title: A new fluorometric assay for neutral endopeptidase (EC 3.4.24.11).
abstract: An intramolecularly quenched fluorogenic peptide structurally related to Leu-enkephalin, Abz-GGDFLRRV-EDDnp, was selectively hydrolyzed at the R-V bond by neutral endopeptidase (NEP, enkephalinase, neprilysin, EC 3.4.24.11) with kinetic parameters (Km = 3 microM, kcat = 127/min and kcat/Km = 42/min microM) similar to those of Leu-enkephalin. The specificity of the assay for NEP was demonstrated by incubating Abz-GGDFLRRV-EDDnp with a kidney homogenate and with crude membrane preparations of brain and lung. For all three homogenates the complementary fragment Abz-GGDFLRR and V-EDDnp accounted for more than 95% of the products which are totally inhibited by 1 microM thiorphan, a highly specific NEP inhibitor. A continuous fluorometric assay for only 15 min was sufficient to quantify the NEP activity with a minimum sensitivity of 5 ng of purified NEP or the equivalent enzymatic activity in crude tissue preparations.
authors.affiliation: Departamento de Fisiologia e Farmacologia, Universidade Federal do Ceará, Fortaleza, CE, Brasil.
authors.forename: K M
authors.initials: KM
authors.lastname: Carvalho
authors.forename: G
authors.initials: G
authors.lastname: Boileau
authors.forename: M S
authors.initials: MS
authors.lastname: França
authors.forename: M A
authors.initials: MA
authors.lastname: Medeiros
authors.forename: A C
authors.initials: AC
authors.lastname: Camargo
authors.forename: L
authors.initials: L
authors.lastname: Juliano
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid; D005470:Fluorometry / Q000379:methods*; D015260:Neprilysin / Q000032:analysis*; D009479:Neuropeptides / Q000032:analysis*; D051381:Rats
chemical_list: D009479:Neuropeptides; D015260:Neprilysin