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A new fluorometric assay for neutral endopeptidase (EC 3.4.24.11).

Karine Maria Martins Bezerra Carvalho, Guy Boileau, Marcelo Santucci França, Marco Alberto Medeiros, Antônio Carlos Martins de Camargo, Luiz Juliano

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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.

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What this paper is about

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.

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Available 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.

Key concepts: Enkephalinase, Thiorphan, Neprilysin, Chemistry, Enzyme kinetics, Endopeptidase, Chromatography, Enzyme

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