Addition of tunicamycin to the culture medium of growing Saccharomyces cerevisiae protoplasts or cells resulted in the formation of a modified exo-1,3-β-d-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 Km values of both enzyme forms were identical. Antibodies raised against the native protein readily precipitated the exo-1,3-β-d-glucanase produced after tunicamycin treatment. The latter enzyme was comparable, in terms of molecular size and lack of affinity for concanavalin A. to the β-d-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-β-d 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-β-d-glucanase secretion, may play a role in the conformation of the protein and in stabilizing the enzymic activity.
AminoffD.,
GathmannW. D.,
McLeanC. M.,
YadomaeT.1980; Quantitation of oligosaccharides released by the β-elimination reaction. Analytical Biochemistry 101:44–53
GoldsteinI. J.,
HayG. W.,
LewisB. A.,
SmithF.1965; Controlled degradation of polysaccharides by periodate oxidation, reduction and hydrolysis. Methods in Carbohydrate Chemistry 5:361–369
HickmanS.,
KulcyckyA.,
LynchR. G.,
KornfeldS.1977; Studies on the mechanism of tunicamycin. Inhibition of IgA and IgE secretion by plasma cells. Journal of Biological Chemistry 253:4402–4408
KilkerR. D.,
SaunierB.,
TkaczJ. S.,
Hersco-vicsA.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
KlenkH.,
ChristophS.,
RottR.1972; Inhibition of glycoprotein biosynthesis of influenza virus by D-glucosamine and 2-deoxy-d-glucose. Virology 49:723–734
LloydK. 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
NotarioV.,
VillaT. G.,
VillanuevaJ. R.1976; Purification of an exo-β-d-glucanase from cell free extracts of Candida utilis. Biochemical Journal 159:555–562
OldenK.,
PrattR. M.,
YamadaK. 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
OnishiH. R.,
TkaczJ. S.,
LampenJ. O.1979; Glycoprotein nature of yeast alkaline phosphatase. Formation of active enzyme in the presence of tunicamycin. Journal of Biological Chemistry 254:11943–11952
SanchezA.,
LarribaG.,
VillanuevaJ. R.,
VillaT. 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
SchultzA. M.,
OroszlanS.1979; Tunicamycin inhibits glycosylation of precursor polyprotein encoded by env gene of Rauscher murine leukemia virus. Biochemical and Biophysical Research Communications 86:1206–1213
SchwarzR. T.,
RohrschneiderJ. M.,
SchmidtM. F. G.1976; Suppression of glycoprotein formation of semliki forest influenza and avian sarcoma virus by tunicamycin. Journal of Virology 19:782–791
TarentinoA. L.,
PlummerJr,
MaleyF.1974; The release of intact oligosaccharides from specific glycoproteins by endo-β-N-acetyl-glucosaminidase H. Journal of Biological Chemistry 249:818–824
TkaczJ. S.,
LampenJ. O.1975; Tunicamycin inhibition of polyisoprenol-N-acetyl-glucosaminyl phosphatase formation in calf-liver microsomes. Biochemical and Biophysical Research Communications 65:248–257
VillaT. G.,
NotarioV.,
VillanuevaJ. 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
VillaT. G.,
LachanceM. A.,
PhaffH. J.1978b; β-Glucanases of the yeast Kluyveromyces phaseolosporus: partial purification and characterization. Experimental Mycology 2:12–25
VillanuevaJ. R.,
NotarioV.,
SantosT.,
VillaT. 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. 323–355PeberdyJ. F.,
RousA. H.,
Rogers.H. J.
Edited by London &New York: Academic Press;
ZacchariusR. M.,
ZellT. E.,
MorrisonJ. H.,
WoodlockJ. J.1969; Glycoprotein staining following electrophoresis on acrylamide gels. Analytical Biochemistry 30:148–152