Purification and tissue level of the beta-amyloid peptide precursor of rat brain.
The beta-amyloid peptide precursor (beta-APP) exists in brain tissue as a membrane-associated protein extractable with 1% Triton X-100. beta-APP has been purified to near homogeneity by the following procedure: 1) anion exchange chromatography, 2) affinity chromatography on heparin agarose, and 3) immunoaffinity adsorption on matrix-bound antibodies directed to a synthetic peptide corresponding to the last 24 amino acids of the cDNA derived amino acid sequence of beta-APP. Conditions were chosen to minimize denaturation of the protein. The identity of the protein was confirmed by its immunoreactivity with antisera directed to five subsequences derived from the cDNA sequence. The amino-terminal sequence of beta-APP was found to be Leu-Glu-Val-Pro-Thr-Asp-Gly-Asn-Ala-Gly-Leu-Leu-Ala-Glu-Pro, which commences at residue 18 of the cDNA-derived primary structure. The procedure resulted in a 2000-fold purification of beta-APP. The purified protein migrated on polyacrylamide gels as a doublet of apparent molecular mass 100-120 kDa, although the predicted molecular mass of its constituent amino acids is 76 kDa. beta-APP clearly behaves anomalously in gel electrophoresis. The beta-APP content of rat brain amounted to 46 micrograms/g tissue. The half-life of the protein was calculated to be about 10 h, which is 30 times as long as that observed by others in transfected PC-12 cells. We conclude that transfected cell systems may not be adequate models for beta-APP processing.
New York State Institute for Basic Research in Developmental Disabilities, Staten Island 10314.
A
A
Potempska
J
J
Styles
P
P
Mehta
K S
KS
Kim
D L
DL
Miller
D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000302:isolation & purification* / Q000378:metabolism*; D016564:Amyloid beta-Protein Precursor; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism*; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000302:isolation & purification* / Q000378:metabolism*; D051381:Rats
D016229:Amyloid beta-Peptides; D016564:Amyloid beta-Protein Precursor; D011498:Protein Precursors