Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1986 May 15;236(1):177–184. doi: 10.1042/bj2360177

The purification and properties of yeast proteinase B from Candida albicans.

P C Farley, M G Shepherd, P A Sullivan
PMCID: PMC1146803  PMID: 3539100

Abstract

A serine proteinase (ycaB) from the yeast Candida albicans A.T.C.C. 10261 was purified to near homogeneity. The enzyme was almost indistinguishable from yeast proteinase B (EC 3.4.21.48), and an Mr of 30,000 for the proteinase was determined by SDS/polyacrylamide-gel electrophoresis. The initial site of hydrolysis of the oxidized B-chain of insulin, by the purified proteinase, was the Leu-Tyr peptide bond. The preferential degradation at this site, analysed further with N-blocked amino acid ester and amide substrates, demonstrated that the specificity of the proteinase is determined by an extended substrate-binding site, consisting of at least three subsites (S1, S2 and S'1). The best p-nitrophenyl ester substrates were benzyloxycarbonyl-Tyr p-nitrophenyl ester (kcat./Km 3,536,000 M-1 X S-1), benzyloxycarbonyl-Leu p-nitrophenyl ester (kcat./Km 2,250,000 M-1 X S-1) and benzyloxycarbonyl-Phe p-nitrophenyl ester (kcat./Km 1,000,000 M-1 X S-1) consistent with a preference for aliphatic or aromatic amino acids at subsite S1. The specificity for benzyloxycarbonyl-Tyr p-nitrophenyl ester probably reflects the binding of the p-nitrophenyl group in subsite S'1. The presence of S2 was demonstrated by comparison of the proteolytic coefficients (kcat./Km) for benzyloxycarbonyl-Ala p-nitrophenyl ester (825,000 M-1 X S-1) and t-butyloxycarbonyl-Ala p-nitrophenyl ester (333,000 M-1 X S-1). Cell-free extracts contain a heat-stable inhibitor of the proteinase.

Full text

PDF
184

Selected References

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

  1. Achstetter T., Emter O., Ehmann C., Wolf D. H. Proteolysis in eukaryotic cells. Identification of multiple proteolytic enzymes in yeast. J Biol Chem. 1984 Nov 10;259(21):13334–13343. [PubMed] [Google Scholar]
  2. Atlas D., Berger A. On the specificity of elastase. Hydrolysis of peptide p-nitrobenzyl esters. Biochemistry. 1972 Dec 5;11(25):4719–4723. doi: 10.1021/bi00775a013. [DOI] [PubMed] [Google Scholar]
  3. 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: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  4. Chattaway F. W., Odds F. C., Barlow A. J. An examination of the production of hydrolytic enzymes and toxins by pathogenic strains of Candida albicans. J Gen Microbiol. 1971 Aug;67(3):255–263. doi: 10.1099/00221287-67-3-255. [DOI] [PubMed] [Google Scholar]
  5. Eisenthal R., Cornish-Bowden A. The direct linear plot. A new graphical procedure for estimating enzyme kinetic parameters. Biochem J. 1974 Jun;139(3):715–720. doi: 10.1042/bj1390715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Every D. Quantitative measurement of protease activities in slab polyacrylamide gel electrophoretograms. Anal Biochem. 1981 Sep 15;116(2):519–523. doi: 10.1016/0003-2697(81)90396-1. [DOI] [PubMed] [Google Scholar]
  7. Gaucher G. M., Stevenson K. J. Thermomycolin. Methods Enzymol. 1976;45:415–433. [PubMed] [Google Scholar]
  8. Gopal P., Sullivan P. A., Shepherd M. G. Enzymes of N-acetylglucosamine metabolism during germ-tube formation in Candida albicans. J Gen Microbiol. 1982 Oct;128(10):2319–2326. doi: 10.1099/00221287-128-10-2319. [DOI] [PubMed] [Google Scholar]
  9. Kominami E., Hoffschulte H., Holzer H. Purification and properties of proteinase B from yeast. Biochim Biophys Acta. 1981 Sep 15;661(1):124–135. doi: 10.1016/0005-2744(81)90091-7. [DOI] [PubMed] [Google Scholar]
  10. Kominami E., Hoffschulte H., Leuschel L., Maier K., Holzer H. The substrate specificity of proteinase B from baker's yeast. Biochim Biophys Acta. 1981 Sep 15;661(1):136–141. doi: 10.1016/0005-2744(81)90092-9. [DOI] [PubMed] [Google Scholar]
  11. Laemmli U. K., Favre M. Maturation of the head of bacteriophage T4. I. DNA packaging events. J Mol Biol. 1973 Nov 15;80(4):575–599. doi: 10.1016/0022-2836(73)90198-8. [DOI] [PubMed] [Google Scholar]
  12. Levine N., Hatcher V. B., Lazarus G. S. Proteinases of human epidermis; a possible mechanism for polymorphonuclear leukocyte chemotaxis. Biochim Biophys Acta. 1976 Dec 8;452(2):458–467. doi: 10.1016/0005-2744(76)90196-0. [DOI] [PubMed] [Google Scholar]
  13. Magni G., Natalini P., Santarelli I., Vita A. Bakers' yeast protease A purification and enzymatic and molecular properties. Arch Biochem Biophys. 1982 Feb;213(2):426–433. doi: 10.1016/0003-9861(82)90568-9. [DOI] [PubMed] [Google Scholar]
  14. Matsudaira P. T., Burgess D. R. SDS microslab linear gradient polyacrylamide gel electrophoresis. Anal Biochem. 1978 Jul 1;87(2):386–396. doi: 10.1016/0003-2697(78)90688-7. [DOI] [PubMed] [Google Scholar]
  15. Morihara K. Comparative specificity of microbial proteinases. Adv Enzymol Relat Areas Mol Biol. 1974;41(0):179–243. doi: 10.1002/9780470122860.ch5. [DOI] [PubMed] [Google Scholar]
  16. Morrissey J. H. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem. 1981 Nov 1;117(2):307–310. doi: 10.1016/0003-2697(81)90783-1. [DOI] [PubMed] [Google Scholar]
  17. Murphy C. J., Shepherd M. G., Sullivan P. A. Chemical modification of L-lactate 2-monooxygenase with fluorodinitrobenzene: evidence for two essential histidine residues. Biochemistry. 1983 Mar 29;22(7):1665–1669. doi: 10.1021/bi00276a022. [DOI] [PubMed] [Google Scholar]
  18. Narayanan A. S., Anwar R. A. The specificity of purified porcine pancreatic elastase. Biochem J. 1969 Aug;114(1):11–17. doi: 10.1042/bj1140011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Negi M., Tsuboi R., Matsui T., Ogawa H. Isolation and characterization of proteinase from Candida albicans: substrate specificity. J Invest Dermatol. 1984 Jul;83(1):32–36. doi: 10.1111/1523-1747.ep12261656. [DOI] [PubMed] [Google Scholar]
  20. Peterson G. L. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem. 1977 Dec;83(2):346–356. doi: 10.1016/0003-2697(77)90043-4. [DOI] [PubMed] [Google Scholar]
  21. Remold H., Fasold H., Staib F. Purification and characterization of a proteolytic enzyme from Candida albicans. Biochim Biophys Acta. 1968 Oct 8;167(2):399–406. doi: 10.1016/0005-2744(68)90219-2. [DOI] [PubMed] [Google Scholar]
  22. Rüchel R. Properties of a purified proteinase from the yeast Candida albicans. Biochim Biophys Acta. 1981 May 14;659(1):99–113. doi: 10.1016/0005-2744(81)90274-6. [DOI] [PubMed] [Google Scholar]
  23. SANGER F., TUPPY H. The amino-acid sequence in the phenylalanyl chain of insulin. 2. The investigation of peptides from enzymic hydrolysates. Biochem J. 1951 Sep;49(4):481–490. doi: 10.1042/bj0490481. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Saheki T., Holzer H. Proteolytic activities in yeast. Biochim Biophys Acta. 1975 Mar 28;384(1):203–214. doi: 10.1016/0005-2744(75)90109-6. [DOI] [PubMed] [Google Scholar]
  25. Schechter I., Berger A. On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun. 1967 Apr 20;27(2):157–162. doi: 10.1016/s0006-291x(67)80055-x. [DOI] [PubMed] [Google Scholar]
  26. Schep G. P., Shepherd M. G., Sullivan P. A. Purification and properties of a beta-1,6-glucanase from Penicillium brefeldianum. Biochem J. 1984 Nov 1;223(3):707–714. doi: 10.1042/bj2230707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sluyterman L. A., Wijdenes J. Organomercurial agarose. Methods Enzymol. 1974;34:544–547. doi: 10.1016/s0076-6879(74)34070-0. [DOI] [PubMed] [Google Scholar]
  28. Tatemoto K. Neuropeptide Y: complete amino acid sequence of the brain peptide. Proc Natl Acad Sci U S A. 1982 Sep;79(18):5485–5489. doi: 10.1073/pnas.79.18.5485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Templeton M. D., Sullivan P. A., Shepherd M. G. The inhibition of ornithine transcarbamoylase from Escherichia coli W by phaseolotoxin. Biochem J. 1984 Dec 1;224(2):379–388. doi: 10.1042/bj2240379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Visser L., Blout E. R. The use of p-nitrophenyl N-tert-butyloxycarbonyl-L-alaninate as substrate for elastase. Biochim Biophys Acta. 1972 Apr 7;268(1):257–260. doi: 10.1016/0005-2744(72)90223-9. [DOI] [PubMed] [Google Scholar]
  31. Wang S. S., Carpenter F. H. Kinetics of the tryptic hydrolysis of the oxidized B chain of bovine insulin. Biochemistry. 1967 Jan;6(1):215–224. doi: 10.1021/bi00853a034. [DOI] [PubMed] [Google Scholar]
  32. Wolf D. H. Control of metabolism in yeast and other lower eukaryotes through action of proteinases. Adv Microb Physiol. 1980;21:267–338. doi: 10.1016/s0065-2911(08)60358-6. [DOI] [PubMed] [Google Scholar]
  33. Zubenko G. S., Jones E. W. Protein degradation, meiosis and sporulation in proteinase-deficient mutants of Saccharomyces cerevisiae. Genetics. 1981 Jan;97(1):45–64. doi: 10.1093/genetics/97.1.45. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES