Cell-free assays for gamma-secretase activity.

C. McLendon, T. Xin, C. Ziani-Cherif, M. P. Murphy, K. A. Findlay, P. A. Lewis, I. Pinnix, K. Sambamurti, R. Wang, A. Fauq, T. E. Golde

Research output: Contribution to journalArticlepeer-review

105 Scopus citations


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.

Original languageEnglish
Pages (from-to)2383-2386
Number of pages4
JournalFASEB Journal
Issue number15
StatePublished - Dec 2000

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics


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