The kininogenase activities of mouse (mK1), rat (rK1) and human (hK1) tissue kallikreins were assayed with the bradykinin-containing synthetic peptides Abz-MTEMARRPPGFSPFRSVTVQNH2 (where Abz stands for o-aminobenzoyl) and Abz-MTSVIRRPPGFSPFRAPRV-NH2, which correspond to fragments Met374-Gln393 and Met375-Val393 of mouse and rat LMWKs (low-molecular-mass kininogens) with the addition of Abz. Bradykinin was released from these peptides by the mK1- and rK1-mediated hydrolysis of Arg–Arg and Arg–Ser (or Arg–Ala) peptide bonds. However, owing to preferential hydrolysis of Phe–Arg compared with the Arg–Ala bond in the peptide derived from rat LMWK, hK1 released bradykinin only from the mouse LMWK fragment and preferentially released des-[Arg9]bradykinin from the rat LMWK fragment (Abz-MTSVIRRPPGFSPFRAPRV-NH2). The formation of these hydrolysis products was examined in more detail by determining the kinetic parameters for the hydrolysis of synthetic, internally quenched fluorescent peptides containing six N- or C-terminal amino acids of bradykinin added to the five downstream or upstream residues of mouse and rat kininogens respectively. One of these peptides, Abz-GFSPFRAPRVQ-EDDnp (where EDDnp stands for ethylenediamine 2,4-dinitrophenyl), was preferentially hydrolysed at the Phe–Arg bond, confirming the potential des-[Arg9]bradykinin-releasing activity of hK1 on rat kininogen. The proline residue that is two residues upstream of bradykinin in rat kininogen is, in part, responsible for this pattern of hydrolysis, since the peptide Abz-GFSPFRASRVQ-EDDnp was preferentially cleaved at the Arg–Ala bond by hK1. Since this peptidase accepts the arginine or phenylalanine residue at its S1 subsite, this preference seems to be determined by the prime site of the substrates. These findings also suggested that the effects observed in rats overexpressing hK1 should consider the activation of B1 receptors by des-[Arg9]bradykinin. For further comparison, two short internally quenched fluorescent peptides that bind to hK1 with affinity in the nM range and some inhibitors described previously for hK1 were also assayed with mK1 and rK1.
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Research Article|
June 15 2004
Differences in substrate and inhibitor sequence specificity of human, mouse and rat tissue kallikreins
Sandro E. FOGAÇA;
Sandro E. FOGAÇA
*Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Tres de Maio 100, São Paulo 04044-20, Brazil
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Robson L. MELO;
Robson L. MELO
*Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Tres de Maio 100, São Paulo 04044-20, Brazil
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Daniel C. PIMENTA;
Daniel C. PIMENTA
*Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Tres de Maio 100, São Paulo 04044-20, Brazil
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Kazuo HOSOI;
Kazuo HOSOI
†Department of Physiology and Oral Physiology, Tokushima University School of Dentistry, 3-Kuramoto-cho, Tokushima-shi, Tokushima 770-8504, Japan
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Luiz JULIANO;
Luiz JULIANO
*Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Tres de Maio 100, São Paulo 04044-20, Brazil
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Maria A. JULIANO
Maria A. JULIANO
1
*Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Tres de Maio 100, São Paulo 04044-20, Brazil
1To whom correspondence should be addressed (e-mail juliano.biof@epm.br).
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Publisher: Portland Press Ltd
Received:
July 11 2003
Revision Received:
February 23 2004
Accepted:
March 25 2004
Accepted Manuscript online:
March 25 2004
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2004
2004
Biochem J (2004) 380 (3): 775–781.
Article history
Received:
July 11 2003
Revision Received:
February 23 2004
Accepted:
March 25 2004
Accepted Manuscript online:
March 25 2004
Citation
Sandro E. FOGAÇA, Robson L. MELO, Daniel C. PIMENTA, Kazuo HOSOI, Luiz JULIANO, Maria A. JULIANO; Differences in substrate and inhibitor sequence specificity of human, mouse and rat tissue kallikreins. Biochem J 15 June 2004; 380 (3): 775–781. doi: https://doi.org/10.1042/bj20031047
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