Purification and characterization of a plasminogen activator secreted by a pig kidney cell line.
The plasminogen activator secreted by calcitonin-treated pig kidney cells was purified, characterized and compared with human urinary urokinase. The purification procedure was based on the following steps: sulphopropyl-Sephadex chromatography, p-aminobenzamidine-Sepharose chromatography, preparative sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and isoelectrofocusing. The purified enzyme was obtained from the conditioned medium with a yield of 13% and a purification factor of 390-fold. Analysis by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under non-reducing conditions showed one closely spaced doublet with an Mr of 50 000; in the presence of reducing agents, two additional bands of Mr 30 000 and 20 000 appeared. The purified enzyme resembles the 53 000-Mr components of human urinary urokinase in amino acid composition and two-dimensional tryptic peptide maps and in its catalytic properties, and the two enzymes cross-react immunologically with rabbit antibodies raised against either. The enzyme appears to be different from tissue plasminogen activator secreted by HeLa cells.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002460:Cell Line; D011232:Chemical Precipitation; D002852:Chromatography, Ion Exchange; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D007668:Kidney / Q000032:analysis*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D010960:Plasminogen Activators / Q000037:antagonists & inhibitors / Q000276:immunology / Q000302:isolation & purification*; D015849:Plasminogen Inactivators; D011480:Protease Inhibitors / Q000494:pharmacology; D013552:Swine; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors / Q000276:immunology
D000596:Amino Acids; D010446:Peptide Fragments; D015849:Plasminogen Inactivators; D011480:Protease Inhibitors; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator