Isolation of mannan-protein complexes from viable cells of Saccharomyces cerevisiae X2180-1A wild type and Saccharomyces cerevisiae X2180-1 A-5 mutant strains by the action of Zymolyase-60,000.
The viable whole cells of Saccharomyces cerevisiae X2180-1A wild type and its mannan mutant strain S. cerevisiae X2180-1A-5, were treated with an Arthrobacter sp. beta-1,3-glucanase in the presence of a serine protease inhibitor, phenyl-methylsulfonyl fluoride. Fractionation of the solubilized materials of each strain with Cetavlon (cetyltrimethylammonium bromide) yielded one mannan-protein complex. Molecular weights of these complexes were almost the same as that of the mannoprotein of the mutant strain prepared by Nakajima and Ballou, which had a molecular weight of 133,000 and were approximately three times larger than those of the mannans isolated from the same cells by hot-water extraction. Each mannan-protein complex contained up to 2% glucose residue, which was not removed by specific precipitation with anti-mannan sera or by affinity chromatography on a column of concanavalin A-Sepharose. Treatment of these complexes with alkaline NaBH4 produced peptide-free mannan containing small amounts of glucose nearly identical to those of the parent complexes. The above findings provide evidence that the glucose residues exist in a covalently linked form to the mannan moiety. Fractionation of the mannan-protein complex of the S. cerevisiae wild-type strain by DEAE-Sephadex chromatography yielded five subfractions of different phosphate content, indicating that these highly intact mannan-protein complexes were of heterogeneous material consisting of many molecular species of different phosphate content.
D000596:Amino Acids / Q000032:analysis; D002241:Carbohydrates / Q000032:analysis; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods; D037222:Collectins; D004798:Enzymes / Q000633:toxicity*; D006867:Hydrolases; D008351:Mannans / Q000302:isolation & purification*; D008970:Molecular Weight; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics*; D013045:Species Specificity
D000596:Amino Acids; D002241:Carbohydrates; D002352:Carrier Proteins; D037222:Collectins; D004798:Enzymes; D008351:Mannans; C037833:zymolyase; D006867:Hydrolases