PROTON-INDUCED FUSION OF OLEIC ACID PHOSPHATIDYLETHANOLAMINE LIPOSOMES
PROTON-INDUCED FUSION OF OLEIC ACID PHOSPHATIDYLETHANOLAMINE LIPOSOMES
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DOI：
10.1021/bi00334a004
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发表时间：
1985-01-01
期刊：
影响因子：
2.9
通讯作者：
PAPAHADJOPOULOS, D
中科院分区：
文献类型：
--
作者：
DUZGUNES, N;STRAUBINGER, RM;PAPAHADJOPOULOS, D
由油酸和磷脂酰乙醇胺（3：7摩尔比）组成的脂质体聚集，变得不稳定，并在150 mM NaCl中融合（< pH 6.5）。利用N，N“-对亚二甲苯双猝灭氨基萘-3，6，8-三磺酸（ANTS），通过脂质体内部含水内容物的相互混合来监测融合（溴化吡啶）（DPX），用于将荧光磷脂从标记的脂质体稀释至未标记的脂质体的共振能量转移测定，浊度的不可逆变化，和快速冷冻断裂电子显微镜。去稳定化之后是由共包封的ANTS/DPX或钙黄绿素的泄漏引起的荧光增加。Ca 2+和Mg 2+也分别在3和4 mM下诱导这些囊泡的融合。融合的阈值是在低浓度（亚融合）的这些二价阳离子的存在下，在较高的pH值。由磷脂酰丝氨酸/磷脂酰乙醇胺或油酸/磷脂酰胆碱（3：7摩尔比）组成的囊泡在pH 7-4范围内不聚集、不稳定或融合，表明磷脂酰丝氨酸和磷脂酰胆碱不能分别取代油酸和磷脂酰乙醇胺用于质子诱导的膜融合。冷冻断裂复制品的油酸/磷脂酰乙醇胺脂质体冷冻1秒内的刺激与pH 5.3显示较大的囊泡和囊泡进行融合，与膜脊和区域之间的双层连续性。pH敏感性脂质体的构建可用作研究生物系统中质子诱导的膜融合和大分子细胞质递送的分子要求的模型。
Liposomes composed of oleic acid and phosphatidylethanolamine (3:7 mole ratio) aggregate, become destabilized and fuse < pH 6.5 in 150 mM NaCl. Fusion is monitored by the intermixing of internal aqueous contents of liposomes, utilizing the quenching of aminonaphthalene-3,6,8-trisulfonic acid (ANTS) by N,N''-p-xylylenebis(pyridinium bromide) (DPX) encapsulated in 2 separate populations of vesicles, a resonance energy transfer assay for the dilution of fluorescent phospholipids from labeled to unlabeled liposomes, irreversible changes in turbidity, and quick-freezing freeze-fracture electron microscopy. Destabilization is followed by the fluorescence increase caused by the leakage of coencapsulated ANTS/DPX or of calcein. Ca2+ and Mg2+ also induce fusion of these vesicles at 3 and 4 mM, respectively. The threshold for fusion is at a higher pH in the presence of low (subfusogenic) concentrations of these divalent cations. Vesicles composed of phosphatidylserine/phosphatidylethanolamine or of oleic acid/phosphatidylcholine (3:7 mole ratio) do not aggregate, destabilize, or fuse in the pH range 7-4, indicating that phosphatidylserine and phosphatidylcholine cannot be substituted for oleic acid and phosphatidylethanolamine, respectively, for proton-induced membrane fusion. Freeze-fracture replicas of oleic acid/phosphatidylethanolamine liposomes frozen within 1 s of stimulation with pH 5.3 display larger vesicles and vesicles undergoing fusion, with membrane ridges and areas of bilayer continuity between them. The construction of pH-sensitive liposomes is useful as a model for studying the molecular requirements for proton-induced membrane fusion in biological systems and for the cytoplasmic delivery of macromolecules.