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

Objective Previous analysis of the angiotensin I-converting enzyme (ACE) ge ne in this laboratory showed that primary mesangial cells in culture are ab le 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 angiot ensin II production on glomerular microcirculation. The aim of this study w as 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 w as submitted to gel filtration on an AcA-34 column and two peaks (ACE(1), m ol. wt 130 000 and ACE(2), 60 000) with ACE on activity Hippuryl-His-Leu an d Z-Phe-His-Leu were separated. The mesangial cells were collected and ACE enzyme was extracted using Triton X-114, followed by centrifugation and con centration. The supernatant was submitted to the same chromatography as des cribed above and two peaks with ACE activity (ACE(Int1), mol. wt 130 000 an d ACE(Int2), 68 000) were separated. The purified ACE were inhibited by ena laprilat and captopril, two potent competitive inhibitors of ACE and by EDT A, 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 optimu m pH of 7.5. Conclusion The activities of full-length wild-type and N-domain ACE were ch aracterized by the ratio of the hydrolysis of Z-Phe-His-Leu/Hippuryl-His-Le u, which was 1 and 4, respectively. The ratios found for ACE(1), ACE(2), AC E(Int1) and ACE(Int2) in the present study were similar to those described above, suggesting that mesangial cells, besides showing the presence of int racellular ACE, are able to secret both full-length wild-type ACE and N-dom ain ACE. Thus, they may potentially have an effect, not only on bradykinin and angiotensin I (ACE wild-type), but also on substance P, luteinizing hor mone-releasing hormone and Met-enkephalin to interfere with glomerular haem odynamics and with the renal microcirculation. I Hypertens 1998, 16:2063-20 74 (C) 1998 Lippincott Williams & Wilkins.