An inhibitor of plasminogen activation from human placenta. Purification and characterization.
Placental extracts contain inhibitors of human urinary urokinase. These extracts form a heterogeneous population of complexes with 125I-urokinase that are recognizable by changes in gel filtration profile and mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment with reducing agents eliminated the size heterogeneity without loss of activity, thereby allowing the placental inhibitor to be purified. Active inhibitor has been isolated in apparently homogeneous form after an eight-step procedure that included salt extraction, ammonium sulfate fractionation, column chromatography on CM-cellulose, DEAE-Sepharose, and hydroxylapatite, chromatofocusing, preparative gel electrophoresis, and hydrophobic chromatography. The purified inhibitor has Mr = 47,000. The inhibitor is relatively specific for plasminogen activators since it does not inhibit the action of plasmin, factor XIIa, plasma kallikrein, or thrombin. The inhibitor forms complexes with 1:1 stoichiometry that block the active sites of urokinase (but not prourokinase) and both one- and two-chain forms of tissue plasminogen activator. The stability of these complexes in sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggest that they are based on covalently bonded structures. Although both types of plasminogen activator are inhibited, the rate of interaction is significantly faster with urokinase, tissue plasminogen activator being inhibited less efficiently. The complexes formed can be dissociated by mild alkali or hydroxylamine, thereby regenerating both enzymes and inhibitor at their original molecular weights. The results suggest that the complexes are stabilized by ester-like bonds; these might involve the hydroxyl of serine at the active site of the proteases and a carboxyl group in the inhibitor.
D000596:Amino Acids / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006023:Glycoproteins / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D005779:Immunodiffusion; D007700:Kinetics; D008970:Molecular Weight; D010920:Placenta / Q000502:physiology*; D015849:Plasminogen Inactivators; D011247:Pregnancy; D011485:Protein Binding; D010959:Tissue Plasminogen Activator / Q000037:antagonists & inhibitors*; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors*
D000596:Amino Acids; D006023:Glycoproteins; D015849:Plasminogen Inactivators; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator