Purification of an active plasminogen activator inhibitor immunologically related to the endothelial type plasminogen activator inhibitor from the conditioned media of a human melanoma cell line.
24 established melanoma cell cultures were screened for their secretion of plasminogen activators and plasminogen activator inhibitors into the culture medium by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by conventional and reverse fibrin autography. Among the cell lines investigated, 22 cell lines predominantly secreting tissue type plasminogen activator (t-PA) and four cell lines additionally secreting urokinase were found. The conditioned media of two cell lines (KRFM and MJZJ) were found to contain plasminogen activator inhibitor (PAI) activity at a Mr position of approximately 50,000. The PAI of one of the two melanoma cell (MJZJ)-conditioned media found to contain PAI activity was purified to apparent homogeneity employing concanavalin A-Sepharose chromatography, gel filtration on Sephadex G-150, chromatography on Affi-Gel blue, and affinity chromatography on a Sepharose 4B immobilized monoclonal anti-t-PA IgG column. The purified melanoma PAI was found to be a single chain protein, acid stable, immunologically related to the endothelial derived PAI. In contrast to endothelial PAI, melanoma PAI presented itself in the conditioned media of the melanoma cells and in the purified preparation to an appreciable extent in its active form.
D002460:Cell Line; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D004727:Endothelium / Q000502:physiology*; D006023:Glycoproteins / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D008545:Melanoma / Q000378:metabolism*; D008970:Molecular Weight; D010960:Plasminogen Activators / Q000378:metabolism; D015849:Plasminogen Inactivators
D003470:Culture Media; D006023:Glycoproteins; D015849:Plasminogen Inactivators; D010960:Plasminogen Activators