1887

Abstract

Addition of tunicamycin to the culture medium of growing protoplasts or cells resulted in the formation of a modified exo-1,3---glucanase which was detectable in both extracellular and intracellular fractions. This modified enzyme had a lower molecular weight than the native form and did not bind to concanavalin A. The activation energy and values of both enzyme forms were identical. Antibodies raised against the native protein readily precipitated the exo-1,3---glucanase produced after tunicamycin treatment. The latter enzyme was comparable, in terms of molecular size and lack of affinity for concanavalin A. to the --glucanase obtained by treatment of the native form with endoglycosidase H; both lacked the carbohydrate moiety present in the native enzyme. The exo-1,3-- glucanase obtained in the presence of the antibiotic was more sensitive to variations in temperature and pH than both endoglycosidase H-treated and non-treated enzymes. Our results suggest that the carbohydrate moiety, if not necessary for exo-1,3---glucanase secretion, may play a role in the conformation of the protein and in stabilizing the enzymic activity.

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1982-12-01
2024-12-10
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References

  1. Aminoff D., Gathmann W. D., McLean C. M., Yadomae T. 1980; Quantitation of oligosaccharides released by the β-elimination reaction. Analytical Biochemistry 101:44–53
    [Google Scholar]
  2. Farkas V. P., Biely P., Bauer S. 1973; Extracellular β-glucanases of the yeast Saccharomyces cerevisiae. Biochimica et biophysica acta 321:246–255
    [Google Scholar]
  3. Garvey J. S., Cremer N. E., Sussdorf D. H. 1977 Methods in Immunology, 3. pp. 313–335 Massachusetts: W. A. Benjamin;
    [Google Scholar]
  4. Goldstein I. J., Hay G. W., Lewis B. A., Smith F. 1965; Controlled degradation of polysaccharides by periodate oxidation, reduction and hydrolysis. Methods in Carbohydrate Chemistry 5:361–369
    [Google Scholar]
  5. Hickman S., Kornfeld S. 1978; Effect of tunicamycin on IgM, IgA and IgG secretion by mouse plasmacytoma cells. Journal of Immunology 121:990–996
    [Google Scholar]
  6. Hickman S., Kulcycky A., Lynch R. G., Kornfeld S. 1977; Studies on the mechanism of tunicamycin. Inhibition of IgA and IgE secretion by plasma cells. Journal of Biological Chemistry 253:4402–4408
    [Google Scholar]
  7. Kilker R. D., Saunier B., Tkacz J. S., Hersco-vics A. 1981; Partial purification from S. cerevisiae of a soluble glucosidase which removes the terminal glucose from the oligosaccharide Glc3 Manq GlcNAc2 . Journal of Biological Chemistry 256:5299–5303
    [Google Scholar]
  8. Klenk H., Christoph S., Rott R. 1972; Inhibition of glycoprotein biosynthesis of influenza virus by D-glucosamine and 2-deoxy-d-glucose. Virology 49:723–734
    [Google Scholar]
  9. Kuo S. C., Lampen J. O. 1974; Tunicamycin, an inhibitor of yeast glycoprotein synthesis. Biochemical and Biophysical Research Communications 58:287–295
    [Google Scholar]
  10. Laurell C. G. 1966; Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Analytical Chemistry 15:45–50
    [Google Scholar]
  11. Lloyd K. O. 1970; The preparation of two insoluble forms of the phytohemagglutinin Concanavalin A and their interactions with polysaccharides and glycoproteins. Archives of Biochemistry and Biophysics 137:460–466
    [Google Scholar]
  12. Notario V., Villa T. G., Villanueva J. R. 1976; Purification of an exo-β-d-glucanase from cell free extracts of Candida utilis. Biochemical Journal 159:555–562
    [Google Scholar]
  13. Olden K., Pratt R. M., Yamada K. M. 1978; Role of carbohydrates in protein secretion and turnover: effects of tunicamycin on the major cell surface glycoprotein of chick embryo fibroblasts. Cell 13:461–473
    [Google Scholar]
  14. Onishi H. R., Tkacz J. S., Lampen J. O. 1979; Glycoprotein nature of yeast alkaline phosphatase. Formation of active enzyme in the presence of tunicamycin. Journal of Biological Chemistry 254:11943–11952
    [Google Scholar]
  15. Ruiz-Herrera J., Sentandreu R. 1975; Site of initial glycosylation of mannoproteins from Saccharomyces cerevisiae. Journal of Bacteriology 124:127–133
    [Google Scholar]
  16. Sanchez A., Larriba G., Villanueva J. R., Villa T. G. 1980; Glycosylation is not necessary for the secretion of exo-l,3-β-d-glucanase by Saccharomyces cerevisiae protoplasts. FEBS Letters 121:283–286
    [Google Scholar]
  17. Schultz A. M., Oroszlan S. 1979; Tunicamycin inhibits glycosylation of precursor polyprotein encoded by env gene of Rauscher murine leukemia virus. Biochemical and Biophysical Research Communications 86:1206–1213
    [Google Scholar]
  18. Schwarz R. T., Klenk H. D. 1974; Inhibition of glycosylation of the influenza virus hemagglutinin. Journal of Virology 14:1023–1034
    [Google Scholar]
  19. Schwarz R. T., Rohrschneider J. M., Schmidt M. F. G. 1976; Suppression of glycoprotein formation of semliki forest influenza and avian sarcoma virus by tunicamycin. Journal of Virology 19:782–791
    [Google Scholar]
  20. Takatsuki A., Arima K., Tamura G. 1971; Tunicamycin, a new antibiotic. I. Isolation and characterization of tunicamycin. Journal of Antibiotics 24:215–223
    [Google Scholar]
  21. Tarentino A. L., Plummer Jr, Maley F. 1974; The release of intact oligosaccharides from specific glycoproteins by endo-β-N-acetyl-glucosaminidase H. Journal of Biological Chemistry 249:818–824
    [Google Scholar]
  22. Tkacz J. S., Lampen J. O. 1975; Tunicamycin inhibition of polyisoprenol-N-acetyl-glucosaminyl phosphatase formation in calf-liver microsomes. Biochemical and Biophysical Research Communications 65:248–257
    [Google Scholar]
  23. Villa T. G., Notario V., Villanueva J. R. 1978a; Direct chemical proof of different glycosylation patterns for yeast exo- and endo-l,3-β-d-glucanases. Journal of General Microbiology 109:371–374
    [Google Scholar]
  24. Villa T. G., Lachance M. A., Phaff H. J. 1978b; β-Glucanases of the yeast Kluyveromyces phaseolosporus: partial purification and characterization. Experimental Mycology 2:12–25
    [Google Scholar]
  25. Villanueva J. R., Notario V., Santos T., Villa T. G. 1976; βGlucanases in nature. Biochemistry and function of β-glucanases in yeast. In Proceedings in the 4th International Symposium on Yeast and other Protoplasts pp. 323–355 Peberdy J. F., Rous A. H., Rogers. H. J. Edited by London &New York: Academic Press;
    [Google Scholar]
  26. Zaccharius 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]
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