Navegando por Palavras-chave "fluorescent peptide"

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    Cathepsin B carboxydipeptidase specificity analysis using internally quenched fluorescent peptides
    (Portland Press, 2002-11-15) Cezari, Maria Helena S [UNIFESP]; Puzer, Luciano [UNIFESP]; Juliano, Maria Aparecida [UNIFESP]; Carmona, Adriana Karaoglanovic [UNIFESP]; Juliano, Luiz [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    We have examined in detail the specificity of the subsites S-1, S-2, S-1' and S-2' for the carboxydipeptidase activity of cathepsin B by synthesizing and assaying four series of internally quenched fluorescent peptides based on the sequence Dnp-GFRFW-OH, where Drip (2,4-dinitrophenyl) is the quenching group of the fluorescence of the tryptophan residue. Each position, except the glycine, was substituted with 15 different naturally occurring amino acids. Based on the results we obtained, we also synthesized efficient and sensitive substrates that contained o-aminobenzoic acid and 3-Dnp-(2,3-diaminopropionic acid), or epsilon-amino-Dnp-Lys, as the fluorescence donor-receptor pair. the higher kinetic parameter values for the carboxydipeptidase compared with the endopeptidase activity of cathepsin B allowed an accurate analysis of its specificity. the subsite S-1 accepted preferentially basic amino acids for hydrolysis; however, substrates with phenylalanine and aliphatic side-chain-containing amino acids at P-1 had lower K-m values. Despite the presence of Glu(245) at S-2, this subsite presented clear preference for aromatic amino acid residues, and the substrate with a lysine residue at P-2 was hydrolysed better than that containing an arginine residue. S-1' is essentially a hydrophobic subsite, and S-2' has particular preference for phenylalanine or tryptophan residues.
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    Differences in substrate and inhibitor sequence specificity of human, mouse and rat tissue kallikreins
    (Portland Press, 2004-06-15) Fogaça, Sandro E. [UNIFESP]; Melo, Robson L. [UNIFESP]; Pimenta, Daniel C. [UNIFESP]; Hosoi, Kazuo; Juliano, Luiz [UNIFESP]; Juliano, Maria Aparecida [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Univ Tokushima
    The kininogenase activities of mouse (mK1), rat (rK1) and human (hK1) tissue kallikrems were assayed with the bradykinin-containing synthetic peptides NH2-MTEMARRPPGFSPFRSVTVQ-NH2 (where Abz stands for o-aminobenzoyl) and Abz-MTS-VIRRPPGFSPFRAPRV-NH2, which correspond to fragments Met(374)-Gln(393) and Met(375)-Val(393) 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-[Arg(9)]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-[Arg(9)]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 S-1 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-[Arg(9)] 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.