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

摘要: Objective Previous analysis of the angiotensin I-converting enzyme (ACE) gene in this laboratory showed that primary mesangial cells culture are able to express ACE mRNA. Moreover, is produced as an ectoenzyme and a secreted form enzyme, indicating potential effect local II production on glomerular microcirculation. The aim study was purify characterize intracellular forms from culture. Methods results Medium Wistar rats collected (third passage), incubated for 20 h with RPMI without fetal bovine serum concentrated 29 times Amicon concentrator. medium submitted gel filtration AcA-34 column two peaks (ACE 1 , mol. wt 130 000 2 60 000) activity Hippuryl-His-Leu Z-Phe-His-Leu were separated. extracted using Triton X-114, followed by centrifugation concentration. supernatant same chromatography described above int1 int2 68 purified inhibited enalaprilat captopril, potent competitive inhibitors EDTA, substrate. K m values mM 3 . enzymes presented optimum pH 8.0 7.5. Conclusion activities full-length wild-type N-domain characterized ratio hydrolysis Z-Phe-His-Leu/Hippuryl-His-Leu, which 4, respectively. ratios found present similar those above, suggesting cells, besides showing presence ACE, secret both ACE. Thus, they may potentially have effect, not only bradykinin I wild-type), but also substance P, luteinizing hormone-releasing hormone Met-enkephalin interfere haemodynamics renal