Koppaka V, Talbot W F, Zhai X, Lentz B R
Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, 27599-7260, USA.
Biophys J. 1997 Nov;73(5):2638-52. doi: 10.1016/S0006-3495(97)78293-6.
Factor Va is an essential protein cofactor of the enzyme factor Xa, which activates prothrombin to thrombin during blood coagulation. Peptides with an apparent Mr of approximately 94,000 (heavy chain; HC) and approximately 74,000 or 72,000 (light chain; LC) interact in the presence of Ca2+ to form active Va. The two forms of Va-LC differ in their carboxyl-terminal C2 domain. Using Va reconstituted with either LC form, we examined the effects of the two LC species on membrane binding and on the activity of membrane-bound Va. We found that 1) Va composed of the 72,000 LC bound only slightly more tightly to membranes composed of a mixture of neutral and acidic lipids, the Kd being reduced by a factor of approximately 3 at 5 mM and by a factor of 6 at 2 mM Ca2+. 2) The two forms of Va seemed to undergo different conformational changes when bound to a membrane. 3) The activity of bovine Va varied somewhat with LC species, the difference being greatest at limiting Xa concentration. We have also addressed the role of the two Va peptides in membrane lipid rearrangements and binding: 1) Va binding increased lateral packing density in mixed neutral/acidic lipid membranes. In the solid phase, Va-HC had no effect, whereas Va-LC and whole Va had similar but small effects. In the fluid phase, Va-HC and whole Va both altered membrane packing, with Va-HC having the largest effect. 2) Va-HC bound reversibly and in a Ca2+-independent fashion to membranes composed of neutral phospholipid (Kd, approximately 0.3 microM; stoichiometry approximately 91). High ionic strength had little effect on binding. 3) The substantial effect of Va on packing within neutral phospholipid membranes was mimicked by Va-HC. 4) Based on measurements of membrane phase behavior, binding of Va or its peptide components did not induce thermodynamically discernible lateral membrane domains. These results suggest that the membrane association of factor Va is a complex process involving both chains of Va, changes in lipid packing, and changes in protein structure.
因子Va是酶因子Xa的一种必需蛋白辅因子，在血液凝固过程中，因子Xa将凝血酶原激活为凝血酶。表观分子量约为94,000的肽段（重链；HC）与约74,000或72,000的肽段（轻链；LC）在Ca2+存在下相互作用形成活性Va。两种形式的Va-LC在其羧基末端C2结构域有所不同。使用与任一轻链形式重构的Va，我们研究了两种轻链种类对膜结合以及膜结合型Va活性的影响。我们发现：1）由72,000轻链组成的Va与由中性和酸性脂质混合物构成的膜的结合仅略微紧密一些，在5 mM Ca2+时解离常数（Kd）降低约3倍，在2 mM Ca2+时降低6倍。2）两种形式的Va在与膜结合时似乎经历了不同的构象变化。3）牛源Va的活性随轻链种类略有变化，在Xa浓度极限时差异最大。我们还探讨了两种Va肽段在膜脂重排和结合中的作用：1）Va的结合增加了中性/酸性混合脂质膜中的侧向堆积密度。在固相时，Va-HC没有作用，而Va-LC和完整Va有相似但较小的作用。在液相时，Va-HC和完整Va都改变了膜的堆积，其中Va-HC的作用最大。2）Va-HC以Ca2+非依赖性方式可逆地结合到由中性磷脂构成的膜上（Kd约为0.3 microM；化学计量比约为91）。高离子强度对结合影响很小。3）Va-HC模拟了Va对中性磷脂膜内堆积的显著影响。4）基于膜相行为的测量，Va或其肽段成分的结合并未诱导出热力学上可辨别的侧向膜结构域。这些结果表明，因子Va与膜的结合是一个复杂的过程，涉及Va的两条链、脂质堆积的变化以及蛋白质结构的变化。
