Purification and characterization of single-chain urokinase-type plasminogen activator (pro-urokinase) from human A431 cells.
A single-chain urokinase-type plasminogen activator (A431sc-uPA) was purified approximately 18,000-fold from A431 human epidermoid carcinoma cell supernatants by monoclonal antibody immunoaffinity chromatography on 5B4-agarose and ion-exchange FPLC (overall yield 63%). More than 100 micrograms of A431sc-uPA can be recovered per liter of supernatant. The product is homogeneous by SDS-PAGE and reverse phase FPLC analysis while two main isoelectric forms of pI 9.05 and pI 9.20 were observed by IEF. SDS-PAGE in reducing and non-reducing conditions, Western blot analysis and zymography showed that A431sc-uPA is a single-chain protein of about 50,000 Mr immunologically related to urokinase (uPA) and distinct from tissue plasminogen activator (tPA). The N-terminal aminoacid sequence of A431sc-uPA (27 residues) is identical to that of human kidney single-chain uPA. A431sc-uPA does not incorporate 3H-diisopropylfluorophosphate and is virtually inactive on the synthetic substrate S-2444. Plasmin treatment converts A431sc-uPA into a two-chain active form with a fibrinolytic specific activity of 123,000 I. U./mg.
D002294:Carcinoma, Squamous Cell; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; 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