Facile and sensitive assay for monitoring proteolytic activities with defined specificities: studies on amyloid beta-protein processing in Alzheimer's disease.
A novel approach was taken to screen for the presence of highly specific proteases in tissue extracts and to monitor their purification. We successfully used this method to identify proteolytic activity in brain which may be responsible for the generation of the N-terminus of the beta-protein, the major protein in the brain amyloid deposits of Alzheimer's disease. A radiolabeled synthetic decapeptide, spanning this putative cleavage site, was used as an artificial substrate. Digestion was monitored by TLC separation of the reaction products followed by autoradiography. Mobility shifts indicate cleavage; the identity of the fragments can be inferred from synthetic markers or from comparison to control digests with known proteases. When using highly purified or semi-purified proteases, larger amounts of unlabeled peptide substrates can be used and the resulting fragments identified by sequencing. The assay is fast, sensitive (femtomole level), compatible with crude extracts, allows simultaneous analysis of multiple samples and is highly selective with respect to the specificity of the enzyme. The method is generally applicable to the search for proteases with known, putative or even desired specificity.
Abraham
C R
CR
Boston University School of Medicine, Arthritis Center, MA 02118-2394.
Bem Meir
A
A
Tempst
P
P
D000544:Alzheimer Disease / Q000378:metabolism*; D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000302:isolation & purification / Q000378:metabolism*; D001921:Brain / Q000378:metabolism; D002855:Chromatography, Thin Layer; D006801:Humans; D008722:Methods; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D012680:Sensitivity and Specificity
D016229:Amyloid beta-Peptides