Table 1. Activity and rate of ACE release from CHO cells transfected with various ACEs.

ACE	ACE activity (mU/ml)		Rate of ACE release
	Soluble ACE	Membrane-bound	% per 24 h
Wild-type*	3.4±0.2	82.0±9.1	11.3±0.8
Wild-type#	0.06±0.2	2.3±0.1	7.8±0.4
Pro1199Leu#	6.4±0.4	29.7±2.1	48.9±5.1
Trp1197Stop#	12.0±0.4	7.7±2.1	83.5±2.0
Ser1211Stop#+	4.4±0.3	3.8±0.3	80.1±0.6
Gln1230StopX*&	628±39	195±10	89.8±2.1

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).