Specific fluorogenic substrates for neprilysin (neutral endopeptidase, EC 3.4.24.11) which are highly resistant to serine- and metalloproteases

Abstract

Two intramolecularly quenched fluorogenic peptides containing oaminobenzoyl (Abz) and ethylenediamine 2,4-dinitrophenyl (EDDnp) groups at amino-and carboxyl-terminal amino acid residues, AbzDArg-Arg-Leu-EDDnp (Abz-DRRL-EDDnp) and Abz-DArg-Arg-PheEDDnp (Abz-DRRF-EDDnp), were selectively hydrolyzed by neutral endopeptidase (NEP, enkephalinase, neprilysin, 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-DRRFEDDnp were K m = 2.8 µM, k cat = 5.3 min -1 , k cat /K m = 2 min -1 µM -1 and K m = 5.0 µM, k cat = 7.0 min -1 , k cat /K m = 1.4 min -1 µM -1 , respectively. The high specificity of these substrates was demonstrated by their resistance to hydrolysis by metalloproteases [thermolysin (EC 3.4.24.2), angiotensin-converting enzyme (ACE; EC 3.4.24.15)], serineproteases [trypsin (EC 3.4.21.4), α-chymotrypsin (EC 3.4.21.1)] and proteases present in tissue homogenates from kidney, lung, brain and testis. The blocked amino-and carboxyl-terminal amino acids protected these substrates against the action of aminopeptidases, carboxypeptidases and ACE. Furthermore, DR amino 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 α-chymotrypsin. The high specifity of these substrates suggests their use for specific NEP assays in crude enzyme preparations.

Expression of Metallopeptidases and Kinin Receptors in Swine Oropharyngeal Tissues: Effects of Angiotensin I-Converting Enzyme Inhibition and Inflammation

“…The ACEi enalaprilat (Vasotec I.V., 1.25 mg/ml) and enalapril (Vasotec) were from Merck Frosst Canada (Kirkland, QC, Canada); RNAlater was from Ambion (Austin, TX); bradykinin and des-Arg 9 -BK were acquired from Peninsula Laboratories (Belmont, CA); LPS extracted from Escherichia coli serotype O111:B4, L-arginine, and o-phthalaldehyde were from Sigma-Aldrich (Oakville, ON, Canada); and TRIzol reagent was purchased from Invitrogen (Burlington, ON, Canada). The internally quenched fluorescent substrates (Abz)RGL(EDDnp) for NEP (Medeiros et al, 1997), K(Dnp)P-PGK(Abz) for mAPP , and (Abz)YRK(Dnp)P for ACE (Araujo et al, 2000) were gifts from Prof. A. Carmona (Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de Sã o Paulo, Sã o Paulo, Brazil). PCR primers were synthesized by Biocorp Inc. (Montreal, QC, Canada).…”

Angiotensin-(1–7) Induces Peripheral Antinociception through Mas Receptor Activation in an Opioid-Independent Pathway

“…It is formed by an angiotensin-converting enzyme-independent pathway and may have similar or opposite actions to those of Ang II [1] . The physiological role of Ang-(1-7) was firmly established by two recent discoveries: (1) identification of the ability of angiotensin to convert enzyme-2, an enzyme that generates Ang-(1-7) from Ang I or Ang II [2][3][4][5] , and (2) characterization of the G proteincoupled receptor Mas as a receptor that is associated with several actions of Ang-(1-7) [6] .…”

The use of Fluorescence Resonance Energy Transfer (FRET) peptidesfor measurement of clinically important proteolytic enzymes

“…Several methods have been described for assaying NEP activity. However, each of these techniques has its own limitations, like being overly laborious and requiring two steps, not selective or not sufficiently sensitivity (Florentin et al 1984, Malfroy and Burnier 1987, Goudreau et al 1994, Medeiros et al 1997.…”

“…To overcome this limitation, we explored NEP endopeptidase activity and obtained sensitive and selective NEP substrates. Previously, a work from our group (Medeiros et al 1997) described the FRET peptide Abz-rRL-EDDnp (r = DArg) as very selective for NEP, being resistant to ACE and other peptidases activity. However this compound had a low k cat /K m (32 mM -1 .s -1 ) mainly due to the low k cat value (0.088 s -1 ), restricting its use for NEP determinations on continuous basis mainly when the enzyme concentration is low.…”

“…Peptides up to 20 residues can provide significant increases in florescence (de Souza et al 2000), allowing the measurement of the enzymatic activity on continuous base. The FRET peptides introduced by Chagas et al (1991) was a breakthrough in the study of proteases' specificity, and the synthesis of different Abzpeptidyl-EDDnp sequences provided the opportunity for us to study the activity of various endopeptidases such as human renin (Oliveira et al 1992), kallikreins (Chagas et al 1995, Del Nery et al 1995, Portaro et al 1997, Angelo et al 2006), cathepsin G (Réhault et al 1999, Korkmaz et al 2008), cathepsin D (Pimenta et al 2000, pro hormone convertase (Johanning et al 1998), lysosomal cathepsins (Portaro et al 2000, Alves et al 2003, Puzer et al 2004) and neprilysin (Medeiros et al 1997).…”