ANALYSIS OF FISSION YEAST CELL WALL

裂殖酵母细胞壁的分析

• 批准号: 7369269
• 负责人: PHILLIPS W ROBBINS
• 金额: $ 0.09万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7369269
• 关键词: ANALYSIS; FISSION; YEAST; CELL; WALL

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.

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。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构适用于该中心，这不一定是调查员的机构。纯化的内糖苷酶已用于确定精神分裂症pombe细胞壁的组成。传统上研究了该结构的成分（甘露蛋白，Alpha-1,3葡聚糖，β-1,3Glucan和一个分支的β-葡聚糖）用热碱进行了研究。取而代之的是，我们通过使用内托β-1,3葡萄糖酶以及新型的内鼠α-1,3葡萄糖酶（Mutanase）消化来依次去除多糖。该方法使用顺序酶促水解，这比热碱处理更为外邦。该方法是可重现的，并且比以前的方法更具体地，更具体地将细胞壁的主要组成部分分为定义的馏分。在这种温和的隔离之后，我们观察到分支的β-葡聚糖比以前描述的要丰富得多。通过扩展新协议，我们准备了大量高度分支的葡聚糖，并通过化学，MALDI-TOF MS，13C和1H高场NMR分析来确定其结构特征。这些分析使我们能够清楚地定义了对已知葡萄糖酶具有抗性的高度分支细胞壁聚合物。最后，为了证明其应用，我们已经确定了已知突变菌株的细胞壁组成。