1887

Microbiology Society logo

Volume 128, Issue 4

An Acid Protease Produced by and in Infected Apple Fruits, and its Possible Role in Pathogenesis Free

Abstract

SUMMARY: The fungus grown in a liquid medium containing protein secreted a protease with many similarities to one extractable from apple fruits infected with this pathogen but absent from healthy fruit. Enzymes from both sources degraded several protein substrates optimally at pH 3·4. At this pH, haemoglobin was degraded optimally at 47 °C, while at 37 °C both enzymes were most stable at pH 5. The pI values for the enzymes were close to 4·0, and their action was inhibited by pepstatin and diazoacetyl-norleucine methyl ester plus copper ions but unaffected by p-hydroxymercuribenzoate, EDTA or trypsin inhibitors. Mercaptoethanol was essential for the recovery of enzyme activity from acrylamide gels in which the and enzymes were electrophoretically homogeneous. The enzyme gave a positive reaction for glycoproteins after purification with a Sepharose-haemoglobin affinity medium. Both enzymes were single units with a molecular weight of 37000–40000 on sodium dodecyl sulphate (SDS)-poryacrylamide gels; gel filtration indicated a molecular weight of 34000–38000. The purified enzyme had no aminopeptidase or carboxypeptidase activity; the products of haemoglobin digestion were peptides with molecular weights on SDS-polyacrylamide gels of 2000–12000. Purified protease did not damage apple fruit tissue or cultured apple cells, lyse plant protoplasts or release proteins from isolated fruit cell walls. The protease had no effect on active fungal or host enzymes that might have a role in host/pathogen interactions, and did not induce phaseollin formation in bean cotyledons. The plant proteins vicilin, legumin and heat-denatured peroxidase were hydrolysed at pH 3·5 and spinach nitrate reductase was inactivated. No natural protease inhibitors were detected in healthy or infected apples. We suggest that the protease might be involved in the nutrition of the pathogen.

  • Received:
  • Revised:
  • Published Online:
© Society for General Microbiology 1982
Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-128-4-799
1982-04-01
2024-04-26
Loading full text...

Full text loading...

/deliver/fulltext/micro/128/4/mic-128-4-799.html?itemId=/content/journal/micro/10.1099/00221287-128-4-799&mimeType=html&fmt=ahah

References

  1. Andrews P. 1964; Estimation of the molecular weights of proteins by Sephadex gel-filtration. Biochemical Journal 91:222–233
     [Google Scholar]
  2. Anson M. L. 1938; Estimation of pepsin, trypsin, papain and cathepsin with haemoglobin. Journal of General Physiology 22:79–89
     [Google Scholar]
  3. Aoyagi T., Kunimoto S., Morishima H., Takeuchi T., Umezawa H. 1971; Effect of pepstatin on acid proteases. Journal of Antibiotics 24:687–694
     [Google Scholar]
  4. Bateman D. F., Basham H. G. 1976; Degradation of plant cell walls and membranes by microbial enzymes. In Physiological Plant Pathology pp. 316–355 Heitefuss R., Williams P. H. Edited by Berlin:: Springer Verlag.;
     [Google Scholar]
  5. Bradford M. 1976; A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye-binding. Analytical Biochemistry 72:248–254
     [Google Scholar]
  6. Byrde R. J. W. 1979; Role of polysaccharidedegrading enzymes in microbial pathogenicity. In Microbial Polysaccharides and Polysaccharases pp. 417–436 Berkeley R. C. W., Gooday G. W., Ellwood D. C. Edited by London:: Academic Press.;
     [Google Scholar]
  7. Byrde R. J. W., Fielding A. H. 1968; Pectin methyl-frarts-eliminase as the maceration factor of Sclerotinia fructigena and its significance in brown rot of apple. Journal of General Microbiology 52:287–297
     [Google Scholar]
  8. Chua G. K., Bushuk W. 1969; Purification of wheat proteases by affinity chromatography on haemoglobin-Sepharose column. Biochemical and Biophysical Research Communications 37:545–550
     [Google Scholar]
  9. Drivdahl R. H., Thimann K. V. 1977; Proteases of senescing oat leaves. I. Purification and general properties. Plant Physiology 59:1059–1063
     [Google Scholar]
  10. Hislop E. C., Hoad G. V., Archer S. A. 1973; The involvement of ethylene in plant diseases. In Fungal Pathogenicity and the Plant’s Response pp. 87–117 Byrde R. J. W., Cutting C. V. Edited by London:: Academic Press.;
     [Google Scholar]
  11. Hislop E. C., Keon J. P. R., Fielding A. H. 1979; Effects of pectin lyase from Monilinia fructigena on viability, ultrastructure and localization of acid phosphatase of cultured apple cells. Physiological Plant Pathology 14:371–381
     [Google Scholar]
  12. Hjerten S., Jerstedt S., Tiselius A. 1965; Some aspects of the use of “continuous” and “discontinuous” buffer systems in polyacrylamide gel electrophoresis. Analytical Biochemistry 11:219–223
     [Google Scholar]
  13. Khare K. B., Bompeix G. 1976; Activités protéolytiques des Sclerotinia sclerotiorum et S. minor: rôle possible de la pathogènese. Revue de mycologie 40:65–84
     [Google Scholar]
  14. Mosolov V. V., Loginova M. D., Fedurkina N. V., Benken I. I. 1976; The biological significance of proteinase inhibitors in plants. Plant Science Letters 7:77–80
     [Google Scholar]
  15. Mosolov V. V., Loginova M. D., Malova E. L., Benken I. I. 1979; A specific inhibitor of Colletotrichum lindemuthianum protease from kidney bean (Phaseolus vulgaris) seeds. Planta 144:265–269
     [Google Scholar]
  16. Mussell H., Strand L. L. 1977; Pectic enzyme involvement in pathogenesis and possible relevance to tolerance and specificity. In Cell Wall Biochemistry Related to Specificity in Host-Plant Pathogen Interactions pp. 31–70 Solheim B., Raa J. Edited by Tromso:: Universitets Forlaget.;
     [Google Scholar]
  17. Nakadi T., Nasuno S., Iguchi N. 1972; Purification and properties of acid carboxypeptidase I from Aspergillus oryzae. Agricultural and Biological Chemistry 36:1343–1352
     [Google Scholar]
  18. Nakadi T., Nasuno S., Iguchi N. 1973; Purification and properties of leucineaminopeptidase I from Aspergillus oryzae. Agricultural and Biological Chemistry 37:757–765
     [Google Scholar]
  19. Peng J. H., Black L. L. 1976; Increased proteinase inhibitor activity in response to infection of resistant tomato plants by Phytophthora infestans. Phytopathology 66:958–963
     [Google Scholar]
  20. Rajagopalan T. G., Stein W. H., Moore S. 1966; The inactivation of pepsin by diazoacetyl-norleucine methyl ester. Journal of Biological Chemistry 24:4295–4297
     [Google Scholar]
  21. Reisner A. H., Nemes P., Bucholtz C. 1975; The use of Coomassie brilliant blue G250 perchloric acid solution for staining in electrophoresis and isoelectric focusing on polyacrylamide gels. Analytical Biochemistry 64:509–516
     [Google Scholar]
  22. Richardson M. 1977; The proteinase inhibitors of plants and microorganisms. Phytochemistry 16:159–169
     [Google Scholar]
  23. Ries S. M., Albersheim P. 1973; Purification of a protease secreted by Colletotrichum lindemuthianum. Phytopathology 63:625–629
     [Google Scholar]
  24. Rinderknecht H., Geokas M. C., Silverman P., Haverback B. J. 1968; A new ultrasensitive method for the determination of proteolytic activity. Clinica chimica acta 21:197–203
     [Google Scholar]
  25. Ryan C. A. 1973; Proteolytic enzymes and their inhibitors in plants. Annual Review of Plant Physiology 24:173–196
     [Google Scholar]
  26. Salmia M. A., Nyman S. A., Mikola J. J. 1978; Characterisation of the proteinase present in germinating seeds of Scots Pine, Pinus sylvestris. Physiologia plantarum 42:252–256
     [Google Scholar]
  27. Shapiro A. C., Vinuela E., Maizel J. V. Jr 1967; Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochemical and Biophysical Research Communications 28:4295–4297
     [Google Scholar]
  28. Sherrard J. H., Kennedy H. A., Dalling M. J. 1979; In vitro stability of nitrate reductase from wheat leaves. III. Isolation and partial characterisation of a nitrate reductase-inactivating factor. Plant Physiology 64:640–645
     [Google Scholar]
  29. Swank R. T., Munkres K. D. 1971; Molecular weight analysis of oligopeptides by electrophoresis on polyacrylamide gel with sodium dodecyl sulphate. Analytical Biochemistry 39:462–477
     [Google Scholar]
  30. Swinburne T. R. 1975; Microbial proteases as elicitors of benzoic acid accumulation in apples. Phytopathologische Zeitschrift 82:152–162
     [Google Scholar]
  31. Tsai M. S., Hseu T. H., Shen Y. S. 1978; Purification and characterization of an acid protease from Monascus kaoliang. International Journal of Peptide and Protein Research 12:293–302
     [Google Scholar]
  32. Tseng T. C., Mount M. S. 1974; Toxicity of endo-polygalacturonate trans-eliminase, phosphatidase and protease to potato and cucumber tissue. Phytopathology 64:229–236
     [Google Scholar]
  33. Urbanek H., Yirdaw G. 1978; Acid protease produced by Fusarium species in cultures and in infected seedlings. Physiological Plant Pathology 13:81–87
     [Google Scholar]
  34. Vesterberg O., Svensson H. 1966; Isoelectric fractionation, analysis and characterisation of ampholytes in natural pH gradients. Acta chemica scandinavica 20:820–834
     [Google Scholar]
  35. Wallace W. 1974; Purification and properties of a nitrate reductase-inactivating enzyme. Biochimica et biophysica acta 341:265–276
     [Google Scholar]
  36. Weber K., Osborn M. 1969; The reliability of molecular weight determinations by dodecyl sulphate-polyacrylamide gel electrophoresis. Journal of Biological Chemistry 244:4406–4412
     [Google Scholar]
  37. Wood R. K. S. 1978; Enzymes produced by fungi and bacteria. Their role in pathogenicity. Annals of Phytopathology 10:127–135
     [Google Scholar]
  38. Yemm E. W., Cocking E. C. 1955; The determination of amino-acids with ninhydrin. Analyst 80:209–213
     [Google Scholar]
  39. Zacharius R. M., Zell T. E., Morrison J. H., Woodlock J. J. 1969; Glycoprotein staining following electrophoresis on acrylamide gels. Analytical Biochemistry 30:148–152
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-128-4-799
Loading
An Acid Protease Produced by Monilinia fructigena in vitro and in Infected Apple Fruits, and its Possible Role in Pathogenesis
Microbiology 128, 799 (1982); https://doi.org/10.1099/00221287-128-4-799
/content/journal/micro/10.1099/00221287-128-4-799
/content/journal/micro/10.1099/00221287-128-4-799
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