Establishment of ?-secretes assay and functional analysis of Presenilin

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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.