Purification of high and low molecular weight plasminogen activator inhibitor 1 from fibrosarcoma cell-line HT 1080 conditioned medium.
Functionally active (high-Mr) and inactive (low-Mr) plasminogen activator inhibitor 1 (PAI) have been purified from fibrosarcoma cell-line HT 1080 conditioned medium, containing 1% fetal calf serum. The two forms were first purified by affinity chromatography on heparin-Sepharose and then separated from each other by gel filtration on Sephadex G-150. The final purification was achieved by affinity chromatography on insolubilized monoclonal antibodies towards human PAI. Alternatively, the low-Mr form was purified by chromatography on carboxymethyl-cellulose. Low-Mr PAI purified in this way, could be almost fully reactivated by treatment with guanidinium chloride. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by immunoblotting revealed that the low-Mr form contained nothing but PAI at an Mr of about 50,000. In addition to PAI, the high-Mr form contained a component, which was not antigenically related to PAI. This compound had a molecular weight of about 75,000 and its NH2-terminal amino acid sequence corresponded to that of human vitronectin. We conclude that the high-Mr form of PAI constitutes a complex between 50,000 Mr PAI and vitronectin from fetal calf serum.
Lindahl
T
T
Department of Clinical Chemistry, Karolinska Hospital, Stockholm, Sweden.
Wiman
B
B
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005354:Fibrosarcoma / Q000378:metabolism*; D006023:Glycoproteins / Q000302:isolation & purification*; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D008970:Molecular Weight; D010960:Plasminogen Activators / Q000037:antagonists & inhibitors; D015849:Plasminogen Inactivators; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D006023:Glycoproteins; D006146:Guanidines; D015849:Plasminogen Inactivators; D010960:Plasminogen Activators; D019791:Guanidine