title: Purification and characterization of angiotensin converting enzyme 2 (ACE2) from murine model of mesangial cell in culture.
abstract: Angiotensin converting enzyme 2 (ACE2) is a component of the renin-angiotensin system (RAS) which converts Ang II, a potent vasoconstrictor peptide into Ang 1-7, a vasodilator peptide which may act as a negative feedback hormone to the actions of Ang II. The discovery of this enzyme added a new level of complexity to this system. The mesangial cells (MC) have multiple functions in glomerular physiology and pathophysiology and are able to express all components of the RAS. Despite of being localized in these cells, ACE2 has not yet been purified or characterized. In this study ACE2 from mice immortalized MC (IMC) was purified by ion-exchange chromatography. The purified enzyme was identified as a single band around 60-70 kDa on SDS-polyacrylamide gel and by Western blotting using a specific antibody. The optima pH and chloride concentrations were 7.5 and 200 mM, respectively. The N-terminal sequence was homologous with many species ACE2 N-terminal sequences as described in the literature. ACE2 purified from IMC was able to hydrolyze Ang II into Ang 1-7 and the K(m) value for Ang II was determined to be 2.87 ± 0.76 μM. In conclusion, we purified and localized, for the first time, ACE2 in MC, which was able to generate Ang 1-7 from Ang II. Ang 1-7 production associated to Ang II degradation by ACE2 may exert a protective effect in the renal hemodynamic.
authors.affiliation: Department of Medicine, Nephrology Division, Federal University of São Paulo, Brazil.
authors.forename: Danielle S
authors.initials: DS
authors.lastname: Aragão
authors.forename: Tatiana S
authors.initials: TS
authors.lastname: Cunha
authors.forename: Danielle Yuri
authors.initials: DY
authors.lastname: Arita
authors.forename: Maria Claudina C
authors.initials: MC
authors.lastname: Andrade
authors.forename: Adriana B
authors.initials: AB
authors.lastname: Fernandes
authors.forename: Ingrid K M
authors.initials: IK
authors.lastname: Watanabe
authors.forename: Renato A
authors.initials: RA
authors.lastname: Mortara
authors.forename: Dulce Elena
authors.initials: DE
authors.lastname: Casarini
mesh_terms: D000595:Amino Acid Sequence; D000803:Angiotensin I / Q000378:metabolism; D000804:Angiotensin II / Q000378:metabolism; D000085962:Angiotensin-Converting Enzyme 2; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D050527:Mesangial Cells / Q000201:enzymology*; D051379:Mice; D008856:Microscopy, Fluorescence; D010446:Peptide Fragments / Q000378:metabolism; D007703:Peptidyl-Dipeptidase A / Q000737:chemistry* / Q000302:isolation & purification*; D017422:Sequence Analysis, DNA
chemical_list: D010446:Peptide Fragments; D000804:Angiotensin II; D000803:Angiotensin I; D007703:Peptidyl-Dipeptidase A; C000705328:Ace2 protein, mouse; D000085962:Angiotensin-Converting Enzyme 2; C118790:angiotensin I (1-7)