van de Waart P, Visser A J, Hemker H C, Lindhout T
Eur J Biochem. 1987 Apr 15;164(2):337-43. doi: 10.1111/j.1432-1033.1987.tb11063.x.
We have monitored the thermotropic behavior of mixed dimyristoylglycerophosphoserine (Myr2GroPSer)/dimyristoylglycerophosphocholine (Myr2GroPCho) and Myr2GroPSer/dipalmitoylglycerophosphocholine (Pam2GroPCho) vesicles in the presence of blood-clotting factor Va, using 1,6-diphenyl-1,3, 5-hexatriene as a lipid probe. The Ca2+-independent interaction of factor Va with these vesicles caused a small increase (1-2 degrees C) in the phase transition temperature, regardless of whether Myr2GroPChe was the lower or higher-melting component of the mixed vesicles. The major effect of factor Va was to increase the polarization of diphenylhexatriene when the mixed vesicles were in the liquid crystalline phase. The protein did not change the anisotropy in the bilayer gel state. The increase in the polarization value above the transition temperature closely correlated with the amount of phospholipid-bound factor Va, as verified by a direct binding technique. In addition, we found that the affinity of factor Va for Myr2GroPSer/Myr2GroPCho and Myr2GroPSer/Pam2GroPCho greatly increased at temperatures above the transition temperatures. Time-dependent fluorescence anisotropy measurements of diphenylhexatriene embedded in vesicles in the liquid crystalline state give fluorescence decay curves which can best be fitted by two exponential functions with two rotational correlation times and a constant term. Vesicles composed of Myr2GroPSer exhibit more ordering than Myr2GroPCho vesicles. However, the order parameter of mixed vesicles composed of 40% Myr2GroPSer and 60% Myr2GroPCho (mol/mol) approached that of Myr2GroPCho. Factor Va dramatically increased the longer rotational correlation time of diphenylhexatriene embedded in mixed vesicles in the liquid crystalline state from 3.7 ns to about 17 ns. The second rank-order parameter increased only slightly, but the calculated steady-state anisotropy increased by twofold. These results indicate that the acidic phospholipid-dependent binding of factor Va to mixed vesicles has an ordering effect on the acyl chains of the acidic phospholipids in the outer layer, but leaves the bulk of the phospholipids, mainly phosphatidylcholine, unaltered. None of the factor-Va-induced alterations in the anisotropy parameters point to the occurrence of lateral phase separation.
我们使用1,6 - 二苯基 - 1,3,5 - 己三烯作为脂质探针，监测了在凝血因子Va存在下，二肉豆蔻酰甘油磷酸丝氨酸（Myr2GroPSer）/二肉豆蔻酰甘油磷酸胆碱（Myr2GroPCho）以及Myr2GroPSer/二棕榈酰甘油磷酸胆碱（Pam2GroPCho）混合囊泡的热致行为。无论Myr2GroPCho是混合囊泡中熔点较低还是较高的成分，因子Va与这些囊泡的Ca2 +非依赖性相互作用都会使相变温度小幅升高（1 - 2摄氏度）。当混合囊泡处于液晶相时，因子Va的主要作用是增加二苯基己三烯的极化。该蛋白质在双层凝胶状态下不会改变各向异性。通过直接结合技术证实，高于转变温度时极化值的增加与磷脂结合的因子Va的量密切相关。此外，我们发现因子Va对Myr2GroPSer/Myr2GroPCho和Myr2GroPSer/Pam2GroPCho的亲和力在高于转变温度时大幅增加。对液晶态囊泡中嵌入的二苯基己三烯进行时间依赖性荧光各向异性测量，得到的荧光衰减曲线可以用具有两个旋转相关时间和一个常数项的两个指数函数进行最佳拟合。由Myr2GroPSer组成的囊泡比Myr2GroPCho囊泡表现出更多的有序性。然而，由40% Myr2GroPSer和60% Myr2GroPCho（摩尔/摩尔）组成的混合囊泡的序参数接近Myr2GroPCho的序参数。因子Va显著增加了液晶态混合囊泡中嵌入的二苯基己三烯的较长旋转相关时间，从3.7纳秒增加到约17纳秒。二阶序参数仅略有增加，但计算出的稳态各向异性增加了两倍。这些结果表明，因子Va与混合囊泡的酸性磷脂依赖性结合对外层酸性磷脂的酰基链具有有序化作用，但使大部分磷脂（主要是磷脂酰胆碱）保持不变。因子Va引起的各向异性参数变化均未表明发生横向相分离。
