Skip to content
1887

Microbiology Society logo Microbiology

Volume 130, Issue 1

Purification and Characterization of Two Acid Proteinases from Free

Abstract

Two proteinases have been purified from cell extracts of the cellular slime mould and have been identified as proteinase E and proteinase B by electrophoretic analysis on polyacrylamide gels containing haemoglobin. Both were probably glycoproteins, each consisting of a single polypeptide chain with apparent molecular weights of 58000 (proteinase E) and 30000 (proteinase B) as indicated by SDS-PAGE. A higher molecular weight of 38000 was suggested for proteinase B by gel filtration. On isoelectric focusing multiple forms of proteinase E were revealed with isoelectric points ranging from 3·05 to 3·35. There was only a single form of proteinase B with an isoelectric point at pH 3·55. Both enzymes hydrolysed proteins at low pH and activated trypsinogen at pH 3·5. Proteinase B had much lower activity than proteinase E and required a reducing agent such as DTT for maximum activity. The pH optimum for activity of proteinase E towards hide powder azure was 2·5. Proteinase B, but not proteinase E, also hydrolysed a number of derivatives of basic amino acids and related peptide derivatives. -Benzoyl-α--arginine--naphthylamide (Bz-Arg-NNap) was used as substrate during purification, -benzoyl--prolyl--phenylalanyl-L-arginine -nitroanilide (Bz-Pro-Phe-Arg-Nan) was used for proteinase B characterization. Activity was optimal at pH 6 and was dependent on a reducing agent. Inhibitor studies indicated that proteinase E was probably a pepstatin-insensitive aspartic proteinase which was also inhibited by mercurials through binding to a cysteine residue not involved in the catalytic mechanism. Proteinase B was inactivated by a range of typical cysteine proteinase inhibitors. Proteinase B may be identical to proteinase I previously purified from Overall the properties of both proteinases were consistent with their being involved in general protein degradation within the lysosomes of

  • Received:
  • Accepted:
  • Published Online:
© Society for General Microbiology 1984
Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-130-1-123
1984-01-01
2025-01-17
Loading full text...

Full text loading...

/deliver/fulltext/micro/130/1/mic-130-1-123.html?itemId=/content/journal/micro/10.1099/00221287-130-1-123&mimeType=html&fmt=ahah

References

  1. Ansari H., Stevens L. 1983; Purification and properties of two neutral proteinases from Aspergillus nidulans. Journal of General Microbiology 129:1637–1644
     [Google Scholar]
  2. Bai Y., Hayashi R. 1979; Properties of the single sulfydryl group of carboxypeptidase Y. Effects of alkyl and aromatic mercurials toward various synthetic substrates. Journal of Biological Chemistry 254:8473–8479
     [Google Scholar]
  3. Barrett A. J. 1972; A new assay for cathepsin B1 and other thiol proteinases. Analytical Biochemistry 47:280–293
     [Google Scholar]
  4. Barrett A. J. 1973; Human cathepsin Bl. Purification and some properties of the enzyme. Biochemical Journal 131:809–822
     [Google Scholar]
  5. Barrett A. J. 1977; Introduction to the history and classification of tissue proteinases. In Proteinases in Mammalian Cells and Tissues pp. 1–55 Barrett A. J. Edited by Amsterdam: Elsevier/North-Holland Biomedical Press;
     [Google Scholar]
  6. Barrett A. J., Kirschke H. 1981; Cathepsin B, cathepsin H, and cathepsin L. Methods in Enzymology 80:535–561
     [Google Scholar]
  7. Barrett A. J., Mcdonald J. K. 1980 Mammalian Proteases: A Glossary and Bibliography. Volume I Endopeptidases. London: Academic Press;
     [Google Scholar]
  8. Braun V., Hantke K., Wolff , Gerisch G. 1972; Degradation of the murein lipoprotein complex of Escherichia coli cell walls by Dictyosteliumdiscoideum amoebae. European Journal of Biochemistry 27:116–125
     [Google Scholar]
  9. Coukell M. B., Walker I. O. 1973; The basic nuclear proteins of the cellular slime mold Dictyostelium discoideum. Cell Differentiation 2:87–95
     [Google Scholar]
  10. Firtel R. A., Brackenbury R. W. 1972; Partial characterization of several protein and amino acid metabolizing enzymes in the cellular slime mold Dictyostelium discoideum. Developmental Biology 27:307–321
     [Google Scholar]
  11. Fong D., Bonner J. T. 1979; Proteases in cellular slime mold development: evidence for their involvement. Proceedings of the National Academy of Sciences of the United States of America 76:6481–6485
     [Google Scholar]
  12. Fong D., Rutherford C. L. 1978; Protease activity during cell differentiation of the cellular slime mold Dictyosteliumdiscoideum. Journal of Bacteriology 134:521–527
     [Google Scholar]
  13. Gustafson G. L., Milner L. A. 1980; Occurrence of Af-acetylglucosamine-1-phosphate in proteinase I from Dictyosteliumdiscoideum. Journal of Biological Chemistry 255:7208–7210
     [Google Scholar]
  14. Gustafson G. L., Thon L. A. 1979; Purification and characterization of a proteinase from Dictyostelium discoideum. Journal of Biological Chemistry 254:12471–12478
     [Google Scholar]
  15. Holzer H., Heinrich P. C. 1980; Control of proteolysis. Annual Review of Biochemistry 49:63–91
     [Google Scholar]
  16. Kominami E., Hoffschulte H., Holzer H. 1981; Purification and properties of proteinase B from yeast. Biochimica et biophysica acta 661:124–135
     [Google Scholar]
  17. Kost R. G., North M. J., Whyte A. 1981; Acid proteinases in various species of cellular slime mold. Experimental Mycology 5:269–277
     [Google Scholar]
  18. Lah T., Turk V. 1982; Autolysis studies of cathepsin D. Hoppe-Seyler’s Zeitschrift für physiologische Chemie 363:247–254
     [Google Scholar]
  19. Leichtling B. H., Majerfeld I. H., Coffman D. S., Rickenberg H. V. 1982; Identification of the regulatory subunit of a cAMP-dependent protein kinase in Dictyostelium discoideum. Biochemical and Biophysical Research Communications 105:949–955
     [Google Scholar]
  20. North M. J. 1978; Inhibition of acid proteinase from Dictyostelium discoideum. Biochemical Society Transactions 6:400–403
     [Google Scholar]
  21. North M. J. 1982a; Comparative biochemistry of the proteinases of eucaryotic microorganisms. Microbiological Reviews 46:308–340
     [Google Scholar]
  22. North M. J. 1982b; Proteolytic activities in Dictyostelium discoideum detected with chromogenic peptide substrates. Experimental Mycology 6:345–352
     [Google Scholar]
  23. North M. J., Cotter D. A. 1983; Proteolytic enzyme activity during Dictyostelium discoideum spore germination. Experimental Mycology (in the Press)
    [Google Scholar]
  24. North M. J., Harwood J. M. 1979; Multiple acid proteinases in the cellular slime mouldDictyostelium discoideum. Biochimica et biophysica acta 566:222–233
     [Google Scholar]
  25. North M. J., Whyte A. 1981; Purification of proteinase E from Dictyostelium discoideum. Society for General Microbiology Quarterly 8:273
     [Google Scholar]
  26. North M. J., Whyte A., Ventom A. 1982; Chloroquine effects in Dictyostelium discoideum: inhibition of other enzymes besides the cathepsin B. FEMS Microbiology Letters 15:189–192
     [Google Scholar]
  27. Rossomando E. F., Maldonado B., Crean E. V., Kollar E. J. 1978; Protease secretion during onset of development in Dictyostelium discoideum. Journal of Cell Science 30:305–318
     [Google Scholar]
  28. Schechter I., Berger A. 1967; On the size of the active site in proteases. I. Papain. Biochemical and Biophysical Research Communications 27:157–162
     [Google Scholar]
  29. Sedmak J. J., Grossberg S. E. 1977; A rapid, sensitive and versatile assay for protein using Coomassie brilliant blue G250. Analytical Biochemistry 79:544–552
     [Google Scholar]
  30. Smith R., Turk V. 1974; Cathepsin D: rapid isolation by affinity chromatography on haemoglobin-agarose resin. European Journal of Biochemistry 48:245–254
     [Google Scholar]
  31. Sussman M., Sussman R. 1969; Patterns of RNA synthesis and of enzyme accumulation and disappearance during cellular slime mouldcyto-differentiation. Symposium of the Society for General Microbiology 19:403–435
     [Google Scholar]
  32. Watts D. J., Ashworth J. M. 1970; Growth of myxamoebae of the cellular slime mould Dictyostelium discoideum in axenic culture. Biochemical Journal 119:171–174
     [Google Scholar]
  33. Weber K., Pringle J. R., Osborn M. 1972; Measurement of molecular weights by electrophoresis on SDS-acrylamide gel. Methods in Enzymology 26:3–27
     [Google Scholar]
  34. Wiener E., Ashworth J. M. 1970; The isolation and characterization of lysosomal particles from myxamoebae of the cellular slime mould Dictyostelium discoideum. Biochemical Journal 118:505–512
     [Google Scholar]
  35. Winter A., Ek K., Andersson U.-B. 1977; Analytical electrofocussing in thin layers of polyacrylamide gels. LKB Application Note 250
     [Google Scholar]
  36. Zacharius R. M., Zell T. E., Morrison J. H., Woodlock J. J. 1969; Glycoprotein staining following electrophoresis on acrylamide gels. Analytical Biochemistry 31:148–152
     [Google Scholar]
/content/journal/micro/10.1099/00221287-130-1-123
Loading
Purification and Characterization of Two Acid Proteinases from Dictyostelium discoideum
Microbiology 130, 123 (1984); https://doi.org/10.1099/00221287-130-1-123
/content/journal/micro/10.1099/00221287-130-1-123
/content/journal/micro/10.1099/00221287-130-1-123
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