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  • Alzheimer amyloid precursor aspartyl proteinase activity in CHAPSO homogenates of Spodoptera frugiperda cells.

Alzheimer amyloid precursor aspartyl proteinase activity in CHAPSO homogenates of Spodoptera frugiperda cells.

Alzheimer disease and associated disorders (2004-12-14)
Troy L Carter, Giuseppe Verdile, David Groth, Alexey Bogush, Stefani Thomas, Patrick Shen, Paul E Fraser, Paul Mathews, Ralph A Nixon, Michelle E Ehrlich, John B J Kwok, Peter St George-Hyslop, Peter Schofield, Yueming Li, Austin Yang, Ralph N Martins, Sam Gandy
ABSTRACT

Presenilins are polytopic, integral proteins that control intramembranous proteolysis at the "gamma-" and "epsilon-" cleavage sites of the Alzheimer amyloid-beta precursor protein (APP) to yield amyloid-beta peptide (Abeta) and the APP intracellular domain (AICD). We have overexpressed a constitutively active, pathogenic form of PS1 (known as PS1 Delta exon 9) together with its substrate, APP-C99, in Spodoptera frugiperda (Sf9) cells. Sf9 cells have been reported to lack endogenous gamma-secretase, an unexpected finding since there exists an insect homologue of PS1. In our hands, neither intact insect cells coexpressing PS1 Delta exon 9/APP-C99 nor the aqueous homogenates of these cells displayed obvious products of the gamma- or epsilon-secretase reactions, as reported. Surprisingly, when APP-C99-expressing cells were homogenized in 3[(3-cholamidopropyl) dimethylammonio]-2-hydroxypropanesulfonic acid (CHAPSO), a detergent known to support gamma-secretase activity, subsequent incubation led to the accumulation of an AICD-like peptide (AICD-L). Aspartyl proteinase inhibitors were effective in preventing the appearance of AICD-L, but inhibitors of other classes of proteinases were ineffective. Immunoprecipitation-mass spectrometry of AICD-L revealed its identity as the minor of the two known AICDs.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
CHAPSO, ≥98%