Activity and rate of ACE release from CHO cells transfected with various ACEs.

CHO cells transfected (stable or transient) with cDNA of human recombinant wild-type ACE or indicated mutants were cultivated in 100 mm Petri dish in F12 medium with 10% FBS. Once cells become confluent, dishes were washed with PBS and a fresh Ultra CHO medium (10 ml) were added. After 24 hours of cultivation culture medium was collected, cells were washed with PBS and lysed with 3 ml HEPES buffer (20 mM, pH 7.5, NaCl 150 mM) with 8 mM CHAPS. ACE activity was determined in culture medium (soluble ACE) and in a lysate (membrane-bound form) with Hip-His-Leu as a substrate and the rate of ACE shedding was calculated (ACE activity in culture medium X 100/ACE activity in culture medium + ACE activity in lysate). *- Stable transfection:, # Transient transfection. + Plasmid with cDNA coding for this mutant was kindly provided by Dr. E. Sturrock (University of Cape Town, South Africa). & Plasmid with cDNA coding for this mutant was kindly provided by Dr. F. Alhenc-Gelas (then INSERM U 367, Paris, France).