Brown R E, Anderson W H, Kulkarni V S
Hormel Institute, University of Minnesota, Austin 55912-3698, USA.
Biophys J. 1995 Apr;68(4):1396-405. doi: 10.1016/S0006-3495(95)80312-7.
As determined by freeze fracture electron microscopy, increasing levels of bovine brain galactosylceramide (GalCer) altered the surface structure of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayers by inducing a striking "macro-ripple" phase in the larger, multilamellar lipid vesicles at GalCer mole fractions between 0.4 and 0.8. The term "macro-ripple" phase was used to distinguish it from the P beta' ripple phase observed in saturated, symmetric-chain length phosphatidylcholines. Whereas the P beta' ripple phase displays two types of corrugations, one with a wavelength of 12-15 nm and the other with a wavelength of 25-35 nm, the macro-ripple phase occurring in GalCer/POPC dispersions was of one type with a wavelength of 100-110 nm. Also, in contrast to the extended linear arrays of adjacent ripples observed in the P beta' ripple phase, the macro-ripple phase of GalCer/POPC dispersions was interrupted frequently by packing defects resulting from double dislocations and various disclinations and, thus, appeared to be continuously twisting and turning. Control experiments verified that the macro-ripple phase was not an artifact of incomplete lipid mixing or demixing during preparation. Three different methods of lipid mixing were compared: a spray method of rapid solvent evaporation, a sublimation method of solvent removal, and solvent removal using a rotary evaporation apparatus. Control experiments also revealed that the macro-ripple phase was observed regardless of whether lipid specimens were prepared by either ultra-rapid or manual plunge freezing methods as well as either in the presence or absence of the cryo-protectant glycerol. The macro-ripple phase was always observed in mixtures that were fully annealed by incubation above the main thermal transition of both POPC and bovine brain GalCer before rapid freezing. If the GalCer mixed with POPC contained only nonhydroxy acyl chains or only 2-hydroxy acyl chains, then the occurrence of macro-ripple phase decreased dramatically.
通过冷冻断裂电子显微镜测定，随着牛脑半乳糖神经酰胺（GalCer）水平的增加，在GalCer摩尔分数为0.4至0.8的较大的多层脂质囊泡中，1-棕榈酰-2-油酰基磷脂酰胆碱（POPC）双层膜的表面结构发生改变，诱导出显著的“大波纹”相。术语“大波纹”相用于将其与在饱和对称链长磷脂酰胆碱中观察到的Pβ'波纹相区分开来。虽然Pβ'波纹相显示出两种类型的波纹，一种波长为12 - 15纳米，另一种波长为25 - 35纳米，但在GalCer/POPC分散体中出现的大波纹相是一种波长为100 - 110纳米的类型。此外，与在Pβ'波纹相中观察到的相邻波纹的延伸线性阵列不同，GalCer/POPC分散体的大波纹相经常被双位错和各种位错引起的堆积缺陷打断，因此似乎在不断地扭曲和转动。对照实验证实，大波纹相不是制备过程中脂质不完全混合或分离的假象。比较了三种不同的脂质混合方法：快速溶剂蒸发的喷雾法、溶剂去除的升华法以及使用旋转蒸发仪去除溶剂的方法。对照实验还表明，无论脂质标本是通过超快速还是手动骤冷冷冻方法制备的，以及是否存在冷冻保护剂甘油，都能观察到大波纹相。在快速冷冻之前，通过在高于POPC和牛脑GalCer的主要热转变温度下孵育使混合物完全退火的情况下，总是能观察到大波纹相。如果与POPC混合的GalCer仅包含非羟基酰基链或仅包含2 - 羟基酰基链，那么大波纹相的出现会显著减少。
