权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Molecular interaction between the components in lysosomal sialidase complex

溶酶体唾液酸酶复合物成分之间的分子相互作用

基本信息

批准号：
12672130
负责人：
UDA Yutaka
金额：
$ 1.92万
依托单位：
Niigata University of Pharmacy and Applied Life Science
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12672130/
关键词：
sialidase β-galactosidase protective protein β-galactosidase complex molecular interaction carboxypeptidase

项目摘要

Recent studies has shown that the mammalian lysosomal sialidase exists as a complex with β-galactosidase and protective protein. The protective protein is a 48kDa heterodimer of 30kDa- and 20kDa protomers and is required for multimerization of β-galactosidase as well as the expression and stabilization of sialidase activity. To understand the function of β-galactosidase complex, we examined the molecular interaction between β-galactosidase components in the complex using Biacore surface plasmon resonance. Each of the β-galactosidase components was separated and purified as described previously. The biotinized β-galactosidase monomer(64kDa) was coupled to a streptoavidine group on the senser tip surface. The 48kDa, 30kDa and 20kDa protein components in protective protein were tested for their interaction with β-galactosidase monomer fixed on the sensor tip. Among the components, 48kDa and 20kDa proteins had strong affinity with β-galactosidase monomer at acidic pH, but 30kDa protein did not show any interaction. This result suggests that β-galactosidase bind to 20kDa protein component in protective protein and forms a β-galactosidase hetero polymer complex, in contrast with 30kDa protein which possess carboxypeptidase activity. To elucidate the molecular interaction between sialidase and β-galactosidase complex, further studies are necessary.We highly purified a sialidase from the ovary of the starfish, Asterina pectinifera. The analysis of the N-terminal amino acid sequences of the purified enzyme showed high homology with that of cathepsin D. On the purification processes, the specific activities of not only sialidase activity but also cathepsin D activity were increased. The two enzymes showed distinct protein band on polyacrylamide gel electrophoresis. It is necessary to study more precisely whether sialidase interact with cathepsin D or not.

最近的研究表明，哺乳动物溶酶体唾液酸酶以与β-半乳糖苷酶和保护性蛋白的复合物形式存在。保护性蛋白是30 kDa和20 kDa原聚体的48 kDa异源二聚体，并且是β-半乳糖苷酶的多聚化以及唾液酸酶活性的表达和稳定所需的。为了了解β-半乳糖苷酶复合物的功能，我们使用Biacore表面等离子体共振检测了复合物中β-半乳糖苷酶组分之间的分子相互作用。如前所述分离和纯化每种β-半乳糖苷酶组分。生物素化的β-半乳糖苷酶单体（64 kDa）与传感器尖端表面上的链霉亲和素基团偶联。检测了保护蛋白中48 kDa、30 kDa和20 kDa蛋白组分与固定在传感器针尖上的β-半乳糖苷酶单体的相互作用。其中48 kDa和20 kDa蛋白在酸性条件下与β-半乳糖苷酶单体有较强的亲和性，而30 kDa蛋白在酸性条件下不与β-半乳糖苷酶单体发生相互作用。这一结果表明，β-半乳糖苷酶与保护蛋白中的20 kDa蛋白组分结合，形成β-半乳糖苷酶异质聚合物复合物，而30 kDa蛋白具有羧肽酶活性。为了阐明唾液酸酶与β-半乳糖苷酶复合物之间的分子相互作用，还需要进一步的研究。N-末端氨基酸序列分析表明，该酶与组织蛋白酶D有很高的同源性。在纯化过程中，不仅唾液酸酶活性的比活性，而且组织蛋白酶D活性的比活性增加。两种酶在聚丙烯酰胺凝胶电泳上均显示出明显的蛋白条带。唾液酸酶是否与组织蛋白酶D相互作用有待进一步研究。