Navegando por Palavras-chave "fluorescence resonance energy transfer (FRET) peptides"

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    Characterization of angiotensin I-converting enzyme N-domain selectivity using positional-scanning combinatorial libraries of fluorescence resonance energy transfer peptides
    (Walter de Gruyter & Co, 2012-12-01) Bersanetti, Patricia A. [UNIFESP]; Sabatini, Regiane A. [UNIFESP]; Matos, Beatriz S. [UNIFESP]; Douglas, Ross G.; Nchinda, Aloysius; Juliano, Maria A. [UNIFESP]; Pesquero, Joao Bosco [UNIFESP]; Sturrock, Edward D.; Carmona, Adriana K. [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); UCT Fac Hlth Sci; Univ Cape Town
    Somatic angiotensin I-converting enzyme (ACE) has two homologous active sites (N and C domains) that show differences in various biochemical properties. in a previous study, we described the use of positional-scanning synthetic combinatorial (PS-SC) libraries of fluorescence resonance energy transfer (FRET) peptides to define the ACE C-domain versus N-domain substrate specificity and developed selective substrates for the C-domain (Bersanetti et al., 2004). in the present work, we used the results from the PS-SC libraries to define the N-domain preferences and designed selective substrates for this domain. the peptide Abz-GDDVAK(Dnp)-OH presented the most favorable residues for N-domain selectivity in the P-3 to P-1' positions. the fluorogenic analog Abz-DVAK(Dnp)-OH (Abz=ortho-aminobenzoic acid; Dnp=2,4-dinitrophenyl) showed the highest selectivity for ACE N-domain (k(cat)/K-m = 1.76 mu M-1.s(-1)). Systematic reduction of the peptide length resulted in a tripeptide that was preferentially hydrolyzed by the C-domain. the binding of Abz-DVAK(Dnp)-OH to the active site of ACE N-domain was examined using a combination of conformational analysis and molecular docking. Our results indicated that the binding energies for the N-domain-substrate complexes were lower than those for the C-domain-substrate, suggesting that the former complexes are more stable.
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    Determination of angiotensin I-converting enzyme activity in cell culture using fluorescence resonance energy transfer peptides
    (Elsevier B.V., 2007-04-15) Sabatini, R. A.; Bersanetti, P. A.; Farias, S. L.; Juliano, L.; Juliano, M. A.; Casarini, D. E.; Carmona, A. K.; Paiva, A. C. M.; Pesquero, J. B.; Universidade Federal de São Paulo (UNIFESP)
    An assay using fluorescence resonance energy transfer peptides was developed to assess angiotensin I-converting enzyme (ACE) activity directly on the membrane of transfected Chinese hamster ovary cells (CHO) stably expressing the full-length somatic form of the enzyme. the advantage of the new method is the possibility of using selective substrates for the two active sites of the enzyme, namely Abz-FRK(Dnp)P-OH for somatic ACE, Abz-SDK(Dnp)P-OH for the N domain, and Abz-LFK(Dnp)-OH for the C domain. Hydrolysis of a peptide bond between the donor/acceptor pair (Abz/Dnp) generates detectable fluorescence, allowing quantitative measurement of the enzymatic activity. the kinetic parameter K-m for the hydrolysis of the three substrates by ACE in this system was also determined and the values are comparable to those obtained using the purified enzyme in solution. the specificity of the activity was demonstrated by the complete inhibition of the hydrolysis by the ACE inhibitor lisinopril. Therefore, the results presented in this work show for the first time that determination of ACE activity directly on the surface of intact CHO cells is feasible and that the method is reliable and sensitive. in conclusion. we describe a methodology that may represent a new tool for the assessment of ACE activity which will open the possibility to study protein interactions in cells in culture. (c) 2007 Elsevier Inc. All rights reserved.