Establishment of ?-secretes assay and functional analysis of Presenilin

γ-分泌物分析的建立在本研究中，我们制备了几种被认为是γ-分泌物的直接底物的融合蛋白，如APPC100-HA、APPC100-FRAG、APPC100-先导肽酶、NotchC100-HA、NotchC100-FRAG和NotchC100-先导肽酶。虽然我们还没有成功地检测到γ分泌活性，但放射性标记的这些多肽是定量检测γ分泌活性的最好底物。目前，我们正在详细优化β-分泌活性的测定条件。分析包括早老素在内的高分子量蛋白质复合体。据报道，早老素与许多其他膜蛋白形成一个大的蛋白质复合体，具有γ-分泌活性。我们用多种洗涤剂对大鼠肝脏微量组分进行了蓝色-天然凝胶-凝胶电泳法(BN-PAGE)和甘油密度梯度离心法的分析，发现洋地黄素增溶的早老素复合体大于其他实验室报道的250 kDa。我们目前正在研究具有γ分泌活性的早老素复合体的最低组分。

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.