Purification and characterization of a novel low molecular weight form of single-chain urokinase-type plasminogen activator.
A low Mr form (Mr 32,000) of single-chain urokinase-type plasminogen activator (scu-PA) was isolated from conditioned culture medium of a human lung adenocarcinoma cell line, CALU-3 (ATCC, HTB-55). The purified material (scu-PA-32k) consists of a single polypeptide chain and is immunologically similar to Mr 33,000 urokinase. Its NH2-terminal sequence is identical to that beginning at Leu-144 of Mr 54,000 urokinase. Whereas low Mr urokinase is derived from mature Mr 54,000 scu-PA by limited hydrolysis by plasmin first of the Lys-158-Ile-159 peptide bond and then of the Lys-136-Lys-137, scu-PA-32k is generated by specific hydrolysis of the Glu-143-Leu-144 peptide bond by an unidentified protease. scu-PA-32k resembles its Mr 54,000 scu-PA counterpart by its very low activity on chromogenic substrates for urokinase, by plasminogen-dependent fibrinolytic activity on fibrin plates, and by the lack of specific binding to fibrin. It activates plasminogen directly with high affinity, Km = 0.9 microM, but low turnover number, kcat = 0.0028 s-1. It is converted to fully active two-chain urokinase by plasmin with Km = 12 microM and kcat = 0.3 s-1. Like Mr 54,000 scu-PA, it causes significant lysis of a 125I-labeled fibrin clot in human plasma with relatively less fibrinogen breakdown as compared to urokinase. scu-PA-32k, which also has conserved fibrin specificity, represents a molecular variant which may be more suitable for large scale production as a fibrin-specific thrombolytic agent by recombinant DNA technology.
D000230:Adenocarcinoma; D000595:Amino Acid Sequence; D002460:Cell Line; D005337:Fibrin / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D005779:Immunodiffusion; D007700:Kinetics; D008175:Lung Neoplasms; D008970:Molecular Weight; D010959:Tissue Plasminogen Activator / Q000302:isolation & purification* / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000302:isolation & purification* / Q000378:metabolism
D005337:Fibrin; D005340:Fibrinogen; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator