Production and characterization of recombinant human plasminogen(S741C-fluorescein). A novel approach to study zymogen activation without generation of active protease.
A variant of recombinant plasminogen with the plasmin active site serine (S741) replaced by cysteine was produced and labeled with fluorescein at this residue to provide the derivative Plg(S741C-fluorescein). Studies of cleavage, conformation, and fibrin-binding properties of the derivative showed it to be a good model substrate to study plasminogen activation. Both in solution and in a fully polymerized fibrin clot, cleavage of the single chain zymogen to the two-chain "plasmin" molecule was accompanied by a 50% quench of fluorescence intensity. This change allows facile, continuous monitoring of the kinetics of cleavage. Measurements of cleavage by single chain t-PA within intact, fully polymerized 3 microM fibrin yielded apparent kcat and Km values of (0.08 s-1, 0.52 microM) and (0.092 s-1, 0.098 microM) for [Glu1]- and [Lys78]Plg(S741C-fluorescein), respectively. These values are similar to those obtained by others with plasma plasminogen. The approach used here might generally be useful in simplifying the analysis of zymogen activation kinetics in cases where the product (protease) has a great influence on its own formation via positive or negative feedback loops.
Horrevoets
A J
AJ
Departments of Biochemistry and Medicine, Queen's University, Kingston, Ontario, Canada, K7L 3N6.
Pannekoek
H
H
Nesheim
M E
ME
D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000737:chemistry*; D005452:Fluoresceins / Q000138:chemical synthesis*; D006801:Humans; D010958:Plasminogen / Q000138:chemical synthesis*; D011994:Recombinant Proteins / Q000138:chemical synthesis*; D013050:Spectrometry, Fluorescence; D010959:Tissue Plasminogen Activator / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism
D004792:Enzyme Precursors; D005452:Fluoresceins; D011994:Recombinant Proteins; C103905:plasminogen (S741C-fluorescein); D010958:Plasminogen; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator