CHARACTERIZATION OF THE INTERACTION BETWEEN PLASMINOGEN AND STAPHYLOKINASE
CHARACTERIZATION OF THE INTERACTION BETWEEN PLASMINOGEN AND STAPHYLOKINASE
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DOI：
10.1111/j.1432-1033.1994.tb20005.x
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发表时间：
1994-08-15
期刊：
影响因子：
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通讯作者：
COLLEN, D
中科院分区：
文献类型：
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作者：
LIJNEN, HR;DECOCK, F;COLLEN, D
通过实时生物特异性相互作用分析测定重组葡激酶（SakSTAR）与纤溶酶（ogen）之间相互作用的结合参数[结合（k（a））和解离（k（d））速率常数和亲和常数（K-a = k（a）/k（d））]。SakSTAR与天然人Glu-纤溶酶原结合的K-a值为0.93 × 10（8）M（-1），而与[S741 A]重组纤溶酶原或Lys-[S741 A]重组纤溶酶原（活性位点Ser 741被丙氨酸替代的完整或蛋白水解降解的纤溶酶原）结合的K-a值分别为2.0 × 10（8）M（-1）和1.6 × 10（8）M（-1）。SakSTAR与活性纤溶酶或活性位点封闭的纤溶酶结合，K-a值分别为4.0x10（8）M（-1）和8.4X10（8）M（-1），而活性位点封闭的LMM-纤溶酶（缺乏Kringles 1-4的纤溶酶衍生物）和纤溶酶B链结合的K-a值分别为1.0x10（8）M（-1）和0.49X10（8）M（-1）。赖氨酸结合位点I（由Kringle 1-3组成的纤溶酶原衍生物）和赖氨酸结合位点II（由Kringle 4组成的纤溶酶原衍生物）以低得多的亲和力结合（K-a值分别为1.2 × 10（5）M（-1）和2.9 × 10（5）M（-1））。这些纤溶酶原衍生物与链激酶的结合以相似的相对K-a值发生。纤溶酶-SakSTAR复合物与链激酶的结合和纤溶酶-链激酶复合物与SakSTAR的结合的K-a值分别比游离纤溶酶的值低44倍和30倍。纤溶酶原与失活突变体[M26 R] Sak 42 D或[M26 A] Sak 42 D结合的K-a这些结果表明：（a）葡激酶对Glu-纤溶酶原和Lysplasminogen的亲和力相当：（B）纤溶酶分子中的活性位点不是结合所必需的;（c）纤溶酶原的Kringle结构1-4对葡激酶的纤溶酶原结合没有显著贡献;（d）葡激酶中的Met 26对于其与纤溶酶原的高亲和力结合是重要的;（e）纤溶酶上葡激酶和链激酶的结合位点至少部分重叠。
Binding parameters [association (k(a)) and dissociation (k(d)) rate constants, and affinity constants (K-a = k(a)/k(d))] for the interaction between recombinant staphylokinase (SakSTAR) and plasmin(ogen) were determined by real-time biospecific interaction analysis. The K-a value for binding of SakSTAR to native human Glu-plasminogen was 0.93X10(8)M(-1) as compared to 2.0X10(8)M(-1) and 1.6X10(8)M(-1), respectively, for the binding to [S741A]recombinant plasminogen or Lys-[S741A]recombinant plasminogen (intact or proteolytically degraded plasminogen with the active site Ser741 replaced by alanine). Binding of SakSTAR to active plasmin or to active-site blocked plasmin occurred with K-a values of 4.0x10(8)M(-1) and 8.4X10(8)M(-1), respectively, whereas active-site blocked LMM-plasmin (a plasmin derivative lacking kringles 1-4) and the plasmin B-chain bound with K-a values of 1.0x10(8)M(-1) and 0.49X10(8)M(-1), respectively. Lysine-binding site I (a plasminogen derivative consisting of kringles 1-3) and lysine-binding site II (a plasminogen derivative consisting of kringle 4) bound with much lower affinity (K-a values of 1.2x10(5)M(-1) and 2.9x10(5)M(-1), respectively). The binding of these plasminogen derivatives to streptokinase occurred with similar relative K-a values. The K-a values for binding of the plasmin-SakSTAR complex to streptokinase and binding of the plasmin-streptokinase complex to SakSTAR, were, respectively, 44-fold and 30-fold lower than the values for free plasmin. The K-a for binding of plasminogen to the inactive mutants [M26R]Sak42D or [M26A]Sak42D (site-specific mutagenesis of Met26 to arginine or alanine) were 10-20-fold lower than that of native staphylokinase.These results indicate that: (a) the affinity of staphylokinase for Glu-plasminogen and Lysplasminogen is comparable; (b) the active site in the plasmin molecule is not required for binding; (c) kringle structures 1-4 of plasminogen do not contribute significantly to plasminogen binding of staphylokinase; (d) Met26 in staphylokinase is important for its high-affinity binding to plasminogen; (e) the binding sites on plasmin for staphylokinase and streptokinase overlap at least partially.