Human factor VIIa and its complex with soluble tissue factor: evaluation of asymmetry and conformational dynamics by ultracentrifugation and fluorescence anisotropy decay methods.
Human factor VIIa and its complex with soluble tissue factor: evaluation of asymmetry and conformational dynamics by ultracentrifugation and fluorescence anisotropy decay methods.
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人因子 VIIa 及其与可溶性组织因子的复合物：通过超速离心和荧光各向异性衰减方法评估不对称性和构象动力学。
DOI：
10.1021/bi00063a011
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发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Ross,JB
中科院分区：
文献类型：
--
作者：
Waxman,E;Laws,WR;Laue,TM;Nemerson,Y;Ross,JB
摘要：超离心和荧光各向异性衰减测量被用来评估人凝血酶Vila（Vila）的不对称性和构象动力学，以及它与作为Vila必需辅因子的人组织因子（sTF）的可溶性截短突变体形成的复合物。沉降速度实验表明Vila和sTF-VIIa复合物都是高度不对称的。在每一种情况下，摩擦比，/球，是一致的家庭一般的椭球体范围从长到扁。荧光各向异性衰减实验用于限制可以描述Vila和sTF-VIIa的流体动力学行为的椭球族。对于Vila和sTF* VIIa复合体，旋转的扁椭圆体被消除。此外，荧光各向异性衰减数据清楚地表明，在结合sTF-VIIa失去了涉及含有酶的活性位点的结构域的节段运动。这表明sTF引起有限范围的Vila构象的稳定。这种稳定化对于TF-VIIa底物，因子X的正确识别可能是重要的。因子Vila（Vila）1与膜结合组织因子（TF）的复合是血液凝固起始中的关键步骤。TF是一种跨膜糖蛋白，由胞外结构域（残基1-219）、单个跨膜结构域（残基220-242）和胞质结构域（残基243-263）组成，胞质结构域含有硫代酯化为棕榈酸酯或硬脂酸酯的半胱氨酸残基[综述参见Bach（1988）]。当与TF复合时，Vila对其天然底物因子X（X）的丝氨酸蛋白酶活性增加许多数量级（Silverberg et al.，
Revised Manuscript Received January 6, 1993 abstract: Ultracentrifugation and fluorescence anisotropy decay measurements were used to evaluate the asymmetry and conformational dynamics of human blood clotting enzyme Vila (Vila) and the complex it forms with a soluble truncation mutant of human tissue factor (sTF) which acts as an essential cofactor for Vila. Sedimentation velocity experimentsshowed that both Vila and the sTF-VIIa complex are highly asymmetric. In each case, the friction ratio,///sphere, is consistent with a family of general ellipsoids ranging from prolate to oblate. Fluorescence anisotropy decay experiments were used to limit the family of ellipsoids which can describe the hydrodynamic behavior of Vila and sTF-VIIa. For both Vila and the sTF* VIIa complex, the oblate ellipsoid of revolution was eliminated. In addition, the fluorescence anisotropy decay data clearly show that upon binding sTF-VIIa loses a segmental motion involving a domain containing the active site of the enzyme. This suggests that sTF causes a stabilization of a limited range of Vila conformations. This stabilization may be important for proper recognition of the TF-VIIa substrate, factor X.The complexation of factor Vila (Vila) 1 with membranebound tissue factor (TF) is a critical step in the initiation of blood coagulation. TF is a transmembrane glycoprotein consisting of an extracellular domain (residues 1-219), a single transmembrane domain (residues 220-242), and a cytoplasmic domain (residues 243-263) which contains a half-cysteine residue thioesterified to palmitate or stearate [for review, see Bach (1988)]. When complexed with TF, the serine protease activity of Vila toward its natural substrate, factor X (X), is increased by many orders of magnitude (Silverberg et al.,