Purification and characterization of angiotensin I-converting enzymes from mesangial cells in culture.
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 and intracellular ACE forms from mesangial cells in culture.


RESULTS
Medium from Wistar 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 (ACE1, mol. wt 130 000 and ACE2, 60000) 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 (ACEInt1, mol. wt 130000 and ACEInt2, 68000) 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 Km values were 2 mM for ACE1 and ACE2 and 3 mM for ACEInt1 and ACEInt2. The enzymes ACE1 and ACE2 presented an optimum pH of 8.0 and ACEInt1 and ACEInt2 an optimum pH of 7.5.


CONCLUSIONS
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 ACE1, ACE2, ACEInt1 and ACEInt2 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.
Andrade
M C
MC
Department of Medicine, EPM, UNIFESP, São Paulo, SP, Brazil.
Quinto
B M
BM
Carmona
A K
AK
Ribas
O S
OS
Boim
M A
MA
Schor
N
N
Casarini
D E
DE
D000803:Angiotensin I / Q000378:metabolism; D000806:Angiotensin-Converting Enzyme Inhibitors / Q000494:pharmacology; D000818:Animals; D001920:Bradykinin / Q000378:metabolism; D002216:Captopril / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D002712:Chlorides / Q000494:pharmacology; D002850:Chromatography, Gel; D015773:Enalaprilat / Q000494:pharmacology; D005920:Glomerular Mesangium / Q000166:cytology / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D009842:Oligopeptides; D007703:Peptidyl-Dipeptidase A / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity; D013696:Temperature
D000806:Angiotensin-Converting Enzyme Inhibitors; D002712:Chlorides; D007527:Isoenzymes; D009842:Oligopeptides; C010980:hippuryl-histidyl-leucine; D000803:Angiotensin I; D002216:Captopril; D007703:Peptidyl-Dipeptidase A; D015773:Enalaprilat; D001920:Bradykinin