tailieunhanh - Báo cáo khóa học: Characterization of presenilin complexes from mouse and human brain using Blue Native gel electrophoresis reveals high expression in embryonic brain and minimal change in complex mobility with pathogenic presenilin mutations

The presenilin proteins are required for intramembrane cleavage of a subset of type 1 membrane proteins including the Alzheimer’s disease amyloid precursor protein. Previous studies indicate presenilin proteins form enzymatically act-ive high molecular mass complexes consisting of hetero-dimers of N- and C-terminal fragments in association with nicastrin, presenilin enhancer-2 and anterior pharynx defective-1 proteins. | Eur. J. Biochem. 271 375-385 2004 FEBS 2003 doi Characterization of presenilin complexes from mouse and human brain using Blue Native gel electrophoresis reveals high expression in embryonic brain and minimal change in complex mobility with pathogenic presenilin mutations Janetta G. Culvenor1 2 3 Nancy T. Ilaya1 2 3 Michael T. Rvan4 Louise Canterford1 3 David E Hoke1 3 . . . . Nicholas A. Williamson1 3 Catriona A. McLean5 Colin L. Masters1 3 and Genevieve Evin1 3 1 Department of Pathology and 2 Centre for Neuroscience The University of Melbourne Australia 3 Mental Health Research Institute of Victoria Australia 4Department of Biochemistry La Trobe University Bundoora Australia 5National Neuroscience Facility University of Melbourne Australia The presenilin proteins are required for intramembrane cleavage of a subset of type 1 membrane proteins including the Alzheimer s disease amyloid precursor protein. Previous studies indicate presenilin proteins form enzymatically active high molecular mass complexes consisting of heterodimers of N- and C-terminal fragments in association with nicastrin presenilin enhancer-2 and anterior pharynx defective-1 proteins. Using Blue Native gel electrophoresis BN PAGE we have studied endogenous presenilin 1 complex mass stability and association with nicastrin presenilin enhancer-2 and anterior pharynx defective-1. Solubilization of mouse or human brain membranes with dodecyl-D-maltoside produced a 360-kDa species reactive with antibodies to presenilin 1. Presenilin 1 complex levels were high in embryonic brain. Complex integrity was sensitive to Triton X-100 and SDS but stable to reducing agent. Addition of 5 M urea caused complex dissolution and nicastrin to migrate as a subcomplex. Nicastrin and pre-senilin enhancer-2 were detected in the presenilin 1 complex following BN PAGE electroelution and second-dimension analysis. Anterior pharynx defective-1 was detected as an 18-kDa form and 9-kDa .