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Biotinylated fluorescent peptide substrates for the sensitive and specific determination of cathepsin D activity
Author(s) -
Baechle D.,
Cansier A.,
Fischer R.,
Brandenburg J.,
Burster T.,
Driessen C.,
Kalbacher H.
Publication year - 2005
Publication title -
journal of peptide science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 66
eISSN - 1099-1387
pISSN - 1075-2617
DOI - 10.1002/psc.607
Subject(s) - biotin , chemistry , peptide , cleavage (geology) , biotinylation , biochemistry , fluorophore , cathepsin d , streptavidin , protease , fluorescence , enzyme , biology , paleontology , physics , quantum mechanics , fracture (geology)
Cathepsin D (CatD) is a member of the mammalian aspartic protease family and is involved in cellular protein degradation and in several pathological processes. A sensitive and specific assay for the determination of CatD activity in biological samples was developed. The peptide amide substrates Amca‐EDKPILF↓FRLGK(biotin)‐CONH 2 (I), Amca‐EEKPIC(Acm)F↓FRLGK(biotin)‐CONH 2 (II) and Amca‐EEKPISF↓FRLGK(biotin)‐CONH 2 (III) contain a CatD cleavage site (F↓F) flanked by a N ‐terminal Amca‐fluorophore (7‐amino‐4‐methylcoumarin‐3‐acetic acid) and a C ‐terminal biotin moiety. Substrates II and III proved to be specific substrates containing only one cleavage site for CatD. After cleavage of the Phe‐Phe bond by CatD all biotin conjugated peptides were removed with streptavidin‐coated magnetic beads. The remaining fluorescent peptides in solution represent the amount of digested substrate. The versatility of this CatD digest and pull down assay was demonstrated by measuring the activity of CatD in different subcellular fractions of human EBV‐transformed B cells and human monocytes. The described method based on the designed CatD substrates represents a valuable tool for routine assays. Copyright © 2004 European Peptide Society and John Wiley & Sons, Ltd.