A new method for quantitative determination of polysaccharides in the yeast cell wall. Application to the cell wall defective mutants of Saccharomyces cerevisiae.
A reliable acid hydrolysis method for quantitative determination of the proportion of beta-glucan, mannan and chitin in Saccharomyces cerevisiae cell wall is reported together with a simple extraction procedure to quantify within a standard error of less than 2% the proportion of the wall per gram of cell dry mass. This method is an optimized version of Saeman's procedure based on sulfuric acid hydrolysis of complex polysaccharides. It resulted in an almost complete release of glucose, mannose and glucosamine residues from cell wall polysaccharides. After complete removal of sulfate ions by precipitation with barium hydroxide, the liberated monosaccharides were separated and quantified by high performance anion-exchange chromatography with pulsed amperometric detection. The superiority of this method over the hydrolysis in either trifluoroacetic or hydrochloric acid resides in its higher efficiency regarding the release of glucose from beta 1,6-glucan and of glucosamine from chitin. The sulfuric acid method was successfully applied to determine the beta-glucan, mannan and chitin contents in cell walls of genetically well-characterized yeast mutants defective in cell wall biosynthesis, and in Schizosaccharomyces pombe cell walls. The simplicity and reliability of this procedure make it the method of choice for the characterization of cell walls from S. cerevisiae mutants generated in the EUROFAN programme, as well as for other pharmacological and biotechnological applications.
Dallies
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Centre de Bioingenierie Gilbert Durand, UMR-CNRS 5504, LA. INRA, Departement de Genie Biochimique et Alimentaire, Institut National des Sciences Appliquees, Toulouse, France.
François
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J
Paquet
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V
D000143:Acids; D002473:Cell Wall / Q000737:chemistry* / Q000235:genetics; D002686:Chitin / Q000032:analysis; D005936:Glucans / Q000032:analysis; D006868:Hydrolysis; D008351:Mannans / Q000032:analysis; D009005:Monosaccharides / Q000737:chemistry; D009154:Mutation; D011134:Polysaccharides / Q000032:analysis*; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics; D012568:Schizosaccharomyces / Q000737:chemistry; D013347:Subcellular Fractions / Q000737:chemistry
D000143:Acids; D005936:Glucans; D008351:Mannans; D009005:Monosaccharides; D011134:Polysaccharides; D002686:Chitin