A highly sensitive peptide substrate for detecting two A beta-degrading enzymes: Neprilysin and insulin-degrading enzyme
Resource
JOURNAL OF NEUROSCIENCE METHODS, 190(1), 57-62
Journal
JOURNAL OF NEUROSCIENCE METHODS
Journal Volume
190
Journal Issue
1
Pages
57-62
Date Issued
2010
Date
2010
Author(s)
Abstract
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.
SDGs
Other Subjects
amyloid beta protein; dipeptidyl carboxypeptidase; endothelin converting enzyme; gelatinase B; insulinase; membrane metalloendopeptidase; plasmin; proteinase; somatostatin; stromelysin; amyloid beta protein; insulinase; membrane metalloendopeptidase; peptide; article; controlled study; enzyme activity; enzyme assay; enzyme kinetics; enzyme specificity; enzyme substrate; fluorescence analysis; human; human cell; priority journal; protein degradation; sensitivity analysis; amino acid sequence; chemistry; fluorescence; fluorometry; high performance liquid chromatography; kinetics; mass spectrometry; metabolism; procedures; Western blotting; Amino Acid Sequence; Amyloid beta-Protein; Blotting, Western; Chromatography, High Pressure Liquid; Fluorescence; Fluorometry; Humans; Insulysin; Kinetics; Neprilysin; Peptides; Somatostatin; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Substrate Specificity; Amino Acid Sequence; Amyloid beta-Peptides; Blotting, Western; Chromatography, High Pressure Liquid; Fluorescence; Fluorometry; Humans; Insulysin; Kinetics; Neprilysin; Peptides; Somatostatin; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Substrate Specificity
Type
journal article
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