Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1995 Jul 1;309(Pt 1):209–214. doi: 10.1042/bj3090209

Characterization of an exocellular serine-thiol proteinase activity in Paracoccidioides brasiliensis.

A K Carmona 1, R Puccia 1, M C Oliveira 1, E G Rodrigues 1, L Juliano 1, L R Travassos 1
PMCID: PMC1135821  PMID: 7619058

Abstract

An exocellular proteinase activity has been characterized in Paracoccidioides brasiliensis culture filtrates. Chromatographic analysis showed that the activity was eluted from an anion-exchange Resource Q column at 0.08-0.1 M NaCl, and by gel filtration near ovalbumin elution, in a single peak. Purification of the proteinase, however, was hampered by the low protein yield, in contrast to the high peptidase activity. Numerous chromogenic peptidyl p-nitroanilide derivatives and internally quenched fluorescent peptides, flanked by Abz (O-aminobenzoyl) and EDDnp (ethylenediaminedinitrophenyl), were tested as substrates. Cleavage was observed with Abz-MKRLTL-EDDnp, Abz-FRLVR-EDDnp, and Abz-PLGLLGR-EDDnp at Leu-Thr, Leu-Val and Leu-Leu/Leu-Gly bonds respectively as determined by isolation of the corresponding fragments by HPLC. Leucine at P1 seemed to be restrictive for the activity of the exocellular enzyme, but threonine (P'1) and leucine (P'2) in Abz-MKRLTL-EDDnp apparently were not essential. Also, a pair of alanines could substitute for lysine (P3) and arginine (P2) in this substrate, with a decrease in the Km values. The exocellular peptidase activity of P. brasiliensis had an optimum pH of > 9.0 and was irreversibly inhibited by PMSF, mercuric acetate and p-hydroxymercuribenzoate. Inhibition of the mercuriate compounds could be partially reversed by Cys/EDTA. E-64 [trans-epoxysuccinyl-L-leucylamido-(4-guanido)butene] was a weak and reversible inhibitor, whereas EDTA and pepstatin were not inhibitory. These results suggest that P. brasiliensis exocellular enzyme belongs to the subfamily of SH-containing serine proteinases.

Full text

PDF
209

Selected References

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

  1. Betzel C., Bellemann M., Pal G. P., Bajorath J., Saenger W., Wilson K. S. X-ray and model-building studies on the specificity of the active site of proteinase K. Proteins. 1988;4(3):157–164. doi: 10.1002/prot.340040302. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Chagas J. R., Juliano L., Prado E. S. Intramolecularly quenched fluorogenic tetrapeptide substrates for tissue and plasma kallikreins. Anal Biochem. 1991 Feb 1;192(2):419–425. doi: 10.1016/0003-2697(91)90558-b. [DOI] [PubMed] [Google Scholar]
  4. Ebeling W., Hennrich N., Klockow M., Metz H., Orth H. D., Lang H. Proteinase K from Tritirachium album Limber. Eur J Biochem. 1974 Aug 15;47(1):91–97. doi: 10.1111/j.1432-1033.1974.tb03671.x. [DOI] [PubMed] [Google Scholar]
  5. Hasnain S., Adeli K., Storer A. C. Purification and characterization of an extracellular thiol-containing serine proteinase from Thermomyces lanuginosus. Biochem Cell Biol. 1992 Feb;70(2):117–122. doi: 10.1139/o92-017. [DOI] [PubMed] [Google Scholar]
  6. Juliano M. A., Juliano L. Synthesis and kinetic parameters of hydrolysis by trypsin of some acyl-arginyl-p-nitroanilides and peptides containing arginyl-p-nitroanilide. Braz J Med Biol Res. 1985;18(4):435–445. [PubMed] [Google Scholar]
  7. Kolattukudy P. E., Lee J. D., Rogers L. M., Zimmerman P., Ceselski S., Fox B., Stein B., Copelan E. A. Evidence for possible involvement of an elastolytic serine protease in aspergillosis. Infect Immun. 1993 Jun;61(6):2357–2368. doi: 10.1128/iai.61.6.2357-2368.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kundu A. K., Manna S. Purification and characterization of extracellular proteinases of Aspergillus oryzae. Appl Microbiol. 1975 Oct;30(4):507–513. doi: 10.1128/am.30.4.507-513.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Mizusawa K., Yoshida F. Thermophilic Streptomyces alkaline proteinase. II. The role of a sulfhydryl group and the conformational stability. J Biol Chem. 1973 Jun 25;248(12):4417–4423. [PubMed] [Google Scholar]
  10. Oliveira M. C., Hirata I. Y., Chagas J. R., Boschcov P., Gomes R. A., Figueiredo A. F., Juliano L. Intramolecularly quenched fluorogenic peptide substrates for human renin. Anal Biochem. 1992 May 15;203(1):39–46. doi: 10.1016/0003-2697(92)90040-e. [DOI] [PubMed] [Google Scholar]
  11. Ong P. S., Gaucher G. M. Protease production by thermophilic fungi. Can J Microbiol. 1973 Jan;19(1):129–133. doi: 10.1139/m73-019. [DOI] [PubMed] [Google Scholar]
  12. Puccia R., Schenkman S., Gorin P. A., Travassos L. R. Exocellular components of Paracoccidioides brasiliensis: identification of a specific antigen. Infect Immun. 1986 Jul;53(1):199–206. doi: 10.1128/iai.53.1.199-206.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Puccia R., Takaoka D. T., Travassos L. R. Purification of the 43 kDa glycoprotein from exocellular components excreted by Paracoccidioides brasiliensis in liquid culture (TOM medium). J Med Vet Mycol. 1991;29(1):57–60. [PubMed] [Google Scholar]
  14. Puccia R., Travassos L. R. 43-kilodalton glycoprotein from Paracoccidioides brasiliensis: immunochemical reactions with sera from patients with paracoccidioidomycosis, histoplasmosis, or Jorge Lobo's disease. J Clin Microbiol. 1991 Aug;29(8):1610–1615. doi: 10.1128/jcm.29.8.1610-1615.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Puccia R., Travassos L. R. The 43-kDa glycoprotein from the human pathogen Paracoccidioides brasiliensis and its deglycosylated form: excretion and susceptibility to proteolysis. Arch Biochem Biophys. 1991 Sep;289(2):298–302. doi: 10.1016/0003-9861(91)90475-x. [DOI] [PubMed] [Google Scholar]
  16. Rinderknecht H., Geokas M. C., Silverman P., Haverback B. J. A new ultrasensitive method for the determination of proteolytic activity. Clin Chim Acta. 1968 Aug;21(2):197–203. doi: 10.1016/0009-8981(68)90127-7. [DOI] [PubMed] [Google Scholar]
  17. Rodrigues E. G., Travassos L. R. Nature of the reactive epitopes in Paracoccidioides brasiliensis polysaccharide antigen. J Med Vet Mycol. 1994;32(1):77–81. [PubMed] [Google Scholar]
  18. Rüchel R., de Bernardis F., Ray T. L., Sullivan P. A., Cole G. T. Candida acid proteinases. J Med Vet Mycol. 1992;30 (Suppl 1):123–132. [PubMed] [Google Scholar]
  19. San-Blas G. The cell wall of fungal human pathogens: its possible role in host-parasite relationships. Mycopathologia. 1982 Sep 17;79(3):159–184. doi: 10.1007/BF01837196. [DOI] [PubMed] [Google Scholar]
  20. Shenolikar S., Stevenson K. J. Purification and partial characterization of a thiol proteinase from the thermophilic fungus Humicola lanuginosa. Biochem J. 1982 Jul 1;205(1):147–152. doi: 10.1042/bj2050147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Stambuk B. U., Puccia R., de Almeida M. L., Travassos L. R., Schenkman S. Secretion of the 43 kDa glycoprotein antigen by Paracoccidioides brasiliensis. J Med Vet Mycol. 1988;26(6):367–373. doi: 10.1080/02681218880000521. [DOI] [PubMed] [Google Scholar]
  22. Stepanov V. M., Chestukhina G. G., Rudenskaya G. N., Epremyan A. S., Osterman A. L., Khodova O. M., Belyanova L. P. A new subfamily of microbial serine proteinase? Structural similarities of Bacillus thuringiensis and Thermoactinomyces vulgaris extracellular serine proteinases. Biochem Biophys Res Commun. 1981 Jun;100(4):1680–1687. doi: 10.1016/0006-291x(81)90712-9. [DOI] [PubMed] [Google Scholar]
  23. Vicentini A. P., Gesztesi J. L., Franco M. F., de Souza W., de Moraes J. Z., Travassos L. R., Lopes J. D. Binding of Paracoccidioides brasiliensis to laminin through surface glycoprotein gp43 leads to enhancement of fungal pathogenesis. Infect Immun. 1994 Apr;62(4):1465–1469. doi: 10.1128/iai.62.4.1465-1469.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES