TAILIEUCHUNG - Báo cáo khoa học: Combined use of selective inhibitors and fluorogenic substrates to study the specificity of somatic wild-type angiotensin-converting enzyme

Somatic angiotensin-converting enzyme (ACE) contains two homologous domains, each bearing a functional active site. Studies on the selectivity of these ACE domains towards either substrates or inhibitors have mostly relied on the use of mutants or isolated domains of ACE. | iFEBS Journal Combined use of selective inhibitors and fluorogenic substrates to study the specificity of somatic wild-type angiotensin-converting enzyme Nicolas D. Jullien1 Philippe Cuniasse1 Dimitris Georgiadis2 Athanasios Yiotakis2 and Vincent Dive1 1 CEA Departement d lngenerie et d Etudes des Proteines Gif Yvette France 2 Department of Chemistry Laboratory of Organic Chemistry University of Athens Greece Keywords active site angiotensin-converting enzyme ACE fluorogenic substrates phosphinic inhibitors Correspondence V. Dive CEA Departement d lngenerie et d Etudes des Proteines 91191 Gif Yvette Cedex France Fax 33 169089071 Tel 33 169083585 E-mail Received 19 December 2005 revised 16 February 2006 accepted 21 February 2006 doi Somatic angiotensin-converting enzyme ACE contains two homologous domains each bearing a functional active site. Studies on the selectivity of these ACE domains towards either substrates or inhibitors have mostly relied on the use of mutants or isolated domains of ACE. To determine directly the selectivity properties of each ACE domain working with wildtype enzyme we developed an approach based on the combined use of N-domain-selective and C-domain-selective ACE inhibitors and fluorogenic substrates. With this approach marked differences in substrate selectivity were revealed between rat mouse and human somatic ACE. In particular the fluorogenic substrate Mca-Ala-Ser-Asp-Lys-DpaOH was shown to be a strict N-domain-selective substrate of mouse ACE whereas with rat ACE it displayed marked C-domain selectivity. Similar differences in selectivity between these ACE species were also observed with a new fluorogenic substrate of ACE Mca-Arg-Pro-Pro-Gly-Phe-Ser-Pro-DpaOH. In support of these results changes in amino-acid composition in the binding site of these three ACE species were pinpointed. Together these data demonstrate that the substrate selectivity of the N-domain and C-domain depends

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