Selective neurotensin-derived internally quenched fluorogenic substrates for neurolysin (EC 3.4.24.16): Comparison with thimet oligopeptidase (EC 3.4.24.15) and neprilysin (EC 3.4.24.11)

Selective neurotensin-derived internally quenched fluorogenic substrates for neurolysin (EC 3.4.24.16): Comparison with thimet oligopeptidase (EC 3.4.24.15) and neprilysin (EC 3.4.24.11)

Author Oliveira, V Google Scholar
Campos, M. Google Scholar
Hemerly, J. P. Google Scholar
Ferro, E. S. Google Scholar
Camargo, ACM Google Scholar
Juliano, M. A. Google Scholar
Juliano, L. Google Scholar
Institution Universidade Federal de São Paulo (UNIFESP)
Universidade de São Paulo (USP)
Inst Butantan
Abstract Internally quenched fluorescent peptides derived from neurotensin (pELYENKPRRPYIL) sequence were synthesized and assayed as substrates for neurolysin (EC 3.4.24.16), thimet oligopeptidase (EC 3.4.24.15 or TOP), and neprilysin (EC 3.4.24.11 or NEP), Abz-LYENKPRRPYILQ-EDDnp (where EDDnp is N-(2,4-dinitrophenyl)ethylenediamine and Abz is ortho-aminobenzoic acid) was derived from neurotensin by the introduction of Q-EDDnp at the C-terminal end of peptide and by the substitution of the pyroglutamic (pE) residue at N-terminus for Abz and a series of shorter peptides was obtained by deletion of amino acids residues from C-terminal, N-terminal, or both sides. Neurolysin and TOP hydrolyzed the substrates at P-Y or Y-I or R-R bonds: depending on the sequence and size of the peptides, while NEP cleaved P-Y or Y-I bonds according to its S'(1) specificity. One of these substrates, Abz-NKPRRPQ-EDDnp was a specific and sensitive substrate for neurolysin (k(cat) = 7.0 s(-l), K(m) = 1.19 muM and k(cat)/K(m) = 5882 mM-(1) . s-(1)), while it was completely resistant to NEP and poorly hydrolyzed by TOP and also by prolyl oligopeptidase (EC 3.4.21.26), Neurolysin concentrations as low as 1 pM were detected using this substrate under our conditions and its analogue Abz-NKPRAPQ-EDDnp was hydrolyzed by neurolysin with k(cat) = 14.03 s(-1), K(m) = 0.82 muM, and k(cat)/K(m) = 17,110 mM(-1) . s(-1), being the best substrate so far described for this peptidase. (C) 2001 Academic Press.
Keywords fluorogenic substrates
neurotensin
neurolysin
thimet oligopeptidase
neprilysin
prolyl oligopeptidase
Language English
Date 2001-05-15
Published in Analytical Biochemistry. San Diego: Academic Press Inc Elsevier Science, v. 292, n. 2, p. 257-265, 2001.
ISSN 0003-2697 (Sherpa/Romeo, impact factor)
Publisher Elsevier B.V.
Extent 257-265
Origin http://dx.doi.org/10.1006/abio.2001.5083
Access rights Closed access
Type Article
Web of Science ID WOS:000168902300012
URI http://repositorio.unifesp.br/handle/11600/26555

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