Establishment of transfected cell lines producing testicular angiotensin-converting enzyme. Structural relationship between its secreted and cellular forms.
Angiotensin-converting enzyme (ACE) is present in endothelial and epithelial cells of various tissues as well as in the circulating plasma. The structural relationship between the cellular and the secreted forms of ACE and the pathways to their biosynthesis have not been determined as yet mainly because of the unavailability of a natural cell line expressing ACE in tissue culture. To circumvent this problem we have permanently transfected a mouse epithelial line with an expression vector containing the recently cloned rabbit testicular ACE cDNA. Clonal derivatives of this line secreted large quantities of enzymatically active ACE. When these cells were cultured in serum-free medium, the only detectable protein in the culture medium was ACE. It has been suggested that a hydrophobic domain near the carboxyl terminus of the enzyme anchors it to the plasma membrane. To test this hypothesis we established cell lines expressing a truncated form of the active enzyme which is missing the putative anchoring domain. Pulse-chase experiments showed that the truncated ACE was secreted from the cells much faster than the native enzyme. Moreover, the secreted form of the native enzyme had a lower molecular weight than the corresponding cellular form. These results are consistent with the hypothesis that the hydrophobic domain is instrumental in keeping the enzyme cell-bound, and secretion is achieved physiologically by removal of this domain from the enzyme by a specific proteolytic cleavage.
Sen
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Department of Heart and Hypertension, Cleveland Clinic Foundation, Ohio 44195.
Samanta
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Livingston
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Sen
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GC
D000818:Animals; D002460:Cell Line; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D015870:Gene Expression; D005822:Genetic Vectors; D007700:Kinetics; D008297:Male; D051379:Mice; D008970:Molecular Weight; D007703:Peptidyl-Dipeptidase A / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D015183:Restriction Mapping; D013737:Testis / Q000201:enzymology*; D014162:Transfection
D012333:RNA, Messenger; D004247:DNA; D007703:Peptidyl-Dipeptidase A