Asymmetric transbilayer distribution of sterol across plasma membranes determined by fluorescence quenching of dehydroergosterol.
Asymmetric transbilayer distribution of sterol across plasma membranes determined by fluorescence quenching of dehydroergosterol.
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通过脱氢麦角甾醇的荧光猝灭确定甾醇跨质膜的不对称跨双层分布。
DOI：
10.1111/j.1432-1033.1982.tb06488.x
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
F. Schroeder
中科院分区：
文献类型：
--
作者：
J. Hale;F. Schroeder
报道了一种测量质膜中甾醇跨双层分布的新方法。该过程使用了荧光甾醇、脱氢麦角甾醇和化学猝灭剂三硝基苯磺酸。由于以下原因，脱氢麦角甾醇可用作甾醇的探针分子：(a)脱氢麦角甾醇不含作为报告基团的庞大侧链。 (b) 脱氢麦角甾醇在结构上与胆固醇和去莫甾醇相似，后者是 LM 成纤维细胞合成的主要甾醇。 (c) 脱氢麦角甾醇与洋地黄皂苷、菲律宾素相互作用，并作为胆固醇氧化酶的底物。 (d)二棕榈酰甘油磷酸胆碱的相变被脱氢麦角甾醇完全消除。 (e) LM 成纤维细胞的天然甾醇去氢麦角甾醇完全被脱氢麦角甾醇取代，对 LM 细胞生长、细胞倍增时间、质膜 (Na+、K+)-ATP 酶和 5'-核苷酸酶活性、微粒体 NADPH 依赖性细胞色素 C 还原酶活性和线粒体琥珀酸依赖性细胞色素 C 还原酶活性没有影响。 (f) LM 成纤维细胞的磷脂组成和甾醇/磷脂比例均未因补充脱氢麦角甾醇而改变。三硝基苯基甘氨酸的三硝基苯基或经三硝基苯磺酸处理的LM成纤维细胞或红细胞表面膜的三硝基苯基是脱氢麦角甾醇荧光的优异猝灭剂。当猝灭剂仅存在于膜的细胞外位点时，小鼠极低密度脂蛋白、LM成纤维细胞质膜、红细胞表面膜和大鼠红细胞膜中的荧光分别猝灭95 +/- 3%、20 +/- 2%、75 +/- 4%和69 +/- 4%。当脱氢麦角甾醇的浓度为 1-85 mol/100 ml 膜甾醇时，三硝基苯基残基可有效地将 LM 细胞质膜中的脱氢麦角甾醇荧光猝灭 20%。当质膜两侧都被三硝基苯基化时，超过 95% 的脱氢麦角甾醇荧光被猝灭。此外，当LM细胞与脱氢麦角甾醇、暴露的乳胶珠和作为吞噬体分离的内吞颗粒一起培养并在非穿透条件下用三硝基苯磺酸处理时，脱氢麦角甾醇的荧光被猝灭近64%。根据这些和其他结果，我们推断LM成纤维细胞质膜的内单层富含脱氢麦角甾醇。相反，红细胞膜中甾醇的分布表明外单层富集。
A new method for measurement of transbilayer distribution of sterol in plasma membranes is reported. The procedure utilized a fluorescent sterol, dehydroergosterol, and a chemical quenching agent, trinitrobenzenesulfonic acid. Dehydroergosterol was useful as a probe molecule for sterols for the following reasons, (a) Dehydroergosterol contained no bulky side chains as reporter groups. (b) Dehydroergosterol structurally resembled cholesterol and desmosterol, the primary sterol synthesized by LM fibroblasts. (c) Dehydroergosterol interacted with digitonin, filipin, and served as a substrate for cholesterol oxidase. (d) The phase transition of dipalmitoylglycerophosphocholine was completely abolished by dehydroergosterol. (e) The native sterol of LM fibroblasts, desmosterol, was completely replaced by dehydroergosterol without effect on LM cell growth, cell doubling time, plasma membrane (Na+, K+)-ATPase and 5'-nucleotidase activity, microsomal NADPH-dependent cytochrome c reductase activity, and mitochondrial succinate-dependent cytochrome c reductase activity. (f) Neither the phospholipid composition nor the sterol/phospholipid ratio of LM fibroblasts were altered by supplementation with dehydroergosterol. The trinitrophenyl group of trinitrophenylglycine or of surface membranes of LM fibroblasts or red blood cells treated with trinitrobenzenesulfonic acid was an excellent quencher of dehydroergosterol fluorescence. Fluorescence in mouse very-low-density lipoproteins, LM fibroblasts plasma membranes, red blood cell surface membranes, and in rat red blood cell membranes was quenched 95 +/- 3%, 20 +/- 2%, 75 +/- 4%, and 69 +/- 4% respectively when the quenching agent was present on only the extracellular site of the membrane. Trinitrophenyl residues effectively quenched the dehydroergosterol fluorescence in the plasma membrane of LM cells by 20% when dehydroergosterol was present from 1-85 mol/100 ml of the membrane sterol. When both sides of the plasma membrane were trinitrophenylated, greater than 95% of the dehydroergosterol fluorescence was quenched. In addition, when LM cells were cultured with dehydroergosterol, exposed latex beads, and the endocytosed particles isolated as phagosomes and treated with trinitrobenzenesulfonic acid under non-penetrating conditions, the fluorescence of the dehydroergosterol was quenched nearly 64%. From these and other results we deduced that the inner monlayer of the LM fibroblasts plasma membrane was enriched with dehydroergosterol. In contrast, the distribution of the sterol in red blood cell membranes indicated an enrichment in the outer monolayer.