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Thromb Haemost 1986; 56(02): 219-224
DOI: 10.1055/s-0038-1661644
Original Article
Schattauer GmbH Stuttgart

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

Angelo Corti
The Lepetit Research Center, Milan, Italy
,
Maria Luisa Nolli
The Lepetit Research Center, Milan, Italy
,
Adolfo Soffientini
The Lepetit Research Center, Milan, Italy
,
Giovanni Cassani
The Lepetit Research Center, Milan, Italy
› Author Affiliations
Further Information

Publication History

Received 12 May 1986

Accepted after revision 22 July 1986

Publication Date:
20 July 2018 (online)

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Summary

A 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.

Key words

Urokinase - Pro-urokinase - Plasminogen activator - Fibrinolysis - Monoclonal antibody
 

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