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 presenilin 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 C‐terminal fragment by standard SDS/PAGE of mouse tissues, and as a predominant 36‐kDa band after presenilin 1 complex second‐dimension analysis. Membranes from brain cortex of Alzheimer's disease patients, or from cases with presenilin 1 missense mutations, indicated no change in presenilin 1 complex mobility. Higher molecular mass presenilin 1‐reactive species were detected in brain containing presenilin 1 exon 9 deletion mutation. This abnormality was confirmed using cells transfected with the same presenilin deletion mutation.

The γ‐secretase complex mediates the final proteolytic event in Alzheimer's disease amyloid‐β biogenesis. This membrane complex of presenilin, anterior pharynx defective, nicastrin, and presenilin enhancer‐2 cleaves the C‐terminal 99‐amino acid fragment of the amyloid precursor protein intramembranously at γ‐sites to form C‐terminally heterogeneous amyloid‐β and cleaves at an ε‐site to release the intracellular domain or ε‐C‐terminal fragment. In this work, two novel in vitroγ‐secretase assays are developed to further explore the biochemical characteristics of γ‐secretase activity. During development of a bacterial expression system for a substrate based on the amyloid precursor protein C‐terminal 99‐amino acid sequence, fragments similar to amyloid‐β and an ε‐C‐terminal fragment were observed. Upon purification this substrate was used in parallel with a transfected source of substrate to measure γ‐secretase activity from detergent extracted membranes. With these systems, it was determined that recovery of size‐fractionated cellular and tissue‐derived γ‐secretase activity is dependent upon detergent concentration and that activity correlates to a subset of high molecular mass presenilin complexes. We also show that by changing the solvent environment with dimethyl sulfoxide, detection of ε‐C‐terminal fragments can be elevated. Lastly, we show that zinc causes an increase in the apparent molecular mass of an amyloid precursor protein γ‐secretase substrate and inhibits its cleavage. These studies further refine our knowledge of the complexes and biochemical factors needed for γ‐secretase activity and suggest a mechanism by which zinc dysregulation may contribute to Alzheimer's disease pathogenesis.

Nicastrin was the first binding partner of presenilin (PS) shown to be a critical component of the presenilin/c-secretase complex essential in development and differentiation, and in generation of Alzheimer's disease Ab amyloid peptide. To investigate the function of this glycoprotein, we compared nicastrin and presenilin protein expression in various mouse tissues. Western blot analysis of PS1, PS2 and nicastrin indicates their expression levels are not coordinated. In adult mouse, nicastrin is highly expressed in muscle membranes, whereas presenilin levels are very low. By Blue Native electrophoresis, a PS1 complex of 400 kDa was detected in lung, brain, thymus and heart; nicastrin was also detected as a 400-kDa complex in brain but in muscle it was detected with a complex mobility of 240 and 290 kDa, suggesting association with alternate protein complexes. Immunocytochemistry confirms strong intracellular expression of nicastrin in skeletal muscle and blood vessel smooth muscle. These findings suggest a function for nicastrin in muscle other than participation in the c-secretase complex.