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. 1975 Apr;147(1):45–53. doi: 10.1042/bj1470045

Aspergillus oryzae acid proteinase. Purification and properties, and formation of pi-chymotrypsin.

R Davidson, A Gertler, T Hofmann
PMCID: PMC1165373  PMID: 239702

Abstract

An acid proteinase from Aspergillus oryzae was isolated from a commercial powder by successive (NH4)2SO4 fractionation, acetone precipitation, and ion-exchange chromatography on phosphate- and DEAE-cellulose columns. The purified enzyme was found to be homogeneous by ultracentrifuge-sedimentation analysis (S20, W equal 3.63S), but electrofocusing in polyacrylamide gels and electrophoresis at pH 3.2 revealed that it consists of two very closely migrating bands. No difference in the amino acid composition and enzymic activities of the two partially separated bands could be detected, and it was concluded that the acid proteinase exists in two molecular forms. The enzyme activates bovine trypsinogen and chymotrypsinogen at pH 3.5 (the kappacat. and Km values at 35degrees C are 11.3S- minus 1, 0.10mM and 1.14S- minus 1, 0.18mM respectively). It hydrolyses the Phe-Phe bond of the synthetic pepsin substrates Z-His-Phe-Phe-OEt (kappacat. equal 1.65S- minus 1, Km equal 0.640mM at pH 3.5, 30degrees C) and Z-Ala-Ala-Phe-Phe-OPy4Pr (kappacat. equal 0.37S- minus 1, Km equal 0.037 mM at pH2.9, 39degrees C), where Z represents benzyloxycarbonyl and OPy4Pr represents 3-(4-pyridyl)-propyl 1-ester. Activation of bovine chymotrypsinogen results from the cleavage of the Arg(15)-Ile(16) bond in the zymogen. No other cleavages were observed. The use of A. oryzae proteinase provides a simple tool for the production of pi-chymotrypsin in good yield and purity.

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Selected References

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  1. Abita J. P., Delaage M., Lazdunski M. The mechanism of activation of trypsinogen. The role of the four N-terminal aspartyl residues. Eur J Biochem. 1969 Apr;8(3):314–324. doi: 10.1111/j.1432-1033.1969.tb00530.x. [DOI] [PubMed] [Google Scholar]
  2. BETTELHEIM F. R., NEURATH H. The rapid activation of chymotrypsinogen. J Biol Chem. 1955 Jan;212(1):241–253. [PubMed] [Google Scholar]
  3. Edman P., Begg G. A protein sequenator. Eur J Biochem. 1967 Mar;1(1):80–91. doi: 10.1007/978-3-662-25813-2_14. [DOI] [PubMed] [Google Scholar]
  4. Fruton J. S. The specificity and mechanism of pepsin action. Adv Enzymol Relat Areas Mol Biol. 1970;33:401–443. doi: 10.1002/9780470122785.ch9. [DOI] [PubMed] [Google Scholar]
  5. GABELOTEAU C., DESNUELLE P. [On the activation of bovine trypsinogen by a crystallized protease from Aspergillus saitoi]. Biochim Biophys Acta. 1960 Aug 12;42:230–237. doi: 10.1016/0006-3002(60)90786-1. [DOI] [PubMed] [Google Scholar]
  6. Graham J. E., Sodek J., Hofmann T. Rhizopus acid proteinases (rhizopus-pepsins): properties and homology with other acid proteinases. Can J Biochem. 1973 Jun;51(6):789–796. doi: 10.1139/o73-098. [DOI] [PubMed] [Google Scholar]
  7. HOFMANN T., SHAW R. PROTEOLYTIC ENZYMES OF PENICILLIUM JANTHINELLUM. I. PURIFICATION AND PROPERTIES OF A TRYPSINOGEN-ACTIVATING ENZYME (PEPTIDASE A). Biochim Biophys Acta. 1964 Dec 23;92:543–557. [PubMed] [Google Scholar]
  8. HOFMANN T. TRYPSINOGEN AKTIVIERENDE ENZYME IN SCHIMMELPILZEN DER GATTUNGEN ASPERGILLUS UND PENICILLIUM. Pharm Acta Helv. 1963 Jul-Aug;38:634–639. [PubMed] [Google Scholar]
  9. Haglund H. Isoelectric focusing in pH gradients--a technique for fractionation and characterization of ampholytes. Methods Biochem Anal. 1971;19:1–104. doi: 10.1002/9780470110386.ch1. [DOI] [PubMed] [Google Scholar]
  10. Hermodson M. A., Ericsson L. H., Titani K., Neurath H., Walsh K. A. Application of sequenator analyses to the study of proteins. Biochemistry. 1972 Nov 21;11(24):4493–4502. doi: 10.1021/bi00774a011. [DOI] [PubMed] [Google Scholar]
  11. Hollands T. R., Fruton J. S. Kinetics of the hydrolysis of synthetic substrates by pepsin and by acetyl-pepsin. Biochemistry. 1968 Jun;7(6):2045–2053. doi: 10.1021/bi00846a005. [DOI] [PubMed] [Google Scholar]
  12. Inouye K., Voynick I. M., Delpierre G. R., Fruton J. S. New synthetic substrates for pepsin. Biochemistry. 1966 Jul;5(7):2473–2483. doi: 10.1021/bi00871a044. [DOI] [PubMed] [Google Scholar]
  13. Kovaleva G. G., Shimanskaya M. P., Stepanov V. M. The site of diazoacetyl inhibitor attachment to acid proteinase of Aspergillus awamori--an analog of penicillopepsin and pepsin. Biochem Biophys Res Commun. 1972 Nov 15;49(4):1075–1081. doi: 10.1016/0006-291x(72)90322-1. [DOI] [PubMed] [Google Scholar]
  14. Matsubara H., Sasaki R. M. High recovery of tryptophan from acid hydrolysates of proteins. Biochem Biophys Res Commun. 1969 Apr 29;35(2):175–181. doi: 10.1016/0006-291x(69)90263-0. [DOI] [PubMed] [Google Scholar]
  15. Miller D. D., Horbett T. A., Teller D. C. Reevaluation of the activation of bovine chymotrypsinogen A. Biochemistry. 1971 Dec 7;10(25):4641–4648. doi: 10.1021/bi00801a008. [DOI] [PubMed] [Google Scholar]
  16. Morihara K., Oka T. Comparative specificity of microbial acid proteinases for synthetic peptides. 3. Relationship with their trypsinogen activating ability. Arch Biochem Biophys. 1973 Aug;157(2):561–572. doi: 10.1016/0003-9861(73)90675-9. [DOI] [PubMed] [Google Scholar]
  17. Robinson N. C., Neurath H., Walsh K. A. Preparation and characterization of guanidinated trypsinogen and -guanidinated trypsin. Biochemistry. 1973 Jan 30;12(3):414–420. doi: 10.1021/bi00727a009. [DOI] [PubMed] [Google Scholar]
  18. Spencer R. L., Wold F. A new convenient method for estimation of total cystine-cysteine in proteins. Anal Biochem. 1969 Oct 15;32(1):185–190. doi: 10.1016/0003-2697(69)90123-7. [DOI] [PubMed] [Google Scholar]
  19. Spiro R. G. Glycoproteins. Annu Rev Biochem. 1970;39:599–638. doi: 10.1146/annurev.bi.39.070170.003123. [DOI] [PubMed] [Google Scholar]
  20. Voynick I. M., Fruton J. S. The comparative specificity of acid proteinases. Proc Natl Acad Sci U S A. 1971 Feb;68(2):257–259. doi: 10.1073/pnas.68.2.257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Wang C. C., Shaw E. A comparison of deacylation rates of para-substituted benzoyl-trypsins and chymotrypsins. Arch Biochem Biophys. 1972 May;150(1):259–268. doi: 10.1016/0003-9861(72)90034-3. [DOI] [PubMed] [Google Scholar]
  22. Wang H. L., Hesseltine C. W. Multiple forms of Rhizopus oligosporus protease. Arch Biochem Biophys. 1970 Oct;140(2):459–463. doi: 10.1016/0003-9861(70)90089-5. [DOI] [PubMed] [Google Scholar]
  23. Weber K., Pringle J. R., Osborn M. Measurement of molecular weights by electrophoresis on SDS-acrylamide gel. Methods Enzymol. 1972;26:3–27. doi: 10.1016/s0076-6879(72)26003-7. [DOI] [PubMed] [Google Scholar]
  24. YPHANTIS D. A. EQUILIBRIUM ULTRACENTRIFUGATION OF DILUTE SOLUTIONS. Biochemistry. 1964 Mar;3:297–317. doi: 10.1021/bi00891a003. [DOI] [PubMed] [Google Scholar]

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