Goins B, Masserini M, Barisas B G, Freire E
Biophys J. 1986 Apr;49(4):849-56. doi: 10.1016/S0006-3495(86)83714-6.
The lateral diffusion coefficient of ganglioside GM1 incorporated into preformed dimyristoylphosphatidylcholine (DMPC) vesicles has been investigated under a variety of conditions using the technique of fluorescence photobleaching recovery. For these studies the fluorescent probe 5-(((2-Carbohydrazino)methyl)thio)acetyl) amino eosin was covalently attached to the periodate-oxidized sialic acid residue of ganglioside GM1. This labeled ganglioside exhibited a behavior similar to that of the intact ganglioside, and was able to bind cholera toxin. The lateral diffusion coefficient of the ganglioside was dependent upon the gel-liquid crystalline transition of DMPC. Above Tm the lateral diffusion coefficient of the ganglioside was 4.7 X 10(-9) cm2 s-1 (with greater than 80% fluorescence recovery). This diffusion coefficient is significantly slower than the one previously observed for phospholipids in DMPC bilayers. The addition of increasing amounts of ganglioside, up to a maximum of 10 mol %, did not have a significant effect on the lateral diffusion coefficient or in the percent recovery. At 30 degrees C, the lateral mobility of ganglioside GM1 was not affected by the presence of 5 mM Ca2+, suggesting that, at least above Tm, Ca2+ does not induce a major perturbation in the lateral organization of the ganglioside molecules. The addition of stoichiometric amounts of cholera toxin to samples containing either 1 or 10 mol % ganglioside GM1 produced only a small decrease in the measured diffusion coefficient. The fluorescence recovery after photobleaching experiments were complemented with excimer formation experiments using pyrene-phosphatidylcholine. Above the transition temperature the presence of 10 mol % ganglioside GMI induced a large decrease in the rate of excimer formation. These results also indicated that the addition of ganglioside GMI to phospholipid bilayer vesicles induces a significant restriction in the lateral mobility parameters of the lipid bilayer and that the presence of Ca2' does not have a further effect in the mobility of the probe molecules.
利用荧光漂白恢复技术，在多种条件下研究了掺入预先形成的二肉豆蔻酰磷脂酰胆碱（DMPC）囊泡中的神经节苷脂GM1的侧向扩散系数。在这些研究中，荧光探针5-(((2-氨基甲酰肼基)甲基)硫代)乙酰基)氨基曙红共价连接到神经节苷脂GM1的高碘酸盐氧化的唾液酸残基上。这种标记的神经节苷脂表现出与完整神经节苷脂相似的行为，并且能够结合霍乱毒素。神经节苷脂的侧向扩散系数取决于DMPC的凝胶-液晶转变。高于熔点（Tm）时，神经节苷脂的侧向扩散系数为4.7×10^(-9) cm² s⁻¹（荧光恢复率大于80%）。该扩散系数明显慢于先前在DMPC双层中观察到的磷脂的扩散系数。添加量增加至最多10 mol%的神经节苷脂，对侧向扩散系数或恢复百分比没有显著影响。在30℃时，5 mM Ca²⁺的存在不影响神经节苷脂GM1的侧向流动性，这表明至少在高于Tm时，Ca²⁺不会在神经节苷脂分子的侧向组织中引起重大扰动。向含有1或10 mol%神经节苷脂GM1的样品中加入化学计量的霍乱毒素，仅使测得的扩散系数略有下降。光漂白实验后的荧光恢复通过使用芘磷脂酰胆碱的准分子形成实验得到补充。在转变温度以上，10 mol%神经节苷脂GMI的存在导致准分子形成速率大幅下降。这些结果还表明，向磷脂双层囊泡中添加神经节苷脂GMI会显著限制脂质双层的侧向流动性参数，并且Ca²⁺的存在对探针分子的流动性没有进一步影响。
