A quantitative receptor assay using Triton X-114 for plasminogen activator binding proteins in solubilized membranes from human liver and placenta.
Cell surface binding proteins play an important role in the localization of plasminogen activator (PA) activity at the cell surface or in the clearance of PAs. We describe a rapid and quantitative receptor assay applicable to the quantification and affinity determination of binding proteins for tissue-type PA and urokinase-type PA in solubilized membranes obtained from human liver, human placenta, or human monocyte-like cells. The method is based on the ability of a solution of the nonionic detergent Triton X-114 to phase separate at temperatures above 20 degrees C. After incubation of integral membrane proteins with radiolabeled ligand, a solution of Triton X-114 is added at 4 degrees C and warmed to 37 degrees C to allow phase partitioning. Radiolabeled ligand bound to membrane protein is recovered in the detergent-rich lower phase which is separated by centrifugation from the detergent-poor upper phase containing free radiolabeled ligand.
Department of Internal Medicine, University Hospital CHUV, Lausanne, Switzerland.
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G
Nguyen
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EK
Kruithof
D002352:Carrier Proteins / Q000032:analysis; D002462:Cell Membrane / Q000378:metabolism; D003902:Detergents; D005069:Evaluation Studies as Topic; D005260:Female; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D017830:Octoxynol; D010920:Placenta / Q000378:metabolism; D011092:Polyethylene Glycols; D011247:Pregnancy; D011956:Receptors, Cell Surface / Q000032:analysis*; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins / Q000378:metabolism; D012995:Solubility; D010959:Tissue Plasminogen Activator / Q000378:metabolism*; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
D002352:Carrier Proteins; D003902:Detergents; C524898:PLAUR protein, human; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; C067262:PLAT protein, human; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator