Isolation of the major serine protease inhibitor from the 5-day serum-free conditioned medium of human embryonic lung cells and demonstration that it is fetuin.
Human embryonic lung (HuEL) cells in culture produce large amounts of the enzyme, plasminogen activator, and thus generate substantial amounts of active plasmin. Despite the presence of plasmin, however, HuEL cells grow in ordered, flattened, adherent sheets. It seemed of interest to characterize protease inhibitors that might be present in HuEL cultures and which might account for this apparent contradiction. This paper reports the isolation and purification of the major serine protease inhibitor in 5-day serum-free conditioned medium (CM) from HuEL cells, and the purification of an identical molecule from fetal bovine serum (FBS). Both the CM-derived inhibitor and the FBS-derived inhibitor are identical with fetuin, the major glycoprotein of FBS. The CM-derived inhibitor is apparently derived from the FBS used to supplement the growth medium of HuEL cells between serum-free CM collection periods. It is not labeled metabolically with 3H-leucine. Its electrophoretic behavior is indistinguishable from that of standard fetuin in SDS-PAGE, non-SDS basic pH,PAGE, and isoelectric focusing. The CM-derived inhibitor and standard fetuin inhibit trypsin and plasmin with similar efficiencies, but neither inhibits chymotrypsin, pancreatic elastase, or plasminogen activator. They are immunologically indistinguishable. The suggestion is made that fetuin, and possibly other protease inhibitors present in HuEL cell cultures, may be concentrated locally by HuEL cells and gradually released back into the medium in the absence of serum. These molecules may serve to protect HuEL cells against proteases they generate.
D002478:Cells, Cultured; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D005110:Extracellular Space / Q000032:analysis; D006801:Humans; D007526:Isoelectric Point; D008168:Lung / Q000166:cytology / Q000196:embryology / Q000201:enzymology*; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000302:isolation & purification; D000509:alpha-Fetoproteins / Q000276:immunology / Q000302:isolation & purification*
D003470:Culture Media; D011480:Protease Inhibitors; D000509:alpha-Fetoproteins