ANALYSIS OF FISSION YEAST CELL WALL

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。纯化的内切葡聚糖酶已被用于测定粟酒裂殖酵母细胞壁的组成。传统上，在用热碱分离其组分（甘露糖蛋白、α-1，3葡聚糖、β-1，3葡聚糖和支链β-葡聚糖）后，对这种结构进行了研究。相反，我们通过用内切β-1，3葡聚糖酶和新型内切α-1，3葡聚糖酶（突变酶）消化来依次去除多糖。这种方法使用连续的酶水解，这比热碱处理更温和。该方法是可重复的，并且允许细胞壁的主要组分比先前的方法更特异性和可重复地分离成限定的级分。在这种温和的分离之后，我们观察到支链β-葡聚糖比先前描述的要丰富得多。通过扩大新方案，我们制备了大量的高度支化的葡聚糖，并通过化学、MALDI-TOF MS、13 C和1H高场NMR分析确定了其结构特征。这些分析使我们能够清楚地定义一种对已知葡聚糖酶具有抗性的高度支化的细胞壁聚合物。最后，为了证明其应用，我们已经确定了已知突变株的细胞壁组成。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Purified endoglucanases have been used to determine the composition of Schizosaccharomyces pombe cell wall. This structure has been traditionally studied after isolating its components (mannoproteins, alpha-1,3 glucan, beta-1,3glucan and a branched beta-glucan) with hot alkali. Instead, we sequentially removed the polysaccharides by digesting with endo beta-1,3 glucanase and with a novel endo alpha-1,3 glucanase (mutanase). This method uses sequential enzymatic hydrolysis, which is more gentile than hot alkali treatment. The method is reproducible and allows for the major components of the cell wall to be more specifically and reproducibly separated into defined fractions than previous methods. After this gentle isolation we observed that a branched beta-glucan is much more abundant than previously described. By scaling-up the new protocol we prepared large amounts of the highly branched glucan and determined its structural features by chemical, MALDI-TOF MS, 13C and 1H high field NMR analyses. These analyses have allowed us to clearly define a highly branched cell wall polymer that is resistant to known glucanases. Finally, to demonstrate its application, we have determined the cell wall composition of known mutant strains.