Cell-free assays for gamma-secretase activity.
The amyloid b-protein (Ab) deposited in Alzheimer's disease (AD) is a normally secreted proteolytic product of the amyloid b-protein precursor ( APP). Generation of Ab from the APP requires two sequential proteolytic events: an initial b-secretase cleavage at the amino terminus of the Ab sequence followed by g-secretase cleavage at the carboxyl terminus of Ab. We describe the development of a robust in vitro assay for g-secretase cleavage by showing de novo Ab production in vitro and establish that this assay monitors authentic gamma-secretase activity by documenting the production of a cognate g-CTF, confirming the size of the Ab produced by mass spectrometry, and inhibiting cleavage in this system with multiple inhibitors that alter g-secretase activity in living cells. Using this assay, we demonstrate that the g-secretase activity 1) is tightly associated with the membrane, 2) can be solubilized, 3) has a pH optimum of 6.8 but is active from pH 6.0 to pH >8.4, and 4) ascertain that activities of the g-40 and g-42 are indeed pharmacologically distinct. These studies should facilitate the purification of the protease or proteases that are responsible for this unusual activity, which is a major therapeutic target for the treatment of AD.[1]References
- Cell-free assays for gamma-secretase activity. McLendon, C., Xin, T., Ziani-Cherif, C., Murphy, M.P., Findlay, K.A., Lewis, P.A., Pinnix, I., Sambamurti, K., Wang, R., Fauq, A., Golde, T.E. FASEB J. (2000) [Pubmed]
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