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Title: Selective neurotensin-derived internally quenched fluorogenic substrates for neurolysin (EC Comparison with thimet oligopeptidase (EC and neprilysin (EC
Authors: Oliveira, V
Campos, M.
Hemerly, J. P.
Ferro, E. S.
Camargo, ACM
Juliano, M. A.
Juliano, L.
Universidade Federal de São Paulo (UNIFESP)
Universidade de São Paulo (USP)
Inst Butantan
Keywords: fluorogenic substrates
thimet oligopeptidase
prolyl oligopeptidase
Issue Date: 15-May-2001
Publisher: Elsevier B.V.
Citation: Analytical Biochemistry. San Diego: Academic Press Inc Elsevier Science, v. 292, n. 2, p. 257-265, 2001.
Abstract: Internally quenched fluorescent peptides derived from neurotensin (pELYENKPRRPYIL) sequence were synthesized and assayed as substrates for neurolysin (EC, thimet oligopeptidase (EC or TOP), and neprilysin (EC 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, 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.
ISSN: 0003-2697
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