Abstract
Abstract Mild alkaline degradation of Saccharomyces cerevisiae X2180 wild type mannan, under conditions that effect β elimination of the substituted serine and threonine residues, releases mannose, mannobiose, mannotriose, and mannotetraose. Methylation analysis demonstrated that these fragments have the same structures as those that are obtained by acetolysis of the polysaccharide component of the yeast mannan. Similar treatment of a mutant mannan that lacks the mannotetraose unit in the polysaccharide component produced only mannose, mannobiose, and mannotriose. Thus, the single mutation alters both the tetrasaccharide units in the polysaccharide component of the mannan and those that are attached to serine and threonine. Because these studies were carried out on the total cell wall mannan, rather than a single molecular species, we cannot say that both the polysaccharide chains and the shorter oligosaccharides linked to serine and threonine are present in the same molecule. Strong alkaline degradation in the presence of sodium borotritide of the mannan residue recovered from the β elimination reaction yielded a protein-free polysaccharide that gave radioactive N-acetylglucosaminitol on acetolysis. Thus, the mannose-containing polysaccharide of the mannan is attached to glucosamine, and it is probable that the linkage to protein is through a di-N-acetylchitobiose to asparagine as in many other glycoproteins. The mannan isolated from the wild type and mutant strains of S. cerevisiae by procedures that avoid exposure to alkali had a molecular weight of 133,000. On treatment with 0.1 n sodium hydroxide at room temperature, the molecular weight was reduced to about 40,000. These conditions could saponify acyl ester bonds but are unlikely to break phosphodiester linkages or any other linkages except those of the oligosaccharides attached to serine and threonine.
Highlights
Our results show that the same fragments, mannose, mannobiose, mannotriose and mannotetraose are released from this mannan by p elimination as are
With S. cerevisiaeX2180, a strain closelyrelated to bakers’yeast, we find the sameoligosaccharidesthat wereearlier obtainedby acetolysisof the mannan [13], namely mannose,a(1-+2)mannobiose,a(l-+2)mannotriose, and a mannotetraosewith the structure aMan(l-+3)
It should be emphasized that, our studies demonstrate that the same gene is involved in synthesis of both kinds of side chains, they do not establish that the polysaccharide component and the short oligosaccharides are attached to the same polypeptide chain
Summary
Carbohydrate was determined by the method of Dubois et al [16], and protein according to Lowry et al [17].Descending paper chromatography was carried out on WhatmanNo 1 filter paper using the following solvents (in volume ratios): Solvent A, 1-butanol-pyridine-water (6:4:3); Solvent B, ethyl acetate-pyridine-water (8:2: 1). Carbohydrate was determined by the method of Dubois et al [16], and protein according to Lowry et al [17]. Descending paper chromatography was carried out on Whatman. No 1 filter paper using the following solvents (in volume ratios): Solvent A, 1-butanol-pyridine-water (6:4:3); Solvent B, ethyl acetate-pyridine-water (8:2: 1). Sugars and sugar alcohols were detected on paper chromatograms with a silver nitrate-sodium hydroxide dip reagent. Paper chromatograms were scanned for radioactivity by cutting 2-cm wide strips into l-cm horizontal bands which were counted in 10 ml of Bray’s solution [18] in a Packard scintillation counter. A Sninco model E analvtical ultracentrifuge was used for measurements of sedimentation velocity
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