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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.
Lijnen, H. R.; Pierard, Luc; Reff, M. E. et al.
1988In Thrombosis Research, 52 (5), p. 431-41
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Keywords :
Urokinase-Type Plasminogen Activator/genetics/isolation & purification/metabolism
Abstract :
[en] 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 (Pierard 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.
Disciplines :
Cardiovascular & respiratory systems
Author, co-author :
Lijnen, H. R.
Pierard, Luc ;  Université de Liège - ULiège > Département des sciences cliniques > Cardiologie - Pathologie spéciale et réhabilitation
Reff, M. E.
Gheysen, D.
Language :
English
Title :
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.
Publication date :
1988
Journal title :
Thrombosis Research
ISSN :
0049-3848
eISSN :
1879-2472
Publisher :
Elsevier, United Kingdom
Volume :
52
Issue :
5
Pages :
431-41
Peer reviewed :
Peer Reviewed verified by ORBi
Available on ORBi :
since 12 July 2014

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