Morrot G, Cribier S, Devaux P F, Geldwerth D, Davoust J, Bureau J F, Fellmann P, Herve P, Frilley B
Proc Natl Acad Sci U S A. 1986 Sep;83(18):6863-7. doi: 10.1073/pnas.83.18.6863.
The fluorescent phospholipid 1-acyl-2-[12-(7-nitrobenz-2-oxa-1,3-diazol-4- yl)aminododecanoyl]phosphatidylcholine (NBD-phosphatidylcholine) and the corresponding aminophospholipid derivatives (NBD-phosphatidylethanolamine and NBD-phosphatidylserine) were introduced in the human erythrocyte membrane by a nonspecific phospholipid exchange protein purified from corn. The lateral mobility of the fluorescent phospholipids was measured by using an extension of the classical photobleaching recovery technique that takes advantage of a modulated fringe pattern and provides a high sensitivity. In intact erythrocytes and in ghosts resealed in the presence of ATP, the fluorescence-contrast curves after photobleaching decayed biexponentially corresponding to two lateral diffusion constants. With NBD-phosphatidylcholine, the majority of the signal corresponded to a "slow" component (1.08 X 10(-9) cm2/sec at 20 degrees C), whereas with the amino derivatives the majority of the signal corresponded to a "fast" component (5.14 X 10(-9) cm2/sec at 20 degrees C). If the ghosts were resealed without ATP, the fast component of the aminophospholipids disappeared. We interpret these results as follows: (i) Provided the cells or the ghosts contain ATP, the three fluorescent phospholipids distribute spontaneously between inner and outer leaflets as endogenous phospholipids, namely NBD-phosphatidylcholine is located in the outer leaflet, while both aminophospholipids are preferentially located in the inner leaflet. (ii) The viscosity of the inner leaflet of human erythrocyte membranes is lower than that of the outer leaflet.
通过从玉米中纯化得到的非特异性磷脂交换蛋白，将荧光磷脂1-酰基-2-[12-(7-硝基苯并-2-恶唑-1,3-二氮杂萘-4-基)氨基十二烷酰基]磷脂酰胆碱（NBD-磷脂酰胆碱）以及相应的氨基磷脂衍生物（NBD-磷脂酰乙醇胺和NBD-磷脂酰丝氨酸）引入人红细胞膜。利用经典光漂白恢复技术的扩展方法测量荧光磷脂的侧向流动性，该方法利用调制条纹图案并提供高灵敏度。在完整的红细胞和在ATP存在下重新密封的血影中，光漂白后的荧光对比曲线呈双指数衰减，对应于两个侧向扩散常数。对于NBD-磷脂酰胆碱，大部分信号对应于一个“慢”成分（20℃时为1.08×10⁻⁹cm²/秒），而对于氨基衍生物，大部分信号对应于一个“快”成分（20℃时为5.14×10⁻⁹cm²/秒）。如果在没有ATP的情况下重新密封血影，氨基磷脂的快成分消失。我们对这些结果的解释如下：(i) 只要细胞或血影含有ATP，这三种荧光磷脂就会像内源性磷脂一样在内侧和外侧小叶之间自发分布，即NBD-磷脂酰胆碱位于外侧小叶，而两种氨基磷脂优先位于内侧小叶。(ii) 人红细胞膜内侧小叶的粘度低于外侧小叶。
