Research Article

Effect of Metal Chelators on γ-Secretase Indicates That Calcium and Magnesium Ions Facilitate Cleavage of Alzheimer Amyloid Precursor Substrate

Figure 1

Characterization of γ-secretase in vitro assay with guinea pig brain enzyme. ~1 μg of purified recombinant C100-3XFLAG was incubated with 2.75 μg of CHAPSO solubilised membranes of guinea pig brain in 20 mM HEPES buffer, pH 7.3, plus 3 mM CaCl2, 3 mM MgCl2 and 150 mM KCl. The reactions were stopped by adding Laemmli buffer, boiled, and separated on Tris-Tricine gels. M2 (anti-FLAG) antibody and WO2 (anti-Aβ 1–16) were used for western blot detection. (a) Production of an AICD fragment in the assay is inhibited by the γ-secretase inhibitors, L-685,458 and DAPT. (b) Both AICD and Aβ are produced in the reaction and inhibited by L-685,458. AICD signal increases in a time dependent manner over 20 h. Aβ signal is increased at 4 h compared to 2 h, but decreased at 20 h, possibly due to degradation. (Inc, incubation at 37°C). (c) Positive effect of phospholipids on AICD production in the γ-secretase assay. PC, phosphatidylcholine; PE, phosphatidylethanolamine. (d) Positive effect of ATP on the γ-secretase assay. ATP was added 1.25 mM concentration. (e) Quantitation of three separate experiments using GeneTools Syngene software shows ~1.6 fold enhancement of γ-secretase activity in the presence of 1.25 mM ATP. The error bars represent SEM.
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