title: Solubilization of full-length amyloid precursor proteins from PC12 cell membranes.
abstract: The amyloid beta protein (A beta) of Alzheimer disease (AD) is derived from the proteolytic processing of the amyloid precursor proteins (APP), which are considered type I transmembrane proteins. Production of A beta from a transmembrane precursor predicts a proteolytic cleavage within the lipid bilayer, a site relatively inaccessible to proteases. Here we show that incubation of a membrane fraction of PC12 cells at 37 degrees C results in the solubilization of an APP species which migrates on SDS-PAGE as full-length APP. The release of this full-length APP was pH-dependent with a peak of activity of pH 9.0. At this pH about 19% of the membrane APP was released from the active subcellular fraction. Under the same conditions other transmembrane proteins remained insoluble. Very little APP was solubilized at 4 degrees C. APP solubilization was specifically inhibited by the serine protease inhibitors aprotinin and pefabloc. Other protease inhibitors, including leupeptin and alpha 1-antitrypsin, had no effect. Several metal cations, including Ca++ and Zn++, also inhibited release of soluble full-length APP. Low levels of full-length APP were also detected in both the soluble fraction of PC12 cell extracts and in the media of PC12 cell cultures. These data suggest the involvement of a serine protease in the solubilization of membrane, full-length APP. The release of this APP could provide a soluble substrate for the proteolytic enzymes involved in the production of A beta.
authors.affiliation: Mount Sinai School of Medicine, Department of Psychiatry, New York, New York.
authors.forename: J A
authors.initials: JA
authors.lastname: Ripellino
authors.forename: D
authors.initials: D
authors.lastname: Vassilacopoulou
authors.forename: N K
authors.initials: NK
authors.lastname: Robakis
mesh_terms: D000682:Amyloid / Q000302:isolation & purification*; D000818:Animals; D007611:Aprotinin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000737:chemistry* / Q000187:drug effects; D017077:Culture Media, Conditioned / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D009363:Neoplasm Proteins / Q000302:isolation & purification / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000302:isolation & purification*; D016716:PC12 Cells / Q000737:chemistry* / Q000187:drug effects; D011328:Prions; D011480:Protease Inhibitors / Q000494:pharmacology; D011498:Protein Precursors / Q000302:isolation & purification*; D051381:Rats; D012697:Serine Endopeptidases / Q000378:metabolism*; D012995:Solubility; D013450:Sulfones / Q000494:pharmacology; D013696:Temperature; D015032:Zinc / Q000494:pharmacology
chemical_list: D000682:Amyloid; D017077:Culture Media, Conditioned; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D011328:Prions; D011480:Protease Inhibitors; D011498:Protein Precursors; D013450:Sulfones; C002010:4-(2-aminoethyl)benzenesulfonylfluoride; D007611:Aprotinin; D012697:Serine Endopeptidases; D015032:Zinc; D002118:Calcium