Sphingomyelinase activity causes transbilayer lipid translocation in model and cell membranes
Sphingomyelinase activity causes transbilayer lipid translocation in model and cell membranes
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DOI：
10.1074/jbc.m303206200
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发表时间：
2003-09-26
影响因子：
4.8
通讯作者：
Goñi, FM
中科院分区：
文献类型：
--
作者：
Contreras, FX;Villar, AV;Goñi, FM
已知神经酰胺诱导膜双层中的结构重排，包括形成富含神经酰胺和缺乏神经酰胺的结构域以及水溶性溶质的流出。这份报告描述了一种新的影响神经酰胺，即诱导跨双层脂质运动。在模型（大单层囊泡）和细胞（红细胞血影）膜中均证实了这种效应，其中神经酰胺生成通过外部添加的鞘磷脂酶的作用原位发生。开发了两种不同的新测定法来检测跨双层脂质运动。其中一项试验需要制备仅在外层单层中含有神经节苷脂的囊泡并包埋神经氨酸酶。鞘磷脂酶活性诱导神经节苷脂水解的条件下，没有神经氨酸酶被释放的囊泡。第二项试验涉及制备脂质体或红细胞血影，其内小叶标记有荧光能量供体。鞘磷脂水解伴随着荧光能量转移到外部水介质中的不可渗透的受体。神经酰胺诱导的transbilayer脂质运动的另一个众所周知的属性的神经酰胺，即促进层状到非层状脂质相变的解释。因此，鞘磷脂酶在膜的一侧产生神经酰胺;神经酰胺然后诱导非层状结构中间体的瞬时形成，这导致双层中脂质不对称性的丧失，即神经酰胺与其他脂质一起的跨双层运动。由于神经酰胺的直接靶点往往是细胞内的，这些观察结果可能与通过鞘磷脂途径的跨膜信号传导机制有关。
Ceramide is known to induce structural rearrangements in membrane bilayers, including the formation of ceramide-rich and -poor domains and the efflux of aqueous solutes. This report describes a novel effect of ceramide, namely the induction of transbilayer lipid movements. This effect was demonstrated in both model (large unilamellar vesicles) and cell (erythrocyte ghost) membranes in which ceramide generation took place in situ through the action of an externally added sphingomyelinase. Two different novel assays were developed to detect transbilayer lipid movement. One of the assays required the preparation of vesicles containing a ganglioside only in the outer monolayer and entrapped neuraminidase. Sphingomyelinase activity induced ganglioside hydrolysis under conditions in which no neuraminidase was released from the vesicles. The second assay involved the preparation of liposomes or erythrocyte ghosts labeled with a fluorescent energy donor in their inner leaflets. Sphingomyelin hydrolysis was accompanied by fluorescence energy transfer to an impermeable acceptor in the outer aqueous medium. Ceramide-induced transbilayer lipid movement is explained in terms of another well known property of ceramide, namely the facilitation of lamellar to non-lamellar lipid-phase transitions. Thus, sphingomyelinase generates ceramide on one side of the membrane; ceramide then induces the transient formation of non-lamellar structural intermediates, which cause the loss of lipid asymmetry in the bilayer, i.e. the transbilayer movement of ceramide together with other lipids. As direct targets for ceramide tend to be intracellular, these observations may be relevant to the mechanism of transmembrane signaling by means of the sphingomyelin pathway.