Olsen P H, Esmon N L, Esmon C T, Laue T M
Department of Biochemistry, University of New Hampshire, Durham 03824.
Biochemistry. 1992 Jan 28;31(3):746-54. doi: 10.1021/bi00118a016.
The two-way and three-way interactions among active-site-blocked bovine thrombin, bovine protein C, and the elastase fragment of rabbit thrombomodulin (elTM) were examined by analytical ultracentrifugation at 23.3 degrees C in 100 mM NaCl, 50 mM Tris (pH 7.65), and 1 mM benzamidine, in the presence of 0 to 5 mM calcium chloride. Thrombin and elTM form a tight (Kd less than 10(-8) M) 1:1 complex in the absence of Ca2+ that weakens with the addition of Ca2+ (Kd approximately 4 microM in 5 mM Ca2+). Without Ca2+, thrombin and protein C form a 1:1 complex (Kd approximately 1 microM) and what appears to be a 1:2 thrombin-protein C complex. The Kd for the 1:1 complex weakens over 100-fold in 5 mM CaCl2. Protein C and elTM form a Ca(2+)-independent 1:1 complex (Kd approximately 80 microM). Nearly identical binding to thrombin and elTM is observed when active-site-blocked activated bovine protein C is substituted for protein C. Thrombin inhibited by diisopropyl fluorophosphate and thrombin inhibited by a tripeptide chloromethyl ketone exhibited identical behavior in binding experiments, suggesting that the accessibility of protein C to the substrate recognition cleft of these two forms of thrombin is nearly equal. Human protein C binds with lower affinity than bovine protein C. Ternary mixtures also were examined. Protein C, elTM, and thrombin form a 1:1:1 complex which dissociates with increasing [Ca2+]. In the absence of Ca2+, protein C binds to the elTM-thrombin complex with an apparent Kd approximately 1 microM.(ABSTRACT TRUNCATED AT 250 WORDS)
在23.3摄氏度下，于含有100 mM氯化钠、50 mM Tris（pH 7.65）和1 mM苯甲脒的溶液中，在0至5 mM氯化钙存在的条件下，通过分析超速离心法研究了活性位点被阻断的牛凝血酶、牛蛋白C和兔血栓调节蛋白的弹性蛋白酶片段（elTM）之间的双向和三向相互作用。在不存在Ca2+的情况下，凝血酶和elTM形成紧密的（解离常数Kd小于10^(-8) M）1:1复合物，随着Ca2+的添加该复合物会减弱（在5 mM Ca2+中Kd约为4 microM）。在没有Ca2+时，凝血酶和蛋白C形成1:1复合物（Kd约为1 microM）以及一种似乎是1:2的凝血酶 - 蛋白C复合物。在5 mM氯化钙中，1:1复合物的Kd减弱超过100倍。蛋白C和elTM形成一种不依赖Ca2+的1:1复合物（Kd约为80 microM）。当用活性位点被阻断的活化牛蛋白C替代蛋白C时，观察到其与凝血酶和elTM的结合几乎相同。被二异丙基氟磷酸抑制的凝血酶和被三肽氯甲基酮抑制的凝血酶在结合实验中表现出相同的行为，这表明蛋白C接近这两种形式凝血酶的底物识别裂隙的可及性几乎相等。人蛋白C的结合亲和力低于牛蛋白C。还研究了三元混合物。蛋白C、elTM和凝血酶形成1:1:1复合物，该复合物会随着[Ca2+]的增加而解离。在没有Ca2+的情况下，蛋白C以约1 microM的表观Kd结合到elTM - 凝血酶复合物上。（摘要截断于250字）
