ANALYSIS OF FISSION YEAST CELL WALL

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 用纯化的内切葡聚糖酶测定了裂殖酵母细胞壁的组成。传统上研究这种结构的方法是用热碱分离甘露糖蛋白、α-1，3-葡聚糖、β-1，3-葡聚糖和支化的β-葡聚糖。取而代之的是，我们通过内切β-1，3葡聚糖酶和一种新的内切α-1，3葡聚糖酶(突变酶)依次去除多糖。这种方法采用顺序酶解，比热碱处理更温和。该方法是可重复性的，并允许细胞壁的主要成分比以前的方法更特异和可重复地分离成定义的组分。在这种温和的分离后，我们观察到支化的β-葡聚糖比之前描述的要丰富得多。通过对新方法的放大，我们制备了大量的高支化葡聚糖，并通过化学、MALDI-TOF MS、13C和1H高场核磁共振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抵抗力的高度分枝的细胞壁聚合物。最后，为了证明它的应用，我们测定了已知突变菌株的细胞壁组成。

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 gentle 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.