Rodríguez P, Fuentes P, Barro M, Alvarez J G, Muñoz E, Collen D, Lijnen H R
Center for Molecular and Vascular Biology, University of Leuven, Belgium.
Eur J Biochem. 1995 Apr 1;229(1):83-90. doi: 10.1111/j.1432-1033.1995.tb20441.x.
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.
克隆了重组链激酶的两个片段，分别包含氨基酸Val143-Lys293（17-kDa rSK）或Val143-Lys386（26-kDa rSK），并在大肠杆菌中表达，纯化至同质，然后评估它们与纤溶酶（原）的相互作用。17-kDa rSK和26-kDa rSK均以1:1的化学计量比与纤溶酶原结合，亲和常数分别为3.0×10⁸ M⁻¹和12×10⁸ M⁻¹，而完整的重组链激酶与纤溶酶原结合的亲和常数为6.3×10⁸ M⁻¹。加入浓度不断增加的重组链激酶可取代17-kDa rSK与纤溶酶原-琼脂糖的结合，而17-kDa rSK不能取代结合的重组链激酶。在纤溶酶原和26-kDa rSK的等摩尔混合物中，用显色底物监测的酰胺水解活性的出现与重组链激酶的等摩尔混合物相比明显延迟（30分钟后为最大活性的60%，而重组链激酶在≤2分钟内达到最大活性）。相反，在纤溶酶原和17-kDa rSK的等摩尔混合物中未产生酰胺水解活性。催化量（1:100摩尔比）的重组链激酶可迅速激活纤溶酶原（10-15分钟内激活60-70%），而26-kDa rSK在60分钟内仅激活4%的纤溶酶原，17-kDa rSK则未产生纤溶酶。纤溶酶与17-kDa rSK或26-kDa rSK的复合物被α₂-抗纤溶酶非常迅速地抑制（表观二级抑制速率常数约为2×10⁷ M⁻¹ s⁻¹），而与重组链激酶的复合物对抑制有抗性。对于26-kDa rSK，α₂-抗纤溶酶的抑制导致复合物解离，功能活性的26-kDa rSK循环至其他纤溶酶原分子；相反，17-kDa rSK仍与纤溶酶-α₂-抗纤溶酶复合物结合。这些发现表明，链激酶分子的不同区域参与与纤溶酶原的结合、活性位点的暴露以及α₂-抗纤溶酶对纤溶酶抑制的损害。因此，链激酶中跨越Val143-Lys293的17-kDa区域介导其与纤溶酶原的结合，但不诱导激活。此外，该区域不干扰α₂-抗纤溶酶对与纤溶酶复合物的抑制。
