A highly sensitive peptide substrate for detecting two Aß-degrading enzymes: neprilysin and insulin-degrading enzyme.
Neprilysin has been singled out as the most promising candidate for use in the degradation of Abeta as a therapy for Alzheimer's disease. In this study, a quenched fluorogenic peptide substrate containing the first seven residues of the Abeta peptide plus a C-terminal Cysteine residue was synthesized to detect neprilysin activity. A fluorophore was attached to the C-terminal Cysteine and its fluorescence was quenched by a quencher linked to the N-terminus of the peptide. When this peptide substrate was degraded by an endopeptidase, fluorescence was produced and proved to be a sensitive detection system for endopeptidase activity. Our results showed that this assay system was extremely sensitive to neprilysin and insulin-degrading enzyme, but insensitive, or much less sensitive, to other Abeta-degrading enzymes. As low as 0.1 nM of neprilysin and 0.2 nM of insulin-degrading enzyme can be detected.
Chen
Po-Ting
PT
Institute of Biochemical Sciences, National Taiwan University, Taipei 10617, Taiwan.
Liao
Tai-Yan
TY
Hu
Chaur-Jong
CJ
Wu
Shu-Ting
ST
Wang
Steven S-S
SS
Chen
Rita P-Y
RP
D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000378:metabolism*; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D005453:Fluorescence; D005470:Fluorometry / Q000379:methods*; D006801:Humans; D007339:Insulysin / Q000378:metabolism*; D007700:Kinetics; D015260:Neprilysin / Q000378:metabolism*; D010455:Peptides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D013004:Somatostatin / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
D016229:Amyloid beta-Peptides; D010455:Peptides; D013004:Somatostatin; D015260:Neprilysin; D007339:Insulysin