title: N-domain angiotensin-I converting enzyme is expressed in immortalized mesangial, proximal tubule and collecting duct cells.
abstract: Somatic ACE (sACE) is found in glomerulus, proximal tubule and excreted in urine. We hypothesized that N-domain ACE can also be found at these sites. ACE profile was analyzed in mesangial (IMC), proximal (LLC-PK1), distal tubule (MDCK) and collecting duct (IMCD) cells. Cell lysate and culture medium were submitted to gel filtration chromatography, which separated two peaks with ACE activity from cells and medium, except from distal tubule. The first had a high molecular weight and the second, a lower one (65 kDa; N-domain ACE). We focused on N-domain ACE purification and characterization from LLC-PK1. Total LLC-PK1 N-domain ACE purification was achieved by ion-exchange chromatography, which presented only one peak with ACE activity, denominated ACE(int2A). ACE(int2A) activity was influenced by pH, NaCl and temperature. The purified enzyme was inhibited by Captopril and hydrolyzed AngI, Ang1-7 and AcSDKP. Its ability to hydrolyze AcSDKP characterized it as an N-domain ACE. ACE(int2A) also presented high amino acid sequence homology with the N-terminal part of sACE from mouse, rat, human and rabbit. The presence of secreted and intracellular N-domain ACE and sACE in IMC, LLC-PK1 and IMCD cells confirmed our studies along the nephron. We identified, purified and characterized N-domain ACE from LLC-PK1.
authors.affiliation: Departamento de Medicina, Disciplina de Nefrologia, Universidade Federal de São Paulo (UNIFESP), Rua Botucatu, 740, São Paulo, Brazil.
authors.forename: Pamella Huey
authors.initials: PH
authors.lastname: Mei Wang
authors.affiliation: Departamento de Medicina, Disciplina de Nefrologia, Universidade Federal de São Paulo (UNIFESP), Rua Botucatu, 740, São Paulo, Brazil. Electronic address: claudinandrade@gmail.com.
authors.forename: Maria Claudina
authors.initials: MC
authors.lastname: Andrade
authors.affiliation: Departamento de Medicina, Disciplina de Nefrologia, Universidade Federal de São Paulo (UNIFESP), Rua Botucatu, 740, São Paulo, Brazil.
authors.forename: Beata Marie Redublo
authors.initials: BM
authors.lastname: Quinto
authors.affiliation: Departamento de Medicina, Disciplina de Nefrologia, Universidade Federal de São Paulo (UNIFESP), Rua Botucatu, 740, São Paulo, Brazil.
authors.forename: Giovana
authors.initials: G
authors.lastname: Di Marco
authors.affiliation: Departamento de Micro-Imuno-Parasitologia, Disciplina de Parasitologia, Universidade Federal de São Paulo (UNIFESP), Rua Botucatu, 862, São Paulo, Brazil.
authors.forename: Renato Arruda
authors.initials: RA
authors.lastname: Mortara
authors.affiliation: Departamento de Fisiologia, Pontifícia Universidade Católica (PUC), Av. Libertador Bernardo O'Higgins 340, Santiago, Chile.
authors.forename: Carlos P
authors.initials: CP
authors.lastname: Vio
authors.affiliation: Departamento de Medicina, Disciplina de Nefrologia, Universidade Federal de São Paulo (UNIFESP), Rua Botucatu, 740, São Paulo, Brazil. Electronic address: casarini.elena@unifesp.br.
authors.forename: Dulce Elena
authors.initials: DE
authors.lastname: Casarini
mesh_terms: D000818:Animals; D002852:Chromatography, Ion Exchange; D004285:Dogs; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D050527:Mesangial Cells / Q000201:enzymology; D051379:Mice; D009842:Oligopeptides / Q000737:chemistry; D007703:Peptidyl-Dipeptidase A / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D017434:Protein Structure, Tertiary; D011817:Rabbits; D051381:Rats; D013379:Substrate Specificity
chemical_list: D009842:Oligopeptides; D007703:Peptidyl-Dipeptidase A; C058504:goralatide
keywords: Angiotensin-I converting enzyme; LLC-PK1; N-domain ACE; Proximal tubule cells