Navegando por Palavras-chave "fluorescence resonance energy transfer peptides"

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    Analysis of the Specificity and Biochemical Characterization of Metalloproteases Isolated from Eupenicillium javanicum Using Fluorescence Resonance Energy Transfer Peptides
    (Frontiers Media Sa, 2017) Hamin Neto, Youssef A. A.; de oliveira, Lilian C. G.[INIFESP]; de oliveira, Juliana R.[INIFESP]; Juliano, Maria A.[INIFESP]; Juliano, Luiz[INIFESP]; Arantes, Eliane C.; Cabral, Hamilton
    Enzymes have important features that may facilitate their application in industrial processes and have been used as alternatives to chemical catalysts. In particular, proteases can be isolated from microorganisms, which provide important sources of advantageous enzymes for industrial processes. For example, Eupenicillium javanicum is a filamentous fungus that has been shown to express industrially applicable enzymes and chemical components, such as antifungal compounds. The biotechnological potential of E. javanicum and proteases made us search a novel protease from this microorganism. The macromolecule was isolated, the main biochemical properties was evaluated, and the specificity of the protease subsites was determined. The protease was produced under solid-state bioprocess with wheat bran and isolated by two chromatography steps with yield of 27.5% and 12.4-fold purification. The molecular mass was estimated at 30 kDa. The N-terminal sequence of the first 20 amino acid residues was AVGAGYNASVALALEKALNN. The enzyme presented higher proteolytic activity at pH 6.0 and 60 degrees C. The protease is stable at wide range of pH values and temperatures and in the presence of surfactants. The primed side of the catalytic site showed the highest catalytic efficiency of the enzyme isolated from E. javanicum. The S'(1) subsite is responsible for catalyzing the protease reaction with substrates with tyrosine in P'(1). These findings provide important insights into the biochemical characterization of a highly active protease from E. javanicum and may facilitate the development of industrial processes involving this protease.
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    Plasminogen hydrolysis by cathepsin S and identification of derived peptides as selective substrate for cathepsin V and cathepsin L inhibitor
    (Walter de Gruyter Gmbh, 2010-05-01) Coppini, Larissa Pereira [UNIFESP]; Barros, Nilana M. T. [UNIFESP]; Oliveira, Marcela [UNIFESP]; Hirata, Izaura Y. [UNIFESP]; Alves, Marcio F. M. [UNIFESP]; Paschoalin, Thaysa [UNIFESP]; Assis, Diego M. [UNIFESP]; Juliano, Maria A. [UNIFESP]; Puzer, Luciano; Broemme, Dieter; Carmona, Adriana K. [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Univ Fed Triangulo Mineiro; Univ British Columbia
    Plasminogen is a glycoprotein implicated in angiogenesis and fibrin clot degradation associated with the release of angiostatin and plasmin activation, respectively We have recently reported that cathepsin V. but not cathepsins L. B, and K. can release angiostatin-like fragments from plasminogen. Here, we extended the investigation to cathepsin S which has been implicated in angiogenesis and tumor cell proliferation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of plasminogen hydrolysis by cathepsin S revealed generation of two fragments (60 and 38 kDa). Amino-terminal sequencing indicated that cleavage occurs at the Leu469-Leu470 peptide bond. in contrast to cathepsin V, which possesses antiangiogenic activity, cathepsin S plasminogen cleavage products were not capable of inhibiting angiogenesis on endothelial cells. Moreover, we explored the different selectivities presented by cathepsins V and S towards plasminogen and synthesized fluorescence resonance energy transfer peptides encompassing the hydrolyzed peptide bonds by both enzymes. the peptide Abz-VLFEKKQ-EDDnp (Abz=ortho-aminobenzoic acid; EDDnp=N-[2,4-dinitrophenyl]ethylenediamine), hydrolyzed by cathepsin V at the Phe-Glu bond. is a selective substrate for the enzyme when compared with cathepsins B. L, and S, whereas Abz-VLFEKKVYLQ-EDDnp is an efficient cathepsin L inhibitor. the demonstrated importance of the S-3'-P-3' interaction indicates the significance of the extended subsites for enzyme specificity and affinity.