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Biochemistry. 1998 Feb 3;37(5):1274-82.

Release of nontransmembrane full-length Alzheimer's amyloid precursor protein from the lumenar surface of chromaffin granule membranes.

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Department of Psychiatry, Mount Sinai School of Medicine, New York 10029, USA.


We previously demonstrated the presence of a soluble form of full-length Alzheimer's amyloid precursor protein (APP) in the lumen of adrenal medullary chromaffin granules (CG). Furthermore, full-length APP is released from CG membranes in vitro at pH 9.0 by an enzymatic mechanism, sensitive to protease inhibitors [Vassilacopoulou et al. (1995) J. Neurochem. 64, 2140-2146]. In this study, we found that when intact CG were subjected to exogenous trypsin, a fraction of APP was not digested, consistent with an intragranular population of APP. To examine the substrate-product relationship between membrane and soluble full-length APP, we labeled CG transmembrane APP with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID), a lipophilic probe, specific for membrane-spanning domains of proteins. APP released from the membranes at pH 9.0 was not labeled with [125I]TID. In addition, this APP was not biotinylated in intact CG. Combined, the results indicate that APP released from CG membranes derives from a unique nontransmembrane population of membrane-associated APP, located in the lumenal side of CG membranes. Dithiobis(succinimidylpropionate) (DSP) cross-linking indicated that APP in CG is situated in close proximity with other proteins, possibly with APP itself. APP complexes were also detected under nonreducing conditions, without DSP cross-linking. These results, combined with our previous studies, indicate that full-length APP within CG exists as three different populations: (I) transmembrane, (II) membrane-associated/nontransmembrane, and (III) soluble. The existence of nontransmembrane populations suggests that putative gamma-secretase cleavage sites of APP, assumed to be buried within the lipid bilayer, could be accessible to proteolysis in a soluble intravesicular environment.

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