146–152. doi: 10.1128/iai.48.1.146-152.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Slots J., Genco R. J. Black-pigmented Bacteroides species, Capnocytophaga species, and Actinobacillus actinomycetemcomitans in human periodontal disease: virulence factors in colonization, survival, and tissue destruction. J Dent Res. 1984 Mar;63(3):412–421. doi: 10.1177/00220345840630031101. [DOI] [PubMed] [Google Scholar]
  25. Sundqvist G., Carlsson J., Herrmann B., Tärnvik A. Degradation of human immunoglobulins G and M and complement factors C3 and C5 by black-pigmented Bacteroides. J Med Microbiol. 1985 Feb;19(1):85–94. doi: 10.1099/00222615-19-1-85. [DOI] [PubMed] [Google Scholar]
  26. Tsai C. M., Frasch C. E. A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal Biochem. 1982 Jan 1;119(1):115–119. doi: 10.1016/0003-2697(82)90673-x. [DOI] [PubMed] [Google Scholar]
  27. Tsutsui H., Kinouchi T., Wakano Y., Ohnishi Y. Purification and characterization of a protease from Bacteroides gingivalis 381. Infect Immun. 1987 Feb;55(2):420–427. doi: 10.1128/iai.55.2.420-427.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. White D., Mayrand D. Association of oral Bacteroides with gingivitis and adult periodontitis. J Periodontal Res. 1981 May;16(3):259–265. doi: 10.1111/j.1600-0765.1981.tb00974.x. [DOI] [PubMed] [Google Scholar]
  29. Yoshimura F., Nishikata M., Suzuki T., Hoover C. I., Newbrun E. Characterization of a trypsin-like protease from the bacterium Bacteroides gingivalis isolated from human dental plaque. Arch Oral Biol. 1984;29(7):559–564. doi: 10.1016/0003-9969(84)90078-5. [DOI] [PubMed] [Google Scholar]
  30. Zacharius R. M., Zell T. E., Morrison J. H., Woodlock J. J. Glycoprotein staining following electrophoresis on acrylamide gels. Anal Biochem. 1969 Jul;30(1):148–152. doi: 10.1016/0003-2697(69)90383-2. [DOI] [PubMed] [Google Scholar]
  31. Zambon J. J., Reynolds H. S., Slots J. Black-pigmented Bacteroides spp. in the human oral cavity. Infect Immun. 1981 Apr;32(1):198–203. doi: 10.1128/iai.32.1.198-203.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]

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