Immunoaffinity purification of HTC rat hepatoma cell plasminogen activator-inhibitor-1.
Incubation of HTC rat hepatoma cells with the synthetic glucocorticoid dexamethasone rapidly inhibits tissue-type plasminogen activator activity by inducing a specific plasminogen activator-inhibitor (PAI-1). Using immobilized polyclonal antibodies raised against HT-1080 human fibrosarcoma PAI-1, we have purified HTC PAI-1 from serum-free medium conditioned by dexamethasone-treated HTC hepatoma cells and shown it to be antigenically related to human PAI-1. Greater than 100-fold purification with greater than 75% yield was achieved in a single step. The purified PAI-1 migrates on SDS-polyacrylamide gels as a single major band of 49 kDa with a minor band of 46 kDa. Digestion of PAI-1 with endoglycosidase F causes a shift toward faster migrating species which retain inhibitory activity. The purified PAI-1 was stable at pH 2.5, lost 50% of its activity after 15 min at 45 degrees C, and showed marked activation after treatment with SDS or guanidine-HCl. Purified PAI-1 rapidly inhibited and formed complexes with both tissue-type and urokinase-type plasminogen activators. Polyclonal rabbit antirat PAI-1 antibodies were raised which immunoprecipitate both free and complexed PAI-1.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000918:Antibody Specificity; D002846:Chromatography, Affinity; D006023:Glycoproteins / Q000276:immunology / Q000302:isolation & purification*; D006358:Hot Temperature; D008114:Liver Neoplasms, Experimental / Q000032:analysis*; D010960:Plasminogen Activators / Q000037:antagonists & inhibitors*; D015849:Plasminogen Inactivators; D051381:Rats; D010959:Tissue Plasminogen Activator / Q000037:antagonists & inhibitors; D014407:Tumor Cells, Cultured / Q000032:analysis
D000596:Amino Acids; D006023:Glycoproteins; D015849:Plasminogen Inactivators; D010960:Plasminogen Activators; D010959:Tissue Plasminogen Activator