On the origin of sphingolipid/cholesterol-rich detergent-insoluble cell membranes: Physiological concentrations of cholesterol and sphingolipid induce formation of a detergent-insoluble, liquid-ordered lipid phase in model membranes
On the origin of sphingolipid/cholesterol-rich detergent-insoluble cell membranes: Physiological concentrations of cholesterol and sphingolipid induce formation of a detergent-insoluble, liquid-ordered lipid phase in model membranes
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DOI：
10.1021/bi971167g
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发表时间：
1997-09-09
期刊：
影响因子：
2.9
通讯作者：
London, E
中科院分区：
文献类型：
--
作者：
Ahmed, SN;Brown, DA;London, E
含有丰富鞘脂和胆固醇的洗涤剂不溶性膜碎片可以从细胞裂解物和模型膜中分离出来。我们已经提出，这些都是由在体内和体外处于液体有序相的膜产生的[Schroeder et al. (1994) Proc. Natl, Acad. Sci， U.S.A. 91, 12130-12134]。为了检测液体有序相的形成，同时避免可能的洗涤剂伪影，我们现在使用荧光猝灭来检查磷脂酰胆碱、鞘脂和胆固醇混合物的相行为。在双棕榈酰磷脂酰胆碱（DPPC）或鞘磷脂（SM）与氮氧标记磷脂酰胆碱（12SLPC）的二元混合物中发现了相分离，在23℃时，DPPC或SM富集的固体状凝胶相与12SLPC富集的液体-无序流体相共存，正如预期的那样，在低浓度的DPPC或SM下未见相分离。相反，只有一个均匀的流体相存在。在模型膜中加入33 mol %的胆固醇大大促进了相分离。在存在胆固醇的情况下，在较高的温度和/或较低浓度的DPPC或SM下，可以观察到相分离。已知DPPC或SM和胆固醇的混合物可以形成液序相。因此，在含胆固醇膜中观察到相分离的事实表明，液有序相域和液无序相域共存。在37℃时，SM/PC比接近0.25时首次发现富含SM的液有序相，此时SM占包括胆固醇在内的总脂质的17%。（这与哺乳动物细胞细胞膜的SM浓度相似或更低）。此外，通过荧光猝灭检测，含胆固醇模型膜的洗涤剂不溶性与液有序相的数量密切相关，因此，从细胞中分离出的洗涤剂不溶性膜很可能在洗涤剂提取之前就存在于液有序相中。促进液体有序相的形成可能是胆固醇和鞘脂在细胞中的重要功能，也可能是富含胆固醇和鞘脂的质膜与大多数其他细胞膜的主要区别。
Detergent-insoluble membrane fragments that are rich in sphingolipid and cholesterol can be isolated from both cell lysates and model membranes. We have proposed that these arise from membranes that are in the liquid-ordered phase both in viva and in vitro [Schroeder et al. (1994) Proc. Natl, Acad. Sci, U.S.A. 91, 12130-12134]. ln order to detect formation of the liquid-ordered phase while avoiding possible detergent artifacts, we have now used fluorescence quenching to examine the phase behavior of mixtures of phosphatidylcholines, sphingolipids, and cholesterol. Phase separation was found in binary mixtures of either dipalmitoylphosphatidylcholine (DPPC) or sphingomyelin (SM) and a nitroxide-labeled phosphatidylcholine (12SLPC), A DPPC- or SM-enriched solidlike gel phase coexisted with a 12SLPC-enriched liquid-disordered fluid phase al 23 degrees C, AS expected, phase separation was not seen at low concentrations of DPPC or SM. Instead, only a uniform fluid phase was present. including 33 mol % cholesterol in model membranes greatly promoted phase separation. Phase separation was seen at higher temperatures and/or at lower concentrations of DPPC or SM in the presence of cholesterol than in its absence, Mixtures of DPPC or SM and cholesterol are known to form the Liquid-ordered phase. Therefore, the fact that phase separation was observed in the cholesterol-containing membranes shows that liquid-ordered and liquid-disordered phase domains coexist, At 37 degrees C, the SM-enriched liquid-ordered phase was first seen at a SM/PC ratio of close to 0.25, when SM made up 17% of the total lipid including cholesterol. (This is similar to or less than the SM concentration of the plasma membranes of mammalian cells.) Furthermore, the detergent insolubility of cholesterol-containing model membranes correlated well with the amount of liquid-ordered phase as detected by fluorescence quenching, Thus, the detergent-insoluble membranes isolated from cells are likely to exist in the liquid-ordered phase prior to detergent extraction, The promotion of liquid-ordered phase formation may be an important function of cholesterol and sphingolipids in cells and may be a major distinction between the cholesterol-and sphingolipid-rich plasma membrane and most other cellular membranes.