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Biochemical studies of mucin cleaving enzyme and gastric mucus glycoprotein

粘蛋白裂解酶和胃粘液糖蛋白的生化研究

基本信息

批准号：
62580129
负责人：
HOTTA Kyoko
金额：
$ 1.22万
依托单位：
Kitasato 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：
1987
资助国家：
日本
起止时间：
1987 至 1989
项目状态：
已结题

项目摘要

Mucus glycoproteins, or mucins, are important components which protect tissues and cells from various, attacking agents. Enzymatical cleavage of oligosaccharides and 0-glycosidic linkage in mucin should greatly expedite structural studies on the protein and sugar part of mucus glycoproteins.The degradation enzymes of mucus glycoproteins were obtained from the culture medium of Streptomyces sp. OH-11242 in which porcine gastric mucus glycoproteins were added as the only source of carbon. A crude enzyme preparation from the culture medium contained endo-glycosidases besides exo-glycosidases and proteases. Detection method of the enzyme activity by HPLC analysis of pyridylaminated(PA) oligosaccharide was developed. The size of oligosaccharides prepared from the medium corresponded to 7-14 glucose oligomers. Reducing end sugars as PA-sugars from PA- oligosaccharide was determined to be galactose, N-acetylglucosamine and N-acetylglucosamine. The endo-glycosidase was partially purified and concluded the presence of a new type of endo-glycosidase liberating not only Gal beta 1-3GalNAc but also other larger oligosaccharides by hydrolysis of the 0-glycosidle linkage between GaINAe and Ser(Thr).Further studies of the structure of rat gastric mucus glycoproteins were carried out. There are two distinct types of mucus glycoprotein subunits in rat stomach ; one is 4.4 X10^5 and the other is 6 X10^6 MW. Their location was differed in the stomach. Sulfate group locations on oligosaccharides of gastric mucus glycoprotein were also differed in the corpus and antrum.

粘液糖蛋白或粘蛋白是保护组织和细胞免受各种攻击的重要成分。酶切黏液蛋白中的低聚糖和0-糖苷键将极大地促进黏液糖蛋白蛋白和糖部分的结构研究。以猪胃黏液糖蛋白为唯一碳源的Streptomyces sp. OH-11242培养基为原料，获得黏液糖蛋白的降解酶。从培养基中制备的粗酶除含有外糖苷酶和蛋白酶外，还含有内糖苷酶。建立了高效液相色谱法测定聚吡啶层合寡糖酶活性的方法。从培养基中制备的低聚糖的大小相当于7-14个葡萄糖低聚物。PA-寡糖的还原糖为PA-糖，分别为半乳糖、n -乙酰氨基葡萄糖和n -乙酰氨基葡萄糖。对内糖苷酶进行了部分纯化，发现存在一种新型内糖苷酶，通过水解GaINAe和Ser（Thr）之间的0-糖苷键，不仅能释放Gal β 1-3GalNAc，还能释放其他更大的低聚糖。对大鼠胃粘液糖蛋白的结构进行了进一步的研究。大鼠胃粘液糖蛋白亚基有两种不同的类型；一个是4.4 X10^5，另一个是6 X10^6兆瓦。它们在胃中的位置不同。胃黏液糖蛋白低聚糖上硫酸基团的位置在胃体和胃窦也不同。