Characterization of a chimaeric plasminogen activator obtained by insertion of the second kringle structure of tissue-type plasminogen activator (amino acids 173 through 262) between residues Asp130 and Ser139 of urokinase-type plasminogen activator
| Autor: | Laurent Piérard, Mitchell E. Reff, H.R. Lijnen, Dirk Gheysen |
|---|---|
| Rok vydání: | 1988 |
| Předmět: |
Plasmin
In Vitro Techniques Tissue plasminogen activator law.invention law medicine Humans chemistry.chemical_classification Urokinase Gel electrophoresis Fibrin Binding Sites Molecular Structure Chimera T-plasminogen activator Hematology Urokinase-Type Plasminogen Activator Molecular biology Recombinant Proteins Amino acid Molecular Weight Kinetics chemistry Tissue Plasminogen Activator Recombinant DNA Plasminogen activator medicine.drug |
| Zdroj: | Thrombosis Research. 52:431-441 |
| ISSN: | 0049-3848 1771-1177 |
| DOI: | 10.1016/0049-3848(88)90027-8 |
| Popis: | A chimaeric recombinant plasminogen activator (rscu-PA- K2) obtained by insertion of the second kringle (K2) of tissue-type plasminogen activator (t-PA) (amino acids 173-262) between residues Asp130 and Ser139 of single chain urokinase-type plasminogen activator (scu-PA) was purified from the conditioned medium of mouse myeloma cells transfected with the previously described plasmid pULB9137 (Piérard et al., J. Biol. Chem. 262, 11771-11778, 1987). Approximately 22 micrograms of purified protein was obtained per liter of conditioned medium with a yield of approximately 25 percent. On sodium dodecylsulfate gel electrophoresis under reducing conditions, rscu-PA- K2 migrated with an apparent Mr of 65,000. Plasmin caused a time- and concentration-dependent conversion to an amidolytically active two chain derivative (rtcu-PA- K2) with a specific activity of 45,000 IU/mg. Both rscu-PA- K2 and rtcu-PA- K2 activated plasminogen directly with Km = 2.0 microM and k2 = 0.00063 s-1 and Km = 100 microM and k2 = 4.1 s-1 respectively. rscu-PA- K2 did not bind extensively to fibrin. It caused concentration-dependent lysis of 125I-fibrin-labeled plasma clots immersed in human plasma with a comparable specific activity and fibrin-specificity as rscu-PA. It is concluded that insertion in scu-PA of the second kringle of t-PA, which is believed to be involved in its fibrin affinity, does not significantly alter the enzymatic properties of scu-PA, but does not confer marked fibrin-affinity to the molecule. |
| Databáze: | OpenAIRE |
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