Purification and properties of a plasminogen activator from cultured rat prostate adenocarcinoma cells.
Zymographic analysis of the supernates from confluent cultures of a rat prostate adenocarcinoma cell line, PA-III, revealed the existence of two molecular forms of specific plasminogen activators, one of molecular weight of approximately 80 000 and another of approximate molecular weight of 45 000, in sodium dodecyl sulfate. The low molecular weight form has been purified 364-fold in 66% yield from the culture medium by a combination of gel filtration on Sephacryl S-200 and affinity chromatography on Sepharose 4B-benzamidine. The purified material possessed a specific activity of 192 000 urokinase CTA units mg-1. This enzyme displayed activity toward human Glu1-plasminogen, characterized by a Km of 1.7 +/- 0.2 microM and a Vmax of 0.53 +/- 0.1 pmol of plasmin min-1 unit-1. A synthetic chromogenic substrate, H-D-Ile-Pro-Arg-p-nitroanilide (S-2288), was found for the activator. The enzyme possessed a Km of 0.33 mM and a kcat of 55 s-1 for S-2288. The activator was found to be a serine protease, inhibited by diisopropyl fluorophosphate (iPr2PF). At a concentration of 1 mM iPr2PF, and 30 nM enzyme, the half-time of this inhibition was 3.8 min. The 45 000 molecular weight enzyme was found to be inhibited by rabbit antibodies to human urokinase, thus characterizing the activator as a member of the urokinase class. The 80 000 molecular weight enzyme was not neutralized by anti-human urokinase but was neutralized by rabbit anti-human melanoma activator, likely allowing it to be classified as the tissue activator type.
D000230:Adenocarcinoma / Q000032:analysis*; D000818:Animals; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D005337:Fibrin / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D010958:Plasminogen / Q000378:metabolism; D010960:Plasminogen Activators / Q000302:isolation & purification* / Q000378:metabolism; D011471:Prostatic Neoplasms / Q000032:analysis*; D011485:Protein Binding; D051381:Rats; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism
D005337:Fibrin; D010958:Plasminogen; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator