Establishment of ?-secretes assay and functional analysis of Presenilin

Research Project

Project/Area Number	13671024
Research Category	Grant-in-Aid for Scientific Research (C)
Allocation Type	Single-year Grants
Section	一般
Research Field	Psychiatric science
Research Institution	YOKOHAMA CITY UNIVERSITY
Principal Investigator	MIURA Satoshi YOKOHAMA CITY UNIV., Medical, Associate Prof., 医学部, 助教授 (60157427)
Co-Investigator(Kenkyū-buntansha)	NAKAI Toshiki YOKOHAMA CITY UNIV., Medical, Assistant Prof., 医学部, 助手 (20270712)
Project Period (FY)	2001 – 2002
Project Status	Completed (Fiscal Year 2002)
Budget Amount *help	¥3,100,000 (Direct Cost: ¥3,100,000) Fiscal Year 2002: ¥1,200,000 (Direct Cost: ¥1,200,000) Fiscal Year 2001: ¥1,900,000 (Direct Cost: ¥1,900,000)
Keywords	presenilin / ?-secretes
Research Abstract	Establishment of γ-secretes assay In 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.