Lentz B R, Barrow D A, Hoechli M
Biochemistry. 1980 Apr 29;19(9):1943-54. doi: 10.1021/bi00550a034.
We have investigated the phase behavior of dipalmitoylphosphatidylcholine-cholesterol bilayers using both the fluorescence of bilayer-associated 1,6-diphenyl-1,3,5-hexatriene (DPH) and freeze-fracture electron microscopy to elucidate specimen structure. Arrhenius analysis of the fluorescence-derived "microviscosity" parameter reveals temperature-induced structural changes in these membranes. In addition, isotherms of DPH fluorescence anisotropy and total intensity are used to detect alterations in membrane structure with varying cholesterol content. Freeze-fracture electron microscopic studies, utilizing rapid "jet-freezing" techniques, show strikingly different fracture-face morphologies for different combinations of sample cholesterol content and temperature. A phase diagram is proposed that offers a unifying interpretation of the fluorescence and freeze-fracture results. In this interpretation, inflections in temperature-scanning and isothermal fluorescence measurements reveal phase lines in the dipalmitoylphosphatidylcholine-cholesterol membranes Two-phase regions of the proposed phase diagram correspond to samples showing two coexisting fracture-face morphologies, while single-phase regions produce membranes having only one clearly identifiable structure. The proposed phase diagram provides an explanation for several conflicting literature proposals of stoichiometries for phosphatidylcholine-cholesterol complexes in membranes. These stoichiometric complexes correspond to the boundaries of two-phase areas in the gel region of the phase diagram. To better approximate the effect of cholesterol on natural membranes, the structure of egg phosphatidylcholine-cholesterol multilamellar vesicles was also investigated by using DPH fluorescence. The results for this complex natural phospholipid system are interpreted by comparison with the synthetic phospholipid results.
我们利用与双层相关的1,6 - 二苯基 - 1,3,5 - 己三烯（DPH）的荧光以及冷冻蚀刻电子显微镜来研究二棕榈酰磷脂酰胆碱 - 胆固醇双层的相行为，以阐明样品结构。对荧光衍生的“微粘度”参数进行阿累尼乌斯分析，揭示了这些膜中温度诱导的结构变化。此外，DPH荧光各向异性和总强度的等温线用于检测不同胆固醇含量下膜结构的变化。利用快速“喷射冷冻”技术进行的冷冻蚀刻电子显微镜研究表明，不同的样品胆固醇含量和温度组合具有截然不同的断裂面形态。提出了一个相图，对荧光和冷冻蚀刻结果提供了统一的解释。在此解释中，温度扫描和等温荧光测量中的拐点揭示了二棕榈酰磷脂酰胆碱 - 胆固醇膜中的相线。所提出的相图的两相区域对应于显示两种共存断裂面形态的样品，而单相区域产生仅具有一种清晰可识别结构的膜。所提出的相图为膜中磷脂酰胆碱 - 胆固醇复合物化学计量的几个相互矛盾的文献提议提供了解释。这些化学计量复合物对应于相图凝胶区域中两相区域的边界。为了更好地近似胆固醇对天然膜的影响，还使用DPH荧光研究了卵磷脂酰胆碱 - 胆固醇多层囊泡的结构。通过与合成磷脂结果比较来解释这个复杂天然磷脂系统的结果。
