Affinity purification of active plasminogen activator inhibitor-1 (PAI-1) using immobilized anhydrourokinase. Demonstration of the binding, stabilization, and activation of PAI-1 by vitronectin.
Human Hep G2 hepatoma and HT 1080 fibrosarcoma cells were cultured in large scale under conditions which allowed enhanced secretion of plasminogen activator inhibitor-1 (PAI-1). A modified urokinase was obtained by reacting urokinase with phenylmethylsulfonyl fluoride followed by alkali treatment. The resulting product, called anhydrourokinase, was found to reversibly bind the PAI-1 when immobilized on cyanogen bromide-activated Sepharose 4B beads. Using this affinity absorbent, we have purified PAI-1 from the cell-conditioned media. A number of differences have been observed during Hep G2 and HT 1080 PAI purification. 1) The PAI activity in Hep G2 medium concentrate is more stable, and the concentrate depleted of active PAI-1 showed spontaneous regeneration of PAI-1 activity. In contrast, the PAI activity in HT 1080 medium concentrate declines rapidly on standing. 2) Hep G2 PAI-1 invariably copurified with an adhesive protein, vitronectin or its NH2-terminal fragment, while pure HT 1080 PAI-1 alone was obtained by affinity purification on anhydrourokinase-Sepharose 4B. 3) Based on specific activity measurement and complex formation analysis using a sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis technique, the purified Hep G2 PAI-1 appears completely active while the HT 1080 PAI-1 is only one-fourth as active. SDS was found to exert dual effects on purified PAI-1s. SDS treatment partially inactivated a fully active Hep G2 PAI-1 and a moderately active HT 1080 PAI-1 but partially activated an HT 1080 PAI-1 whose activity had previously been allowed to decay to a very low level. Purified vitronectin was found to enhance and stabilize the PAI-1 activity of the partially active HT 1080 PAI-1. It is concluded that fully active PAI-1 in association with vitronectin can be isolated by anhydrourokinase-Sepharose 4B chromatography and that vitronectin is a binding protein for PAI-1 which activates and stabilizes PAI-1.
Wun
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Monsanto Co., Chesterfield, Missouri 63198.
Palmier
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Siegel
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Smith
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CE
D006528:Carcinoma, Hepatocellular; D002846:Chromatography, Affinity; D003470:Culture Media; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D004800:Enzymes, Immobilized; D005354:Fibrosarcoma; D006023:Glycoproteins / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D008113:Liver Neoplasms; D008970:Molecular Weight; D010664:Phenylmethylsulfonyl Fluoride; D010960:Plasminogen Activators / Q000037:antagonists & inhibitors; D015849:Plasminogen Inactivators; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014568:Urokinase-Type Plasminogen Activator; D019096:Vitronectin
D003470:Culture Media; D004800:Enzymes, Immobilized; D006023:Glycoproteins; D015849:Plasminogen Inactivators; D019096:Vitronectin; D012967:Sodium Dodecyl Sulfate; D010664:Phenylmethylsulfonyl Fluoride; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator