Characterization of plasminogen activator from two human renal carcinoma cell lines.
Plasminogen activator (PA) activity was identified in the conditioned medium of two human renal carcinoma cell lines, Cur and Caki-1. PA activity of medium, following chromatography on Con A-Sepharose, was divided into effluent and eluate fractions, the latter obtained after elution with methyl mannoside. The ratio of PA activity in effluent:eluate was 90:10 for Caki-1 and 60:40 for Cur. The PA of both effluent fractions and the Caki-1 eluate fraction was of the urokinase (UK) type. Identification rested on molecular weight determination by zymography (major component with Mr 52,000 and a less prominent component of 93,000), lack of binding to fibrin, inhibition by anti-UK antibodies, and lack of inhibitory effect of anti-tissue type PA (TPA) antibodies or the Erythrina trypsin inhibitor, which inhibits TPA but not UK. PA of the Cur eluate fraction gave a more complex pattern in that it bound significantly to fibrin (like TPA), was completely inhibited by both anti-UK and anti-TPA antibodies, but was unaffected by Erythrina trypsin inhibitor. These results raise the possibility of an unusual PA-like enzyme that immunologically cross reacts with anti-UK and anti-TPA. Most of the PA of both cell lines was secreted in a latent form that could be activated by trypsin treatment. The latency appears to result largely from secretion of urokinase proenzyme, which is consistent with the Mr 52,000 of the major PA species and the insensitivity to diisopropyl fluorophosphate inhibition prior to trypsin activation. However, in addition, a UK binding component was found in the conditioned medium, which produced an Mr 93,000 component by reaction with UK.
N F
NF
Nelson
W
W
Cieplak
S C
SC
Dacus
M D
MD
Prager
D007611:Aprotinin; D002460:Cell Line; D002846:Chromatography, Affinity; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D004895:Erythrina; D005337:Fibrin / Q000378:metabolism; D006801:Humans; D007163:Immunosorbent Techniques; D007680:Kidney Neoplasms / Q000032:analysis*; D007700:Kinetics; D010946:Plants, Medicinal; D010960:Plasminogen Activators / Q000032:analysis*; D010959:Tissue Plasminogen Activator / Q000032:analysis; D014361:Trypsin Inhibitors / Q000494:pharmacology; D014568:Urokinase-Type Plasminogen Activator / Q000032:analysis
D003470:Culture Media; D014361:Trypsin Inhibitors; D005337:Fibrin; D007611:Aprotinin; D010960:Plasminogen Activators; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator