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Purification and characterization of a novel low molecular weight form of single-chain urokinase-type plasminogen
Abstract:
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.
Insights
A novel low molecular weight form of single-chain urokinase-type plasminogen activator (scu-PA-32k) was identified. This fibrin-specific variant shows potential for large-scale production as a thrombolytic agent.
Area of Science:
- Biochemistry
- Molecular Biology
- Thrombolysis
Background:
- Single-chain urokinase-type plasminogen activator (scu-PA) is a key enzyme in fibrinolysis.
- Low molecular weight variants of urokinase have been investigated for therapeutic potential.
Purpose of the Study:
- To isolate and characterize a novel low molecular weight form of scu-PA (scu-PA-32k).
- To evaluate the enzymatic properties and fibrinolytic activity of scu-PA-32k.
- To assess its potential as a fibrin-specific thrombolytic agent.
Main Methods:
- Isolation of scu-PA-32k from conditioned culture medium of human lung adenocarcinoma cells (CALU-3).
- Biochemical characterization including NH2-terminal sequencing and immunological comparison.
- Enzymatic assays using chromogenic substrates, fibrin plates, and plasminogen activation.
- Assessment of fibrin clot lysis in human plasma.
Main Results:
- scu-PA-32k, a single polypeptide chain (Mr 32,000), was purified and found to be immunologically similar to urokinase.
- It exhibits low activity on chromogenic substrates but potent plasminogen-dependent fibrinolytic activity.
- scu-PA-32k activates plasminogen with high affinity but low turnover, and is converted to active two-chain urokinase by plasmin.
- It effectively lyses fibrin clots in plasma with minimal fibrinogen breakdown, demonstrating conserved fibrin specificity.
Conclusions:
- scu-PA-32k is a distinct molecular variant of scu-PA generated by specific protease hydrolysis.
- Its fibrin-specific activity and reduced fibrinogenolysis suggest potential as an improved thrombolytic agent.
- scu-PA-32k may be suitable for large-scale production via recombinant DNA technology for therapeutic applications.