Purification and characterization of angiotensin I-converting enzymes from mesangial cells in culture

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dc.contributor.author Andrade, MCC
dc.contributor.author Quinto, BMR
dc.contributor.author Carmona, A. K.
dc.contributor.author Ribas, O. S.
dc.contributor.author Boim, M. A.
dc.contributor.author Schor, N.
dc.contributor.author Casarini, D. E.
dc.date.accessioned 2016-01-24T12:30:43Z
dc.date.available 2016-01-24T12:30:43Z
dc.date.issued 1998-12-01
dc.identifier http://dx.doi.org/10.1097/00004872-199816121-00031
dc.identifier.citation Journal of Hypertension. Philadelphia: Lippincott Williams & Wilkins, v. 16, n. 12, p. 2063-2074, 1998.
dc.identifier.issn 0263-6352
dc.identifier.uri http://repositorio.unifesp.br/handle/11600/25995
dc.description.abstract Objective Previous analysis of the angiotensin I-converting enzyme (ACE) gene in this laboratory showed that primary mesangial cells in culture are able to express ACE mRNA. Moreover, ACE is produced as an ectoenzyme and as a secreted form of the enzyme, indicating a potential effect of local angiotensin II production on glomerular microcirculation. the aim of this study was to purify and characterize the secreted end intracellular ACE forms from mesangial cells in culture.Methods and results Medium from Wister rats mesangial cells was collected (third passage), incubated for 20 h with RPMI without fetal bovine serum and concentrated 29 times in an Amicon concentrator. the concentrated medium was submitted to gel filtration on an AcA-34 column and two peaks (ACE(1), mol. wt 130 000 and ACE(2), 60 000) with ACE on activity Hippuryl-His-Leu and Z-Phe-His-Leu were separated. the mesangial cells were collected and ACE enzyme was extracted using Triton X-114, followed by centrifugation and concentration. the supernatant was submitted to the same chromatography as described above and two peaks with ACE activity (ACE(Int1), mol. wt 130 000 and ACE(Int2), 68 000) were separated. the purified ACE were inhibited by enalaprilat and captopril, two potent competitive inhibitors of ACE and by EDTA, using Hippuryl-His-Leu as a substrate. the K-m values were 2 mM for ACE(1) and ACE(2) and 3 mM for ACE(Int1) end ACE(Int2). the enzymes ACE(1) and ACE(2) presented an optimum pH of 8.0 and ACE(Int1) and ACE(Int2) an optimum pH of 7.5.Conclusion the activities of full-length wild-type and N-domain ACE were characterized by the ratio of the hydrolysis of Z-Phe-His-Leu/Hippuryl-His-Leu, which was 1 and 4, respectively. the ratios found for ACE(1), ACE(2), ACE(Int1) and ACE(Int2) in the present study were similar to those described above, suggesting that mesangial cells, besides showing the presence of intracellular ACE, are able to secret both full-length wild-type ACE and N-domain ACE. Thus, they may potentially have an effect, not only on bradykinin and angiotensin I (ACE wild-type), but also on substance P, luteinizing hormone-releasing hormone and Met-enkephalin to interfere with glomerular haemodynamics and with the renal microcirculation. I Hypertens 1998, 16:2063-2074 (C) 1998 Lippincott Williams & Wilkins. en
dc.format.extent 2063-2074
dc.language.iso eng
dc.publisher Lippincott Williams & Wilkins
dc.relation.ispartof Journal of Hypertension
dc.rights Acesso restrito
dc.subject angiotensin-converting enzyme en
dc.subject N-domain en
dc.subject mesangial cell en
dc.subject bradykinin en
dc.subject angiotensin I en
dc.title Purification and characterization of angiotensin I-converting enzymes from mesangial cells in culture en
dc.type Artigo
dc.contributor.institution Universidade Federal de São Paulo (UNIFESP)
dc.description.affiliation Universidade Federal de São Paulo, Escola Paulista Med, Dept Med, Disciplina Nefrol,Div Nephrol, BR-04023900 São Paulo, Brazil
dc.description.affiliation Universidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-04023900 São Paulo, Brazil
dc.description.affiliationUnifesp Universidade Federal de São Paulo, Escola Paulista Med, Dept Med, Disciplina Nefrol,Div Nephrol, BR-04023900 São Paulo, Brazil
dc.description.affiliationUnifesp Universidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-04023900 São Paulo, Brazil
dc.identifier.doi 10.1097/00004872-199816121-00031
dc.description.source Web of Science
dc.identifier.wos WOS:000077791400031



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