Establishment of ?-secretes assay and functional analysis of Presenilin

早老素β-分泌测定方法的建立及功能分析

• 批准号: 13671024
• 负责人: MIURA Satoshi
• 金额: $ 1.98万
• 依托单位: YOKOHAMA CITY UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13671024/
• 关键词: presenilin; ?-secretes

Establishment of γ-secretes assayIn this study, we made several fusion proteins such as APPC100-HA, APPC100-Frag, APPC100-leader-peptidase, NotchC100-HA, NotchC100-Frag and NotchC100-leader-peptidase which were thought to be direct substrates for γ-secretes. Though we have not succeeded in detecting the γ-secretes activities, radiolabeled these polypeptides are most favorable substrates detecting the γ-secretes activities in quantative fashion. We are now optimizing the assay conditions in detail for ?-secretes activities.Analysis of high molecular weight protein complex including presenilin.Presenilin has been reported to form a large protein complex with many other membrane proteins which has an activity of γ-secretes. We analyzed solubilized rat liver micro some fraction with many kind of detergents by Blue-Native-Gel -Electrophoresis (BN-PAGE) and glycerol-density-gradient centrifugation, and found that digitonin solubilized presenilin complex is larger than 250kDa that is previously reported from another laboratories. We are now investigating the minimum components of presenilin complex having γ-secretes activity.

γ-分泌酶检测方法的建立本研究制备了几种γ-分泌酶直接底物的融合蛋白，如APPC 100-HA、APPC 100-Frag、APPC 100-leader-peptidase、NotchC 100-HA、NotchC 100-Frag和NotchC 100-leader-peptidase。虽然我们还没有成功地检测到γ-分泌活性，放射性标记的这些多肽是最有利的底物检测γ-分泌活性的定量方式。我们现在正在详细优化测定条件，早老素（Presenilin）是一种具有γ-分泌活性的膜蛋白，它与许多膜蛋白形成一个大的蛋白质复合物。用多种去污剂溶解大鼠肝微粒体组分，通过BN-PAGE和甘油密度梯度离心分析，发现毛地黄皂苷溶解的早老素复合物分子量大于250 kDa。我们正在研究具有γ-分泌活性的早老素复合物的最小组分。