Purification of plasminogen activator from Rous sarcoma virus-infected chick embryo fibroblast culture medium.
A new procedure for the purification of plasminogen activator secreted by cultured Rous sarcoma virus-infected chick embryo fibroblasts was described. The enzyme was isolated from culture medium containing 0.75% calf serum depleted of plasminogen by lysine-agarose affinity column chromatography and of high-molecular-weight protease inhibitors by ultracentrifugation. The culture conditions allowed convenient preparation of large amounts of culture fluid with relatively high concentrations of plasminogen activator. The purification of the enzyme was accomplished by affinity chromatography on fibrin-celite and p-aminobenzamidine-agarose columns, and by gel-filtration chromatography in the presence of urea. The activity was recovered in greater than 90% yield, and the enzyme was essentially homogeneous when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Yields from 500 ml culture fluid exceeded 500 micrograms.
Kacian
D L
DL
Harvey
R C
RC
D000818:Animals; D001769:Blood; D002364:Caseins / Q000378:metabolism; D002478:Cells, Cultured; D002642:Chick Embryo; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003470:Culture Media / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts; D008858:Microscopy, Phase-Contrast; D008970:Molecular Weight; D010960:Plasminogen Activators / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D015849:Plasminogen Inactivators; D001357:Sarcoma, Avian / Q000201:enzymology*; D013755:Tetradecanoylphorbol Acetate; D014461:Ultracentrifugation
D002364:Caseins; D003470:Culture Media; D015849:Plasminogen Inactivators; D010960:Plasminogen Activators; D013755:Tetradecanoylphorbol Acetate