The local phospholipid environment modulates the activation of blood clotting
The local phospholipid environment modulates the activation of blood clotting
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DOI：
10.1074/jbc.m607973200
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发表时间：
2007-03-02
影响因子：
4.8
通讯作者：
Morrissey, James H.
中科院分区：
文献类型：
--
作者：
Shaw, Andrew W.;Pureza, Vincent S.;Morrissey, James H.
膜结合酶的例子比比皆是，局部膜环境对其起着重要作用，包括触发血液凝固的胞外酶、血浆丝氨酸蛋白酶、VIIa因子、与完整的膜蛋白结合的组织因子。这种酶复合体的活性明显受到脂质双层组成的影响，并进一步受到组织因子在某些细胞表面进入膜微域的影响。不幸的是，关于膜微域组成如何控制因子VIIa-组织因子活性的知之甚少，因为膜系留酶催化的反应通常是在实验者无法控制酶附近的膜组成的条件下进行的。为了克服这个问题，我们使用了一种纳米级的方法，通过在包含67+/-1个磷脂分子/叶(纳米盘)的稳定双层上组装因子VIIa-组织因子复合体，从而完全控制组织因子周围的膜环境。我们研究了磷脂双层组成的局部变化如何调节因子VIIa-组织因子复合体的活性。我们还讨论了这种酶是否需要一池膜结合的蛋白质底物(因子X)来进行有效的催化，或者它是否可以有效地激活因子X，后者直接结合到与组织因子相邻的膜纳米结构域。我们已经证明，因子VIIa-组织因子复合体的完全蛋白分解活性需要极高的局部阴离子磷脂浓度，而且不需要大量的膜结合因子X来支持持续催化。
Examples abound of membrane-bound enzymes for which the local membrane environment plays an important role, including the ectoenzyme that triggers blood clotting, the plasma serine protease, factor VIIa, bound to the integral membrane protein, tissue factor. The activity of this enzyme complex is markedly influenced by lipid bilayer composition and further by tissue factor partitioning into membrane microdomains on some cell surfaces. Unfortunately, little is known about how membrane microdomain composition controls factor VIIa-tissue factor activity, as reactions catalyzed by membrane-tethered enzymes are typically studied under conditions in which the experimenter cannot control the composition of the membrane in the immediate vicinity of the enzyme. To overcome this problem, we used a nanoscale approach that afforded complete control over the membrane environment surrounding tissue factor by assembling the factor VIIa-tissue factor complex on stable bilayers containing 67 +/- 1 phospholipid molecules/leaflet (Nanodiscs). We investigated how local changes in phospholipid bilayer composition modulate the activity of the factor VIIa-tissue factor complex. We also addressed whether this enzyme requires a pool of membrane-bound protein substrate (factor X) for efficient catalysis, or alternatively if it could efficiently activate factor X, which binds directly to the membrane nanodomain adjacent to tissue factor. We have shown that full proteolytic activity of the factor VIIa-tissue factor complex requires extremely high local concentrations of anionic phospholipids and further that a large pool of membrane-bound factor X is not required to support sustained catalysis.