Cell-free assays for gamma-secretase activity

FASEB J. 2000 Dec;14(15):2383-6. doi: 10.1096/fj.00-0286fje.


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.

MeSH terms

  • Amyloid Precursor Protein Secretases
  • Amyloid beta-Peptides / metabolism*
  • Amyloid beta-Protein Precursor / metabolism*
  • Animals
  • CHO Cells
  • Cell-Free System
  • Cricetinae
  • Endopeptidases / analysis*
  • Hydrogen-Ion Concentration
  • Membrane Proteins / metabolism*
  • Models, Biological
  • Oligopeptides / pharmacology
  • Protease Inhibitors / pharmacology
  • Solubility


  • Amyloid beta-Peptides
  • Amyloid beta-Protein Precursor
  • Membrane Proteins
  • Oligopeptides
  • Protease Inhibitors
  • Amyloid Precursor Protein Secretases
  • Endopeptidases