Angiotensin-converting enzyme in cultured endothelial cells. Synthesis, degradation, and transfer to culture medium.
Cultured endothelial cells from swine aorta possess the ecto-enzyme angiotensin-converting enzyme (E.C.3.4.15.1) in cell-associated form and also release it in soluble form into the culture medium. Using antibody to purified converting enzyme from swine kidney and incorporation of 3H-leucine, we examined the synthesis, degradation, and release of enzyme into the medium. 3H-leucine is incorporated into cellular converting enzyme, and later appears in the enzyme in the culture medium. The amount of cell-associated enzyme activity remains constant, and the amount of activity in the medium increases linearly during this period. These data show unequivocally that the appearance of enzyme activity in the cell culture medium is accompanied by synthesis of new enzyme protein. In pulse-chase experiments, the radioactivity disappeared from the cellular enzyme in two kinetic components with apparent half-lives of 1-2 hours and 20 hours, respectively. The appearance of the radiolabel in the medium enzyme corresponds very closely in rate and amount to the slow disappearance from the cells. There was no apparent uptake of labeled medium enzyme by the cells. The data suggest that the pool of active enzyme on the cell surface is the immediate precursor of the medium enzyme. The effects of culture conditions on the turnover of angiotensin-converting enzyme were also examined. The incorporation of 3H-leucine into both cell and medium enzyme was greater when cells were maintained in medium containing serum than when they were maintained in serum-free medium. The rate of degradation of the cellular enzyme was similar under the two culture conditions.
D000818:Animals; D001011:Aorta / Q000166:cytology*; D001692:Biological Transport; D002455:Cell Division; D002478:Cells, Cultured; D003470:Culture Media; D004727:Endothelium / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D006207:Half-Life; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D007703:Peptidyl-Dipeptidase A / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D014176:Protein Biosynthesis; D013552:Swine
D003470:Culture Media; D007703:Peptidyl-Dipeptidase A; D007930:Leucine