Haverstick D M, Glaser M
Department of Biochemistry, University of Illinois, Urbana 61801.
J Cell Biol. 1988 Jun;106(6):1885-92. doi: 10.1083/jcb.106.6.1885.
Large vesicles (5-10-micron in diameter) were formed in the presence of phospholipids fluorescently labeled on the acyl chain and visualized using a fluorescence microscope, charge-coupled-device camera and digital image processor. When such vesicles contained a fluorescent phosphatidic acid (PA) and were exposed to 2 mM CaCl2 or 0.5 mM PrCl3, it was possible to visualize PA-enriched domains within the vesicles. Calcium-induced domain formation was reversible in the presence of 4 mM EGTA. Vesicles were formed containing fluorescent PA on either the inner or outer leaflet of the bilayer and the patching and dissolution of patching were studied under conditions where calcium was present on the outside of the vesicle and where calcium was distributed across the bilayer. In addition, vesicles were formed with two different fluorescent PA's, one on the inner leaflet and a different one on the outer leaflet of the bilayer. The results of the experiments show that in vesicles formed primarily with naturally occurring phospholipids such as egg phosphatidylcholine or brain phosphatidylethanolamine, there was no coordinate action of the two leaflets of the bilayer. An exception to this was found, however, if the vesicles were formed in the presence of primarily dioleoyl phospholipids (greater than 95 mol %). In these vesicles there was a coordinate or coupled response to calcium by the two leaflets of the bilayer. In most cases, however, the two leaflets of the bilayer showed independent or uncoupled domain formation.
在存在酰基链上荧光标记的磷脂的情况下形成了大囊泡（直径5 - 10微米），并使用荧光显微镜、电荷耦合器件相机和数字图像处理器进行可视化观察。当此类囊泡含有荧光磷脂酸（PA）并暴露于2 mM氯化钙或0.5 mM氯化镨时，有可能观察到囊泡内富含PA的区域。在4 mM乙二醇双四乙酸（EGTA）存在的情况下，钙诱导的区域形成是可逆的。形成的囊泡在双层膜的内叶或外叶上含有荧光PA，并在囊泡外部存在钙以及钙分布在双层膜上的条件下研究了斑块的形成和溶解。此外，形成的囊泡在双层膜的内叶和外叶上分别含有两种不同的荧光PA。实验结果表明，在主要由天然存在的磷脂如鸡蛋磷脂酰胆碱或脑磷脂酰乙醇胺形成的囊泡中，双层膜的两个叶没有协同作用。然而，如果囊泡是在主要存在二油酰磷脂（大于95摩尔%）的情况下形成的，则发现了一个例外。在这些囊泡中，双层膜的两个叶对钙有协同或耦合反应。然而，在大多数情况下，双层膜的两个叶显示出独立或非耦合的区域形成。
