Purification and characterization of single-chain urokinase-type plasminogen activator from human cell cultures.
A urokinase-type plasminogen activator was purified from conditioned media of several human cell cultures, but preferably from the human lung adenocarcinoma line CALU-3 (ATCC, HTB-55), using a combination of chromatography on zinc chelate-Sepharose, SP-Sephadex C-50, and Sephadex G-100. Final yields of 65-100 micrograms/liter of starting material were obtained with a 290-fold purification factor and a recovery of 30%. The purified plasminogen activator consists of a single polypeptide chain with Mr 54,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and is very similar or identical to single-chain urokinase-type plasminogen activator on the basis of immunodiffusion, amino acid composition, and the lack of specific binding to fibrin. It has very low amidolytic activity on Pyroglu-Gly-Arg-rho-nitroanilide and is converted to two-chain urokinase by limited exposure to plasmin. It has a specific activity of 60,000 IU/mg on fibrin plates and directly activates plasminogen following Michaelis-Menten kinetics with Km = 1.1 microM and kappa cat = 0.0026 S-1. It is concluded that the plasminogen activator purified from CALU-3-conditioned media is physically and kinetically identical to single-chain urokinase-type plasminogen activator. With the present straightforward purification method and a readily available source, sufficient amounts of single-chain urokinase-type plasminogen activator can be obtained for more detailed investigations of its biochemical, biological, and thrombolytic properties.
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D002478:Cells, Cultured; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D005779:Immunodiffusion; D007700:Kinetics; D010960:Plasminogen Activators / Q000302:isolation & purification* / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000302:isolation & purification* / Q000378:metabolism
D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator