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cDNA cloning of enzymes involved in the synthesis and degradation of cell wall-matrix polysaccharides

参与细胞壁基质多糖合成和降解的酶的 cDNA 克隆

基本信息

批准号：
03640574
负责人：
KAMISAKA Seiichiro
金额：
$ 1.54万
依托单位：
OSAKA CITY UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1993
项目状态：
已结题

项目摘要

The mechanical properties of cell walls are determined by cell wallconstituting molecules, particularly matrix polysaccharides. The goal of this study is to clone cDNA of enzymes involved in the synthesis and degradation of xyloglucans and arabinoxylans, which are responsible for determining cell wall extensibility. Results are as follows :(1) Matrix polysaccharide-degrading enzymes : Xyloglucan degrading enzymes were solubilized with NaCl from Vigna epicotyl cell walls. The enzymes were fractionated and purified using Con A-Sepharose, Mono S, Superdex 75, and Phenyl Superose column. The enzymes are separated into 3 peaks on Mono S colum. alpha-Fucosidases which eliminate fucos-sede chain of xyloglucans were separated into Mono S-binding and -not binding fractions.(2) Matrix polysaccharide-modifying enzymes : Using Golgi-vesicle fraction, the screening of the enzyme that ester-links ferulic acid to arabinose moieties of arabinoxylans. Oat arabinoxylans and synthesitic feruloyl-CoA were used as the substrates. Unfortunately, the enzyme activity was not detected, because of the high activity of feruloyl-CoA esterase in the enzyme preparation. At present, extensive effort has been done to eliminate the esterase from the enzyme preparation.The present srudy will soon lead to the cloning of cDNA of the enzymes responsible for the synthesis and degradation of matrix polysaccharides determing the mechanical properteis of cell walls, although the cDNA cloning was not done within the research period of 3 years.

细胞壁的机械性质由细胞壁组成分子，特别是基质多糖决定。本研究的目的是克隆参与木葡聚糖和阿拉伯聚糖的合成和降解的酶的cDNA，这些酶负责决定细胞壁的可伸展性。结果如下：（1）基质多糖降解酶系：NaCl溶液可溶解豇豆上胚轴细胞壁中的木葡聚糖降解酶系。用Con A-Sepharose、Mono S、Superdex 75和Phenyl Superose柱对酶进行分级和纯化。在Mono S柱上酶被分离成3个峰。将消除木葡聚糖的岩藻糖-sede链的α-岩藻糖苷酶分离成单S-结合级分和单S-不结合级分。(2)基质多糖修饰酶：使用高尔基体-囊泡组分，筛选将阿魏酸酯连接到阿拉伯聚糖的阿拉伯糖部分的酶。燕麦阿拉伯聚糖和合成的阿魏酰辅酶A被用作底物。不幸的是，由于酶制剂中阿魏酰-CoA酯酶的高活性，未检测到酶活性。目前，人们已经做了大量的工作来消除酶制剂中的酯酶，虽然在3年的研究期间内还没有克隆到负责合成和降解决定细胞壁机械性质的基质多糖的酶的cDNA，但本研究将很快导致该酶的cDNA克隆。