TISSUE FACTOR AND ITS EXTRACELLULAR SOLUBLE DOMAIN - THE RELATIONSHIP BETWEEN INTERMOLECULAR ASSOCIATION WITH FACTOR-VIIA AND ENZYMATIC-ACTIVITY OF THE COMPLEX
TISSUE FACTOR AND ITS EXTRACELLULAR SOLUBLE DOMAIN - THE RELATIONSHIP BETWEEN INTERMOLECULAR ASSOCIATION WITH FACTOR-VIIA AND ENZYMATIC-ACTIVITY OF THE COMPLEX
复制标题
DOI：
10.1021/bi00131a015
复制
发表时间：
1992-04-28
期刊：
影响因子：
2.9
通讯作者：
NEMERSON, Y
中科院分区：
文献类型：
--
作者：
WAXMAN, E;ROSS, JBA;NEMERSON, Y
我们发现，分离的组织因子胞外区(TF1-218；STF)的活性只有野生型跨膜Tf(TF1-263)的4%，在测量Tf：VIIa复合体将X因子转化为Xa的实验中。此外，只有当存在由磷脂酰丝氨酸和磷脂酰胆碱(30/70w/w)组成的囊泡时，STF的活性才明显。为了确定活性降低是否是由于STF对VIIa的亲和力减弱所致，我们使用平衡超速离心法、荧光各向异性和活性滴定研究了它们之间的相互作用。STF：VIIa复合体的超速离心建立了1：1的化学计量比，平衡解离常数(K(D))的上限为1 nM。该值与以丹磺酰荧光各向异性为观察对象，用STF滴定标记为VIIa的丹磺酰-D-Phe-L-Phe-Arg氯甲基酮活性中心(DF-VIIa)相一致。通过压力解离实验获得了结合作用的量化值。这些实验表明，STF与DF-VIIa相互作用的K(D)为0.59 nm(25℃)。这一值可以与用同样方法获得的DF-VIIa与重组成磷脂酰胆碱囊泡的TF1-263相互作用的K(D)为7.3 pM进行比较。DF-VIIa与STF和TF1-263相互作用的摩尔体积变化量分别为63和117mLmol-1。这些结合数据表明，STF：VIIa复合体在数量和质量上都不同于TF1-263和VIIa形成的复合体。
We find that the isolated, extracellular domain of tissue factor (TF1-218; sTF) exhibits only 4% of the activity of wild-type transmembrane TF (TF1-263) in an assay that measures the conversion of factor X to Xa by the TF:VIIa complex. Further, the activity of sTF is manifest only when vesicles consisting of phosphatidylserine and phosphatidylcholine (30/70 w/w) are present. To determine whether the decreased activity results from weakened affinity of sTF for VIIa, we studied their interaction using equilibrium ultracentrifugation, fluorescence anisotropy, and an activity titration. Ultracentrifugation of the sTF:VIIa complex established a stoichiometry of 1:1 and an upper limit of 1 nM for the equilibrium dissociation constant (K(d)). This value is in agreement with titrations of dansyl-D-Phe-L-Phe-Arg chloromethyl ketone active site labeled VIIa (DF-VIIa) with sTF using dansyl fluorescence anisotropy as the observable. Pressure dissociation experiments were used to obtain quantitative values for the binding interaction. These experiments indicate that the K(d) for the interaction of sTF with DF-VIIa is 0.59 nM (25-degrees-C). This value may be compared to a K(d) of 7.3 pM obtained by the same method for the interaction of DF-VIIa with TF1-263 reconstituted into phosphatidylcholine vesicles. The molar volume change of association was found to be 63 and 117 mL mol-1 for the interaction of DF-VIIa with sTF and TF1-263, respectively. These binding data show that the sTF:VIIa complex is quantitatively and qualitatively different from the complex formed by TF1-263 and VIIa.