A novel brain cysteine protease forms an SDS stable complex with the beta-amyloid precursor protein.
Alzheimer's disease (AD) brain accumulates beta-protein (A beta) a peptide proteolytically derived from the beta-amyloid precursor protein (APP). The abnormal production and aggregation of A beta have been implicated in the pathogenesis of the disease. The mechanism of production of A beta in vivo is not yet clear; but endoproteases capable of degrading APP are likely to be involved in the process. We have isolated a protease from AD brain by following its activity in digesting a synthetic peptide of 10 amino acids derived from the APP sequence flanking the N-terminus of A beta. The protease was purified by a fractionation scheme including ammonium sulfate precipitation and column chromatography using hydrophobic interaction, anion exchange, affinity, hydroxyapatite and size exclusion gels. The purity of the final product was assessed on a silver stained SDS gel by the presence of a single band. Microsequencing was performed following trypsin digestion of the sample. Internal peptide sequences were found to have sequence homology to cysteine proteases in the database. The enzyme requires DTT for activity and can be inhibited by specific inhibitors of cysteine but not serine proteases. The purified enzyme has a pI of 5.0 and a native tetrameric structure with subunits of 48 kD each. The enzyme is capable of digesting APP and generating a short peptide recognizable by antibodies specific to the C-terminus of APP. Interestingly, the purified protease also forms heat- and SDS-stable complexes with APP.
Chang
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T
Department of Medicine, Boston University School of Medicine, Massachusetts 02118, USA.
Abraham
C R
CR
D000544:Alzheimer Disease / Q000201:enzymology*; D000595:Amino Acid Sequence; D016564:Amyloid beta-Protein Precursor / Q000378:metabolism*; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology*; D002855:Chromatography, Thin Layer; D003546:Cysteine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data
D016564:Amyloid beta-Protein Precursor; D003546:Cysteine Endopeptidases