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.
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.
Lijnen
H R
HR
Center for Thrombosis and Vascular Research, Leuven, Belgium.
Piérard
L
L
Reff
M E
ME
Gheysen
D
D
D001665:Binding Sites; D002678:Chimera; D005337:Fibrin / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D015394:Molecular Structure; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010959:Tissue Plasminogen Activator / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D011994:Recombinant Proteins; D005337:Fibrin; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator