A capillary electrophoresis method for evaluation of Abeta proteolysis in vitro.
According to the amyloid hypothesis, Abeta peptides are neurotoxic and underlie development and progression of Alzheimer's disease (AD). Multiple Abeta clearance mechanisms, including destruction of the peptides by proteolytic enzymes, are hypothesized to regulate physiological Abeta peptide levels. The insulin-degrading enzyme (IDE) is considered one of the predominant enzymes having Abeta degrading activity. Despite its putative role in protecting against AD, relatively few methods exist for studying IDE activity in vitro. We report the application of capillary electrophoresis (CE) as a novel method for evaluating IDE-mediated Abeta 1-40 proteolysis. This method employs chemically unmodified substrates that are readily obtained from commercial sources. It involves minimal sample preparation, and requires no specialized equipment beyond a CE instrument equipped with a standard fused silica capillary. In the present analysis, we demonstrate that this CE-based method is amenable to kinetic analysis, and show that IDE-mediated Abeta proteolysis is significantly and disproportionately inhibited in the presence of insulin, an alternative IDE substrate.
Alper
Benjamin J
BJ
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, United States.
Schmidt
Walter K
WK
D016229:Amyloid beta-Peptides / Q000032:analysis* / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D019075:Electrophoresis, Capillary / Q000379:methods* / Q000592:standards; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D007339:Insulysin / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis* / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
D016229:Amyloid beta-Peptides; D007328:Insulin; D010446:Peptide Fragments; D011994:Recombinant Proteins; C078592:amyloid beta-protein (1-40); D007339:Insulysin