Characterization of endogenous APP processing in a cell-free system

• Published in: AGE 1998-01, Vol.21 (1), p.15-23
• Main Authors: Brown, Abraham M., Potempska, Anna, Tummolo, Donna, Spruyt, Michael A., Jacobsen, J. Steven, Sonnenberg-Reines, June
• Format: Article
• Language: English
• Subjects: Peptides
• ISSN: 0161-9152; 2509-2715; 1574-4647; 2509-2723
• DOI: 10.1007/s11357-998-0003-y

We have developed a simple in vitro assay using tissue homogenates that allows detection and characterization of several endogenous proteolytic activities which convert Alzheimer's amyloid precursor protein (APP) to the smaller, carboxy-terminal fragments, postulated to be intermediates in the formation of β-amyloid peptide (Aβ). Incubation at 37°C results in the degradation of transmembrane APP and formation of a mixture of carboxy-terminal containing peptides with mass values of 9-12 kDa. Epitope mapping and electrophoretic comparison with a truncated APP standard showed one of these peptides to contain the entire Aβ sequence. Analysis of pH dependence shows that formation of this carboxy-terminal product as well as another fragment, that is the likely product of 'secretase' activity, requires acidic pH. This suggests that cleavage of full-length APP to secreted forms may take place in an acidic intracellular compartment.[PUBLICATION ABSTRACT]