Biotinylated fluorescent peptide substrates for the sensitive and specific determination of cathepsin D activity.
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 downward arrowFRLGK(biotin)-CONH2 (I), Amca-EEKPIC(Acm)F downward arrowFRLGK(biotin)-CONH2 (II) and Amca-EEKPISF downward arrowFRLGK(biotin)-CONH2 (III) contain a CatD cleavage site (F downward arrowF) 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.
Baechle
D
D
Medical and Natural Sciences Research Center, University of Tübingen, Germany.
Cansier
A
A
Fischer
R
R
Brandenburg
J
J
Burster
T
T
Driessen
C
C
Kalbacher
H
H
D000595:Amino Acid Sequence; D001402:B-Lymphocytes / Q000166:cytology / Q000201:enzymology; D019921:Biotinylation; D002402:Cathepsin D / Q000378:metabolism*; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009000:Monocytes / Q000166:cytology / Q000201:enzymology; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
D005456:Fluorescent Dyes; D010455:Peptides; D002402:Cathepsin D