Neprilysin (NEP) is a transmembrane metallopeptidase normally expressed by a variety of tissues. It is also known as neutral endopeptidase, and enkephalinase. NEP cleaves peptides at the N-terminal side of hydrophobic amino acid residues and is responsible for the degradation and inactivation of a variety of physiological substrates. NEP is a major extracellular amyloid beta-peptide degrading enzyme in the brain and targeting NEP is considered a potential therapeutic strategy for the prevention and treatment of Alzheimer's disease. NEP has also been implicated in the pathogenesis of hypertension, diabetes, and cancer.
The SensoLyte® 520 Neprilysin Assay Kit employs a novel internally quenched 5-FAM/QXL™ FRET substrate for the detection of neprilysin activity. The enzyme cleaves the FRET substrate into two separate fragments resulting in the release of 5-FAM fluorescence, which can be monitored at excitation /emission= 490/520 nm. The long wavelength fluorescence of 5-FAM is less interfered by the autofluorescence of components in biological samples and test compounds. The assay can detect as low as 0.78 ng/mL of active neprilysin.
Figure 1. Sensitivity of the assay has been tested using serial dilutions of recombinant human neprilysin. 5-FAM/QXL™ 520 FRET substrate was incubated with the indicated amount of enzyme and fluorescence was measured after 60 min (FlexStation 384II, Molecular Devices). This assay can detect as low as 0.78 ng/mL of active neprilysin.
Figure 2. Inhibition of neprilysin activity by Thiorphan as measured with SensoLyte® 520 Neprilysin Assay Kit.