1887

Abstract

-Glucanase activities were found associated with and their culture fluids. Mild acid treatment of the organisms led to rapid inactivation of -glucanase activities, the degree of loss increasing with the age of the cultures; the results suggested an extracytoplasmic location of the cell-associated enzymes. Most of the -glucanase activities were associated with the cell walls in organisms phenotypically resistant to amphotericin B methyl ester (AME).

Two proteins (I and II) exhibiting -glucanase activity were isolated and purified by conventional procedures from cell-free extracts, cell-wall autolysates and culture fluids of sensitive and phenotypically resistant to AME. The purified enzymes appeared homogeneous on isoelectric focusing, gel electrophoresis and ultracentrifugation, with molecular weights of 150000 (I) and 49000 (II). Both enzymes hydrolysed cell walls purified from AME-sensitive and phenotypically resistant organisms, but showed different substrate specificities and patterns of activity. Enzyme II hydrolysed (1 →3)--glucans by an endolytic mechanism releasing laminaritetraose as the initial product. Glucose was the only product released by enzyme I. The properties of the individual enzymes were unaffected by their localization or the age of the culture of the organisms.

The loosening of the polysaccharide packing by ultrasonic treatment of cell walls purified from AME-resistant organisms increased the -glucanase activities bound to the walls, but did not solubilize them. Autolysis of cell walls released 58 to 66% of their -glucanase activity in 20 h, but no further release was attained on prolonged incubation. The amount of -glucanase activity released by autolysis was increased by a variety of pretreatments. Diethyl pyrocarbonate inhibited -glucanase activity and prevented autolysis. Evidence is presented indicating that interactions with lipids, polysaccharides and other cell wall proteins may be involved in the control of the activity of the cell wall-associated -glucanases in organisms phenotypically resistant to AME.

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1982-04-01
2024-04-18
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References

  1. Aoki K., Kajiwara S., Shinke R., Nishira H. 1979; Tannic acid staining and extraction of enzymes in polyacrylamide gel electrophoresis. Analytical Biochemistry 95:575–578
    [Google Scholar]
  2. Arnold W. N. 1972; The structure of the yeast cell wall. Solubilization of a marker enzyme, β-fructo-furanosidase, by the autolytic enzyme system. Journal of Biological Chemistry 247:1161–1169
    [Google Scholar]
  3. Barras D. R., Stone B. A. 1969; β-l,3-Glucan hydrolases from Euglena gracilis. I. The nature of the hydrolases. Biochimica et biophysica acta 191:329–341
    [Google Scholar]
  4. Bruss M. L., Black A. L. 1978; Enzymatic microdetermination of glycogen. Analytical Biochemistry 84:309–312
    [Google Scholar]
  5. Cortat M., Matile P., Wlemken A. 1972; Isolation of glucanase-containing vesicles from budding yeasts. Archiv für Mikrobiologie 82:189–205
    [Google Scholar]
  6. Davies R., Wayman F. J. 1973; Estimation of glucose and invertase activity in presence of thiols. Analytical Biochemistry 55:143–153
    [Google Scholar]
  7. Dickerson A. G., Baker R. C. F. 1979; The binding of enzymes to fungal β-glucans. Journal of General Microbiology 112:67–75
    [Google Scholar]
  8. Farkas V., Biely P., Bauer S. 1973; Extracellular β-glucanases of the yeast Saccharomyces cerevisiae. . Biochimica et biophysica acta 321:246–255
    [Google Scholar]
  9. Fleet G. H., Phaff H. J. 1974; Glucanases in Schizosaccharomyces. Isolation and properties of the cell wall-associated β-(1 →3)-glucanases. Journal of Biological Chemistry 249:1717–1728
    [Google Scholar]
  10. Folch J., Lees M., Sloane-Stanley G. H. 1957; A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry 226:497–509
    [Google Scholar]
  11. Gale E. F. 1974; The release of potassium ions from Candida albicans in the presence of polyene antibiotics. Journal of General Microbiology 80:451–465
    [Google Scholar]
  12. Gale E. F., Johnson A. M., Kerridge D., Koh T. Y. 1975; Factors affecting the changes in amphotericin sensitivity of Candida albicans during growth. Journal of General Microbiology 87:20–36
    [Google Scholar]
  13. Gale E. F., Ingram J., Kerridge D., Notario V., Wayman F. J. 1980; Reduction of amphotericin resistance in stationary phase cultures of Candida albicans by treatment with enzymes. Journal of General Microbiology 117:383–391
    [Google Scholar]
  14. Hasegawa S., Nordin J. H., Kirkwood S. 1969; Enzymes that hydrolyse fungal cell wall poly-saccharides. I. Purification and properties of an endo-α-d-(l → 3)-glucanase from Trichoderma viride. . Journal of Biological Chemistry 244:5460–5470
    [Google Scholar]
  15. Herbert D., Phipps P. J., Strange R. E. 1971; Chemical analysis of microbial cells. Methods in Microbiology 5B:209–344
    [Google Scholar]
  16. Johnson B. F. 1968; Lysis of yeast cell walls induced by 2-deoxyglucose at their sites of glucan synthesis. Journal of Bacteriology 95:1169–1172
    [Google Scholar]
  17. Kidby D. K., Davies R. 1970; Thiol induced release of invertase from cell walls of Sac-charomyces fragilis. . Biochimica et biophysica acta 201:261–266
    [Google Scholar]
  18. Kritzman G., Chet I., Henis Y. 1978; Location of β-(l,3)-glucanase in the mycelium of Sclerotium rolfsii. . Journal of Bacteriology 134:470–475
    [Google Scholar]
  19. Lampen J. O. 1968; External enzymes of yeasts: their nature and formation. Antonie van Leeuwenhoek 34:1–8
    [Google Scholar]
  20. Nelson N. 1944; A photometric adaptation of the Somogyi method for the determination of glucose. Journal of Biological Chemistry 153:375–380
    [Google Scholar]
  21. Notario V., Villa T. G., Villanueva J. R. 1976; Purification of an exo-β-glucanase from cell-free extracts of Candida utilis. . Biochemical Journal 159:555–562
    [Google Scholar]
  22. Notario V., Villa T. G., Villanueva J. R. 1979; Cell wall-associated l,4-β-d-xylanase in Cryptococcus albidus var.aerius: in situ characterization of the activity. Journal of General Microbiology 114:415–422
    [Google Scholar]
  23. Notario V., Gale E. F., Kerridge D., Wayman F. J. 1982; Phenotypic resistance to amphotericin B in Candida albicans’, relationship to glucan metabolism. Journal of General Microbiology 128:761–777
    [Google Scholar]
  24. Phaff H. J. 1971; Structure and biosynthesis of the yeast cell envelope. In The Yeasts. Volume 2, Physiology and Biochemistry of Yeasts pp. 135–210 Edited by Rose A. H., Harrison J. S. New York and London: Academic Press;
    [Google Scholar]
  25. Polacheck I., Rosenberger R. F. 1978; Distribution of autolysins in hyphae of Aspergillus nidulans: evidence for a lipid-mediated attachment to hyphal walls. Journal of Bacteriology 135:741–747
    [Google Scholar]
  26. Racusen D. 1979; Glycoprotein detection in poly-acrylamide gel with thymol and sulphuric acid. Analytical Biochemistry 99:474–476
    [Google Scholar]
  27. Reese E. T., Mandels M. 1963; Enzymatic hydrolysis of β-glucans. In Proceedings of the Symposium on Advances in Enzymic Hydrolysis of Cellulose and Related Materials pp. 197–234 Edited by Reese E. T. New York: Pergamon Press;
    [Google Scholar]
  28. Sietsma J. H., Wessels J. G. H. 1979; Evidence for covalent linkages between chitin and β-glucan in a fungal wall. Journal of General Microbiology 114:99–108
    [Google Scholar]
  29. Villa T. G., Notario V., Benitez T., Villanueva J. R. 1976; Purification of an exo-1,3-β-glucanase from Candida utilis. . Canadian Journal of Biochemistry 54:927–934
    [Google Scholar]
  30. Villanueva J. R., Gacto M. 1973; Charac-terization of β-(l,3)-glucanases of yeasts. Proceedings of the 3rd International Specialized Symposium on Yeast261–283
    [Google Scholar]
  31. Villanueva J. R., Notario V., Santos T., Villa T. G. 1976; β-Glucanases in nature. Biochemistry and function of β-glucanases in yeast. In Microbial and Plant Protoplasts pp. 323–355 Edited by Peberdy J. F., Rose A. H., Rogers H. J., Cocking E. C. London: Academic Press;
    [Google Scholar]
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