Purification and Characterization of Single-Chain Urokinase-Type Plasminogen Activator (Pro-Urokinase) from Human A431 Cells

Abstract

SummaryA single-chain urokinase-type plasminogen activator (A431sc-uPA) was purified ˜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 jig 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.