SPECIFIC FLUOROGENIC SUBSTRATES FOR NEPRILYSIN (NEUTRAL ENDOPEPTIDASE, EC-3.4.24.11) WHICH ARE HIGHLY RESISTANT TO SERINEPROTEASE AND METALLOPROTEASE

Two intramolecularly quenched fluorogenic peptides containing o-aminob enzoyl (Abz) and ethylenediamine 2,4-dinitrophenyl (EDDnp) groups at a mino- and carboxyl-terminal amino acid residues, Abz-DArg-Arg-Leu-EDDn p (Abz-DRRL-EDDnp) and Abz-DArg-Arg-Phe-EDDnp (Abz-DRRF-EDDnp), were s electively hydrolyzed by neutral endopeptidase (NEP, enkephalinase, ne prilysin, EC 3.4.24.11) at the Arg-Leu and Arg-Phe bonds, respectively . The kinetic parameters for the NEP-catalyzed hydrolysis of Abz-DRRL- EDDnp and Abz-DRRF-EDDnp were K-m = 2.8 mu M, k(cat) = 5.3 min(-1), k( cat)/K-m = 2 min(-1) mu M-1 and K-m = 5.0 mu M, k(cat) = 7.0 min(-1), k(cat)/K-m = 1.4 min(-1) mu M-1, respectively. The high specificity of these substrates was demonstrated by their resistance to hydrolysis b y metalloproteases [thermolysin (EC 3.4.24.2), angiotensin-converting enzyme (ACE; EC 3.4.24.15)], serineproteases [trypsin (EC 3.4.21.4), a lpha-chymotrypsin (EC 3.4.21.1)] and proteases present in tissue homog enates from kidney, lung, brain and testis. The blocked amino- and car boxyl-terminal amino acids protected these substrates against the acti on of aminopeptidases, carboxypeptidases and ACE. Furthermore, DR amin o acids ensured total protection of Abz-DRRL-EDDnp and Abz-DRRF-EDDnp against the action of thermolysin and trypsin. Leu-EDDnp and Phe-EDDnp were resistant to hydrolysis by alpha-chymotrypsin. The high specifit y of these substrates suggests their use for specific NEP assays in cr ude enzyme preparations.