Covalent molecular weight approximately 92 000 hybrid plasminogen activator derived from human plasmin amino-terminal and urokinase carboxyl-terminal domains.
The preparation of a new class of covalent hybrid plasminogen activators containing the fibrin-binding domains of human plasmin(ogen) and the catalytic active center of human urokinase will be described. Hybridization of the sulfhydryl form of the NH2-terminal plasmin-derived heavy (A) chain (PlnA) with the sulfhydryl form of the COOH-terminal urokinase-derived active heavy (B) chain (u-PAB) was carried out; a covalent PlnA-u-PAB hybrid plasminogen activator was prepared. The sulfhydryl form of PlnA (PlnA(SH)2) was isolated from reduced Lys-2-plasmin by L-lysine-substituted Sepharose column chromatography. For the isolation of the sulfhydryl form of u-PAB (u-PAB(SH], high molecular weight urokinase was adsorbed onto a benzamidine-Sepharose column and reduced with 100 mM 2-mercaptoethanol on the column. The urokinase NH2-terminal light (A) chain was washed off the column, and the u-PAB(SH) chain was eluted from the column. The specific activity of the isolated u-PAB(SH) chain was determined to be 242 000 IU/mg of protein. The PlnA(SH)2 and u-PAB(SH) chains were mixed at a molar ratio of PlnA(SH)2 to u-PAB(SH) of 3:2; the reducing agents were then removed by gel filtration. The hybridization (reoxidation) reaction was allowed to proceed for 48 h at 4 degrees C. The covalent hybrid activator, in 40% yield, was purified from the reaction mixture to homogeneity, by a sequential affinity chromatography method with L-lysine-substituted Sepharose followed by anti-low molecular weight urokinase IgG-Sepharose, and then gel filtration through Sephadex G-150.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D005341:Fibrinolysin / Q000378:metabolism*; D006801:Humans; D005779:Immunodiffusion; D007700:Kinetics; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D010960:Plasminogen Activators / Q000378:metabolism*; D055503:Protein Multimerization; D013552:Swine; D013917:Thrombin / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
D010446:Peptide Fragments; D010960:Plasminogen Activators; D013917:Thrombin; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator