ASSOCIATION OF A PROTEIN WITH MEMBRANE-VESICLES AT THE COLLISIONAL LIMIT - STUDIES WITH BLOOD-COAGULATION FACTOR-VA LIGHT CHAIN ALSO SUGGEST MAJOR DIFFERENCES BETWEEN SMALL AND LARGE UNILAMELLAR VESICLES
ASSOCIATION OF A PROTEIN WITH MEMBRANE-VESICLES AT THE COLLISIONAL LIMIT - STUDIES WITH BLOOD-COAGULATION FACTOR-VA LIGHT CHAIN ALSO SUGGEST MAJOR DIFFERENCES BETWEEN SMALL AND LARGE UNILAMELLAR VESICLES
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DOI：
10.1021/bi00398a067
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发表时间：
1987-12-01
期刊：
影响因子：
2.9
通讯作者：
NELSESTUEN, GL
中科院分区：
文献类型：
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作者：
ABBOTT, AJ;NELSESTUEN, GL
囊泡大小可以是蛋白质-膜缔合的非常敏感的调节剂。此外，碰撞极限的反应可能是许多类型的蛋白质-膜或蛋白质-受体相互作用的特征。为了定量探测这些效应，我们分析了凝血因子Va轻链（Va-LC）与半径为15-150 nm的磷脂囊泡的关联。每个囊泡的蛋白质结合位点的数量与囊泡表面积近似成比例。缔合速率接近碰撞极限，缔合反应的活化能为4.5。0.5千卡/摩尔在协议与扩散理论这种类型的相互作用在碰撞极限，所观察到的关联速率常数填充所有网站的囊泡半径的倒数近似成正比。这种一般性质对许多系统具有重要意义，例如血液凝固，包括可能较慢的缔合速率和较高的Km值，涉及整个细胞的反应相对于磷脂囊泡获得的那些。如果扩散参数是影响解离的唯一因素，则接近碰撞极限的反应的解离速率常数也应该与囊泡尺寸的倒数成比例。然而，Va-LC结合到小单层囊泡（SUV，≤ 100%）。15-nm半径）给出比结合至大单层囊泡（LUV，≥ 15-nm半径）的Va-LC更慢的解离速率。35-nm半径）。这表明LUV与SUV的内在蛋白-磷脂亲和常数KI发生了变化。在平衡条件下，缔合和解离速率的累积效应导致Va-LC对SUV的亲和力高于LUV。竞争结合研究证实了后者。此外，这两个速率常数的温度依赖性表明，完全熵驱动的结合LUVs，但很大程度上是熵驱动的结合SUV。在很大程度上是熵的相互作用被认为是离子的性质。在与LUV和SUV结合之间观察到的差异可能反映了这些类型的磷脂结构之间的热力学差异。
Vesicle size can be a very sensitive modulator of protein-membrane association. In addition, reactions at the collisional limit may be characteristic of many types of protein-membrane or protein-receptor interactions. To probe these effects quantitatively, we analyzed the association of blood clotting factor Va light chain (Va-LC) with phospholipid vesicles of 15-150-nm radius. The number of protein binding sites per vesicle was approximately proportional to vesicle surface area. Association rates approached the collisional limit, and the activation energy for the association reaction was 4.5 .+-. 0.5 kcal/mol. In agreement with diffusional theory for this type of interaction at the collisional limit, the observed association rate constant for filling all sites was approximately proportional to the inverse of vesicle radius. This general property has important implications for many systems such as blood coagulation including possible slower association rates and higher Km values for reactions involving whole cells relative to those obtained for phospholipid vesicles. Dissociation rate constants for reactions that are near the collisional limit should also be proportional to the inverse of vesicle size if diffusional parameters are the only factors influencing dissociation. However, Va-LC bound to small unilamellar vesicles (SUVs, .ltoreq. 15-nm radius) gave slower dissociation rates than Va-LC bound to large unilamellar vesicles (LUVs, .gtoreq. 35-nm radius). This indicated a change in KI, the intrinsic protein-phospholipid affinity constant for LUVs vs SUVs. The cumulative effect of association and dissociation rates resulted in higher affinity of Va-LC for SUVs than LUVs under equilibrium conditions. The latter was corroborated by competition binding studies. Furthermore, the temperature dependence of both rate constants indicated an entirely entropy-driven binding to LUVs but a largely enthalpy-driven binding to SUVs. Interactions which are largely entropic are thought to be ionic in nature. The differences observed between binding to LUVs and SUVs may reflect thermodynamic differences between these types of phospholipid structures.