A highly selective assay for neutral endopeptidase based on the cleavage of a fluorogenic substrate related to Leu-enkephalin.
An intramolecularly quenched fluorogenic peptide structurally related to Leu-enkephalin, containing o-aminobenzoyl (Abz) and ethylenediamine 2,4-dinitrophenyl (EDDnp) groups at amino- and carboxyl-terminal amino acid residues, Abz-Gly-Gly-D-Phe-Leu-Arg-Arg-Val-EDDnp (Abz-GGDFLRRV-EDDnp), was selectively hydrolyzed at the Arg-Val bond by neutral endopeptidase (NEP, enkephalinase, neprilysin, EC 3.4.24.11) with kinetic parameters (Km = 3 microM, kcat = 127 min-1 and kcatsolidusKm = 42 min-1 microM-1) similar to those of the Leu-enkephalin. The specificity of the NEP assay was demonstrated by incubating Abz-GGDFLRRV-EDDnp with a kidney homogenate or with crude membrane preparations of brain and lung: more than 95% of all products released were the complementary fragments Abz-GGDFLRR and V-EDDnp which were totally inhibited by 1 microM thiorphan, a highly specific NEP inhibitor. The blocked amino- and carboxyl-terminal amino acids protected this substrate against the action of aminopeptidases as well as of carboxypeptidases. Furthermore, D-Phe amino acid also ensured a very good protection of Abz-GGDFLRRV-EDDnp against the action of other tissue endopeptidases distinct from NEP. A continuous fluorometric assay for only 5 min was sufficient to quantify the NEP activity with a minimum sensitivity of 5 ng of purified enzyme or the equivalent enzymatic activity in crude tissue preparations. Therefore, amounts as little as 0.5 ng of enzyme could be quantified employing longer times of incubation.
Carvalho
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Departamento de Fisiologia e Farmacologia, Universidade Federal do Ceará, Fortaleza, 60430-270, Brazil.
Boileau
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Camargo
A C
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Juliano
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D000595:Amino Acid Sequence; D000818:Animals; D004743:Enkephalin, Leucine / Q000031:analogs & derivatives / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D015260:Neprilysin / Q000032:analysis* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000706:statistics & numerical data; D013379:Substrate Specificity
D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D004743:Enkephalin, Leucine; D015260:Neprilysin