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  • Title: Structural domains of streptokinase involved in the interaction with plasminogen.
    Author: Rodríguez P, Fuentes P, Barro M, Alvarez JG, Muñoz E, Collen D, Lijnen HR.
    Journal: Eur J Biochem; 1995 Apr 01; 229(1):83-90. PubMed ID: 7744053.
    Abstract:
    Two fragments of recombinant streptokinase, comprising amino acids Val143-Lys293 (17-kDa rSK) or Val143-Lys386 (26-kDa rSK), were cloned and expressed in Escherichia coli, purified to homogeneity and their interactions with plasmin(ogen) were evaluated. Both 17-kDa rSK and 26-kDa rSK bound to plasminogen with a 1:1 stoichiometry and with affinity constants of 3.0 x 10(8) M-1 and 12 x 10(8) M-1, respectively, as compared to 6.3 x 10(8) M-1 for the binding of intact recombinant streptokinase to plasminogen. Binding of 17-kDa rSK to plasminogen-Sepharose was displaced by addition of increasing concentrations of recombinant streptokinase, whereas bound recombinant streptokinase was not displayed by 17-kDa rSK. In equimolar mixtures of plasminogen and 26-kDa rSK, the appearance of amidolytic activity as monitored with a chromogenic substrate, was significantly delayed compared to the equimolar mixture with recombinant streptokinase (60% of the maximal activity after 30 min, compared to maximum activity within < or = 2 min). In contrast, no amidolytic activity was generated in equimolar mixtures of plasminogen and 17-kDa rSK. Plasminogen was rapidly activated by catalytic amounts (1:100 molar ratio) of recombinant streptokinase (60-70% within 10-15 min), whereas only 4% of the plasminogen was activated within 60 min with 26-kDa rSK, and no plasmin was generated with 17-kDa rSK. Complexes of plasmin with 17-kDa rSK or with 26-kDa rSK were very rapidly inhibited by alpha 2-antiplasmin (apparent second-order inhibition rate constant of approximately 2 x 10(7) M-1 s-1), whereas the complex with recombinant streptokinase was resistant to inhibition. With 26-kDa rSK, inhibition by alpha 2-antiplasmin resulted in dissociation of the complexes and recycling of functionally active 26-kDa rSK to other plasminogen molecules; 17-kDa rSK, in contrast, remained associated with the plasmin-alpha 2-antiplasmin complex. These findings suggest that different regions of the streptokinase molecule are involved in binding to plasminogen, in active-site exposure, and in impairment of the inhibition of plasmin by alpha 2-antiplasmin. Thus, the 17-kDa region spanning Val143-Lys293 in streptokinase mediates its binding to plasminogen but does not induce activation. Furthermore, this region does not interfere with the inhibition of the complex with plasmin by alpha 2-antiplasmin.
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