Kininogen-derived fluorogenic substrates for investigating the vasoactive properties of rat tissue kallikreins - Identification of a T-kinin-releasing rat kallikrein

Kininogen-derived fluorogenic substrates for investigating the vasoactive properties of rat tissue kallikreins - Identification of a T-kinin-releasing rat kallikrein

Author ElMoujahed, A. Google Scholar
BrillardBourdet, M. Google Scholar
Juliano, M. A. Google Scholar
Moreau, T. Google Scholar
Chagas, JR Google Scholar
Gutman, N. Google Scholar
Prado, E. S. Google Scholar
Gauthier, F. Google Scholar
Institution UNIV TOURS
Universidade Federal de São Paulo (UNIFESP)
Abstract Peptide substrates with intramolecularly quenched fluorescence that reproduce the rat kininogen sequences at both ends of the bradykinin moiety were synthesized and used to investigate the kinin-releasing properties of five rat tissue kallikreins (rK1, rK2, rK7, rK9, rK10). Substrates derived from rat H- and L-kininogen were cleaved best by rK1, especially that including the N-terminal insertion site of bradykinin; Abz-TSVIRRPQ-EDDnp(Abz = O-aminobenzoyl, EDDnp = ethylenediamine 2,4-dinitrophenyl), which was cleaved at the R-R bond with a k(cat)/k(m) of 12 400 mM(-1) s(-1). Replacement of the P2' residue Pro by Val in Abz-TSVIRRPQ-EDDnp gave a far less specific substrate that was rapidly hydrolysed by all five rat kallikreins and human kallikrein hK1. Peptidyl-N-methyl coumarylamide substrates, which lack prime residues, also had low specificities. The importance of the P2' residue for rK1 specificity was further demonstrated using a human-kininogen-derived substrate that included the N-terminal insertion site of bradykinin (Abz-LMKRP-EDDnp). This was cleaved at the M-K bond by hK1 (kallidin-releasing site), but at the K-R bond (bradykinin-releasing site) by rK1. Competition experiments, with Abz-TSVIRRPQ-EDDnp. which is resistant to most kallikreins. and Abz-TSVIRRVQ-EDDnp. a general kallikrein substrate. demonstrated that the former competitively inhibited hydrolysis by rK9 and hK1, with K-i values similar to the K-m, values for the substrate. Thus Pro in P2' does not prevent the peptide binding to the enzyme active site, but impairs cleavage of the scissile bond. The T-kininogen-derived substrate with the T-kinin C-terminal sequence (Abz-FRLVR-EDDnp) was cleaved by rK10 (k(cat)/K-m = 2310 mM(-1) s(-1)) and less rapidly by rK1, rK7 and hK1, at the R-L bond, while that corresponding to the N-terminal (Abz-ALDMMISRP-EDDnp) of T-kinin was resistant to all five kallikreins used, suggesting that none has T-kininogenase activity. But this substrate was hydrolysed by a semi-purified sample of submandibular gland extract. Another kallikrein, identified as kallikrein rK3, was isolated from this fraction and shown to hydrolyze Abz-ALDMMISRP-EDDnp: rK3 also specifically released T-kinin from purified T1/T2-kininogen after HPLC fractionation. Injection of purified rK3 and of Abz-ALDMMISRP-EDDnp-cleaving fractions into the circulation of anesthesized rats caused transient falls in blood pressure, as did purified rK1 but none of the other purified rat or human kallikreins. This effect occurred via activation of the kinin system since it was blocked by Hoe140, a kinin receptor antagonist.
Keywords tissue kallikrein
kininogen
kinin
Language English
Date 1997-07-15
Published in European Journal Of Biochemistry. New York: Springer Verlag, v. 247, n. 2, p. 652-658, 1997.
ISSN 0014-2956 (Sherpa/Romeo, impact factor)
Publisher Springer
Extent 652-658
Origin https://doi.org/10.1111/j.1432-1033.1997.00652.x
Access rights Open access Open Access
Type Article
Web of Science ID WOS:A1997XM61300025
URI http://repositorio.unifesp.br/11600/43582

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