Basáñez G, Ruiz-Argüello M B, Alonso A, Goñi F M, Karlsson G, Edwards K
Departamento de Bioquímica, Universidad del País Vasco, Bilbao, Spain.
Biophys J. 1997 Jun;72(6):2630-7. doi: 10.1016/S0006-3495(97)78906-9.
Cryo-transmission electron microscopy has been applied to the study of the changes induced by phospholipase C on large unilamellar vesicles containing phosphatidylcholine, as well as to the action of sphingomyelinase on vesicles containing sphingomyelin. In both cases vesicle aggregation occurs as the earliest detectable phenomenon; later, each system behaves differently. Phospholipase C induces vesicle fusion through an intermediate consisting of aggregated and closely packed vesicles (the "honeycomb structure") that finally transforms into large spherical vesicles. The same honeycomb structure is also observed in the absence of enzyme when diacylglycerols are mixed with the other lipids in organic solution, before hydration. In this case the sample then evolves toward a cubic phase. The fact that the same honeycomb intermediate can lead to vesicle fusion (with enzyme-generated diacylglycerol) or to a cubic phase (when diacylglycerol is premixed with the lipids) is taken in support of the hypothesis according to which a highly curved lipid structure ("stalk") would act as a structural intermediate in membrane fusion. Sphingomyelinase produces complete leakage of vesicle aqueous contents and an increase in size of about one-third of the vesicles. A mechanism of vesicle opening and reassembling is proposed in this case.
冷冻透射电子显微镜已被应用于研究磷脂酶C对含有磷脂酰胆碱的大单层囊泡所诱导的变化，以及鞘磷脂酶对含有鞘磷脂的囊泡的作用。在这两种情况下，囊泡聚集是最早可检测到的现象；之后，每个系统的行为有所不同。磷脂酶C通过由聚集且紧密堆积的囊泡组成的中间体（“蜂窝结构”）诱导囊泡融合，该中间体最终转变为大的球形囊泡。当在水合前将二酰基甘油与有机溶液中的其他脂质混合时，在没有酶的情况下也观察到相同的蜂窝结构。在这种情况下，样品随后会演变成立方相。相同的蜂窝中间体可导致囊泡融合（由酶产生二酰基甘油时）或立方相（当二酰基甘油与脂质预混合时）这一事实，支持了如下假说：高度弯曲的脂质结构（“柄”）将作为膜融合的结构中间体。鞘磷脂酶会导致囊泡水相内容物完全泄漏，并且囊泡大小增加约三分之一。在这种情况下提出了一种囊泡开放和重新组装的机制。
