Grechishnikova I V, Bergström F, Johansson L B, Brown R E, Molotkovsky J G
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow 117871, Russian Federation.
Biochim Biophys Acta. 1999 Aug 20;1420(1-2):189-202. doi: 10.1016/s0005-2736(99)00088-7.
New fluorescent cholesterol analogs, (22E, 20R)-3beta-hydroxy-23-(9-anthryl)-24-norchola-5,22-die ne (R-AV-Ch), and the 20S-isomer (S-AV-Ch) were synthesized, their spectral and membrane properties were characterized. The probes bear a 9-anthrylvinyl (AV) group instead of C22-C27 segment of the cholesterol alkyl chain. Computer simulations show that both of the probes have bulkier tail regions than cholesterol and predict some perturbation in the packing of membranes, particularly for R-AV-Ch. In monolayer experiments, the force-area behavior of the probes was compared with that of cholesterol, pure and in mixtures with palmitoyloleoyl phosphatidylcholine (POPC) and N-stearoyl sphingomyelin (SSM). The results show that pure R-AV-Ch occupies 35-40% more cross-sectional area than cholesterol at surface pressures below film collapse (0-22 mN/m); whereas S-AV-Ch occupies nearly the same molecular area as cholesterol. Isotherms of POPC or SSM mixed with 0.1 mol fraction of either probe are similar to isotherms of the corresponding mixtures of POPC or SSM with cholesterol. The probes show typical AV absorption (lambda 386, 368, 350 and 256 nm) and fluorescence (lambda 412-435 nm) spectra. Steady-state anisotropies of R-AV-Ch and S-AV-Ch in isotropic medium or liquid-crystalline bilayers are higher than the values obtained for other AV probes reflecting hindered intramolecular mobility of the fluorophore and decreased overall rotational rate of the rigid cholesterol derivatives. This suggestion is confirmed by time-resolved fluorescence experiments which show also, in accordance with monolayer data, that S-AV-Ch is better accommodated in POPC-cholesterol bilayers than R-AV-Ch. Model and natural membranes can be labeled by either injecting the probes via a water-soluble organic solvent or by co-lyophilizing probe and phospholipid prior to vesicle production. Detergent-solubilization studies involving 'raft' lipids showed that S-AV-Ch almost identically mimicked the behavior of cholesterol and that of R-AV-Ch was only slightly inferior. Overall, the data suggest that the AV-labeled cholesterol analogs mimic cholesterol behavior in membrane systems and will be useful in related studies.
合成了新型荧光胆固醇类似物，即(22E, 20R)-3β-羟基-23-(9-蒽基)-24-降胆甾-5,22-二烯（R-AV-Ch）和20S-异构体（S-AV-Ch），并对其光谱和膜性质进行了表征。这些探针带有一个9-蒽基乙烯基（AV）基团，取代了胆固醇烷基链的C22-C27片段。计算机模拟表明，这两种探针的尾部区域都比胆固醇更庞大，并预测膜的堆积会受到一些干扰，尤其是对于R-AV-Ch。在单层实验中，将探针与胆固醇、纯的以及与棕榈酰油酰磷脂酰胆碱（POPC）和N-硬脂酰鞘磷脂（SSM）混合的胆固醇的力-面积行为进行了比较。结果表明，在低于膜塌陷的表面压力（0-22 mN/m）下，纯R-AV-Ch比胆固醇占据的横截面积多35-40%；而S-AV-Ch占据的分子面积与胆固醇几乎相同。POPC或SSM与0.1摩尔分数的任何一种探针混合的等温线与POPC或SSM与胆固醇的相应混合物的等温线相似。探针显示出典型的AV吸收（λ386、368、350和256 nm）和荧光（λ412-435 nm）光谱。R-AV-Ch和S-AV-Ch在各向同性介质或液晶双层中的稳态各向异性高于其他AV探针的值，这反映了荧光团分子内运动受阻以及刚性胆固醇衍生物的整体旋转速率降低。时间分辨荧光实验证实了这一推测，该实验还表明，与单层数据一致，S-AV-Ch在POPC-胆固醇双层中比R-AV-Ch更易容纳。通过水溶性有机溶剂注射探针或在制备囊泡之前将探针与磷脂共冻干，可对模型膜和天然膜进行标记。涉及“筏”脂质的去污剂增溶研究表明，S-AV-Ch几乎完全模拟了胆固醇的行为，而R-AV-Ch仅略逊一筹。总体而言，数据表明AV标记的胆固醇类似物在膜系统中模拟了胆固醇的行为，将有助于相关研究。
