1887

Journal of Medical Microbiology logo

Volume 33, Issue 4

Purification and characterisation of an extracellular serine proteinase from and its detection in tissue Free

Abstract

Surmmary

A serine proteinase (Alp) from the culture supernate of a clinical isolate of was purified to virtual homogeneity at a yield of 41%. The procedure involved affinity chromatography on agarose-ɛ-amino-caproyl-D-trypto-phan methyl ester. Alp had an estimated mol. wt of 32 Kda and the pI was determined at pH 7.9. The enzyme was fully inhibited by phenylmethyl sulphonyl fluoride, chymostatin and α-1-proteinase inhibitor, and it was largely inhibited by α-1-anti-chymotrypsin. Partial inhibition was observed with tosyl-phenylalanine chloromethyl ketone, but tosyl-lysine chloromethyl ketone was ineffective. Thus, Alp may be identical with the major chymotryptic activity of , which has already been described. The N-terminal sequence of 25 amino acids revealed an 88% homology of Alp with the subtilisin-related proteinase of . Alp acted on casein over a broad range from pH 5.5 to 11.5 and also acts to a lesser extent on haemoglobin and serum albumin. The enzyme degraded elastin and a synthetic elastase substrate; hence, it may be identical with the previously described elastinolytic activity of the fungus. At pH 7.3 and a concentration of 1 μg/ml, Alp was not toxic for Vero cells, but it efficiently detached such cells from a plastic surface. Specific antibodies against Alp were detected by enzyme immunoassay in the sera of patients and Alp-antigen was demonstrated by immunofluorescence in mycotic human lung. In addition, a second proteinase (Exalp) with extremely alkaline activity, and an aspartic proteinase of are described.

  • Received:
  • Revised:
  • Published Online:
© Society for General Microbiology, 1990
Loading

Article metrics loading...

/content/journal/jmm/10.1099/00222615-33-4-243
1990-12-01
2024-04-20
Loading full text...

Full text loading...

/deliver/fulltext/jmm/33/4/medmicro-33-4-243.html?itemId=/content/journal/jmm/10.1099/00222615-33-4-243&mimeType=html&fmt=ahah

References

  1. Schønheyder H. Pathogenetic and serological aspects of pulmonary aspergillosis. Scand J Infect Dis 1987 Suppl 51:1–62
     [Google Scholar]
  2. Bodey GP, Vartivarian S. Aspergillosis. Eur J Clin Microbiol Infect Dis 1989; 8:413–437
     [Google Scholar]
  3. Walsh TJ, Dixon DM. Nosocomial aspergillosis: environ mental microbiology, hospital epidemiology, diagnosis and treatment. Eur J Epidemiol 1989; 5:131–142
     [Google Scholar]
  4. Nakagawa Y. Alkaline proteinases from Aspergillus. Meth ods Enzymol 1970; 19:581–591
     [Google Scholar]
  5. Cohen BL. The proteases of aspergilli. In Smith JE, Pateman JA. (eds) Genetics and physiology of Aspergillus London: Academic Press; 1977281–292
     [Google Scholar]
  6. Jönsson AG, Martin SM. Protease production by Aspergillus fumigatus. Agr. Biol Chem 1964; 28:734–739
     [Google Scholar]
  7. Miyaji M, Nishimura K. Relationship between proteolytic activity of Aspergillus fumigatus and the fungus invasiveness of mouse brain. Mycopathologia 1977; 62:161–166
     [Google Scholar]
  8. Staib F. Extracellular proteolytic activity of Aspergillus fumigatus strains with septum-like structures in their phialides in serum-albumin agar. Zentralbl Bakteriol Mikrobiol Hyg [A] 1982; 252:279–285
     [Google Scholar]
  9. Kothary MH, Chase T, Macmillan JD. Correlation of elastase production by some strains of Aspergillus fumigatus with ability to cause pulmonary invasive aspergillosis in mice. Infect Immun 1984; 43:320–325
     [Google Scholar]
  10. Staib F, Mishra SK, Rajendran C. et al. A notable aspergillus from a mortal aspergilloma of the lung. New aspects of the epidemiology, serodiagnosis and taxonomy of Aspergillus fumigatus. Zentralbl Bakteriol [A] 1980; 247:530–536
     [Google Scholar]
  11. Biguet J, Tran van Ky P, Fruit J, Andrieu S. Identification d’une activite chymotrypsique au niveau de fractions remarquables de l’extrait antigenique à’Aspergillus fumigatus. Repercussions sur le diagnostic immunologique de l’aspergillose. Rev Immunol (Paris) 1967; 31:317–328
     [Google Scholar]
  12. Staib F. Pleural fluid as nutrient substratum for Aspergillus fumigatus and Aspergillus fiavus. Submerged growth in pleural fluid and extracellular proteolysis in pleural fluid agar. Zentralbl Bakteriol Mikrobiol Hyg [A] 1985; 260:543–549
     [Google Scholar]
  13. Kunitz M. Crystalline soybean trypsin inhibitor. J Gen Physiol 1947; 30:291–310
     [Google Scholar]
  14. Scharmann W, Balke E. Untersuchungen iiber eine Protease (Elastase) von Pseudomonas aeruginosa. I. Bildung und Reinigung der Protease. Hoppe Seylers Z Physiol Chem 1974; 355:443–450
     [Google Scholar]
  15. Kraus E, Femfert U. Proteinase K from the mold Tritirach- ium album limber. Specificity and mode of action. Hoppe Seylers Z Physiol Chem 1976; 357:937–947
     [Google Scholar]
  16. Martin SM, Jonsson AG. An extracellular protease from Aspergillus fumigatus. Canad J Biochem 1965; 43:1745–1753
     [Google Scholar]
  17. Bout D, Fruit J, Capron A. Application de la chromatogra phie d’affinite à l’isolement des fractions antigeniques à’Aspergillus fumigatus supportant une activite chymotrypsique. C R Acad Sci [D] (Paris) 1973; 276:2341–2344
     [Google Scholar]
  18. Rüchel R, Gross J. Preparations of continuous gradient gel slabs: a simple technique. Anal Biochem 1979; 92:91–98
     [Google Scholar]
  19. Kratzin HD, Wiltfang J, Karas M, Neuhoff V, Hilschmann N. Gas-phase sequencing after electroblotting on polyvinylidene difluoride membranes assigns correct molecular weight to myoglobin molecular weight markers. Anal Biochem 1989; 183:1–8
     [Google Scholar]
  20. Haun U, Rüchel R, Spies A. A series of serological tests for the detection of antigens and specific antibodies in deep-seated candidosis: experimental aspects. Myko-sen 1987; 30:472–482
     [Google Scholar]
  21. Johnson GD, Davidson RS, McNamee KC, Russel G, Goodwin D, Holborow EJ. Fading of immunofluorescence during microscopy: a study of the phenomenon and its remedy. J Immunol Methods 1982; 55:231–242
     [Google Scholar]
  22. Sundsmo JS, Götze O. Human monocyte spreading induced by factor B of the alternative pathway of complement activation. Cell Immunol 1980; 52:1–17
     [Google Scholar]
  23. Dion WM. The proteolyic enzymes of microorganisms. I. Survey of fungi and actinomycetes for protease production in submerged culture. Canad J Res [C] 1950; 28:577–585
     [Google Scholar]
  24. Tran van Ky P, Uriel J, Rose F. Characterisation de types d’activites enzymatiques dans des extraits antigeniques à’Aspergillus fumigatus apres electrophorese et immu-nelectrophorese en agarose. Ann Inst Pasteur (Paris) 1966; 111:161–170
     [Google Scholar]
  25. Schønheyder H, Andersen P. Fractionation of Aspergillus fumigatus antigens by hydrophobie interaction chromatography and gel filtration. Int Archs Allergy Appl Immunol 1984; 73:231–236
     [Google Scholar]
  26. Rhodes JC, Bode RB, McCuan-Kirsch CM. Elastase production in clinical isolates of Aspergillus. Diagn Microbiol Infect Dis 1988; 10:165–170
     [Google Scholar]
  27. Panneerselvam M, Dhar SC. Physico-chemical properties of the acid proteinase from Aspergillus fumigatus. Ital J Biochem 1981; 30:63–74
     [Google Scholar]
  28. Tatsumi H, Ogawa Y, Murakami S. et al. A full length cDNA clone for the alkaline protease from Aspergillus oryzae: structural analysis and expression in Saccharo-myces cerevisiae. Mol Gen Genet 1989; 219:33–38
     [Google Scholar]
  29. Jany KD, Lederer G, Mayer B. Proteinase K—a new subclass of the subtilisins. The amino acid sequence of the enzyme. Biol Chem Hoppe Seyler 1986; 367:87
     [Google Scholar]
  30. Frosco M, Chase T, Macmillan J. Further purification of elastase from Aspergillus fumigatus. Annual Meeting of the American Society for Microbiology 1989, New Orleans. Abstract No. F 88:
     [Google Scholar]
  31. Harvey C, Longbottom JL. Characterization of a second major antigen Ag13 (antigen C) of Aspergillus fumigatus and investigation of its immunological reactivity. Clin Exp Immunol 1987; 70:247–254
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/00222615-33-4-243
Loading
Purification and characterisation of an extracellular serine proteinase from Aspergillus fumigatus and its detection in tissue
J. Med. Microbiol. 33, 243 (1990); https://doi.org/10.1099/00222615-33-4-243
/content/journal/jmm/10.1099/00222615-33-4-243
/content/journal/jmm/10.1099/00222615-33-4-243
Loading

Data & Media loading...

Most cited 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