Zachowski A, Craescu C T, Galacteros F, Devaux P F
J Clin Invest. 1985 May;75(5):1713-7. doi: 10.1172/JCI111880.
We have used spin-labeled analogues of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine to compare the transverse diffusion rates of lipids in normal and sickle erythrocytes. The beta-chain of the spin-labeled lipids was a short chain (five carbons) providing the spin labels with a relative water solubility, and hence permitting their rapid incorporation into cell membranes. The orientation of the labeled lipids in the membranes was assayed by selective chemical reduction of the nitroxide labels embedded in the outer leaflet. We have found that all three spin-labeled phospholipids are initially incorporated in the outer leaflet. Upon incubation at 4 degrees C the aminophospholipids, not the phosphatidylcholine, diffuse toward the inner leaflet within 3 h. The transverse diffusion rate of aminophospholipids is reduced by 41% (phosphatidylserine) and 14% (phosphatidylethanolamine) in homozygote sickle cells (SS) when compared with normal cells (AA) or heterozygote cells (AS or SC). At equilibrium the asymmetric distribution of spin-labeled phospholipids resulting from this selective diffusion is also reduced in SS cells when compared with AA, SC, or AS cells. This reduced asymmetry was not found in a reticulocyte-rich blood sample (hemoglobin A), indicating that the age of the cell cannot be responsible for this phenomenon. Moreover, because at low temperatures the sickling process does not occur, the observed perturbations in phospholipid organization reflect preexisting membrane abnormalities in sickle cells. Ghosts loaded with ATP give the same results. Varying the concentration of intracellular calcium had no effect on lipid diffusion, except at very high free calcium concentrations (3 microM) when diffusion was practically abolished. We suggest that membrane protein alterations may be part of the explanation of the observed abnormalities.
我们使用了磷脂酰胆碱、磷脂酰乙醇胺和磷脂酰丝氨酸的自旋标记类似物，来比较正常红细胞和镰状红细胞中脂质的横向扩散速率。自旋标记脂质的β链是短链（五个碳原子），使自旋标记物具有相对的水溶性，从而使其能够快速掺入细胞膜。通过对嵌入外叶的氮氧化物标记物进行选择性化学还原，测定标记脂质在膜中的取向。我们发现，所有三种自旋标记的磷脂最初都掺入外叶。在4℃孵育时，氨基磷脂而非磷脂酰胆碱在3小时内扩散到内叶。与正常细胞（AA）或杂合子细胞（AS或SC）相比，纯合子镰状细胞（SS）中氨基磷脂的横向扩散速率降低了41%（磷脂酰丝氨酸）和14%（磷脂酰乙醇胺）。与AA、SC或AS细胞相比，SS细胞中由这种选择性扩散导致的自旋标记磷脂的不对称分布在平衡时也减少。在富含网织红细胞的血样（血红蛋白A）中未发现这种不对称性降低，这表明细胞年龄不是造成这种现象的原因。此外，由于在低温下不会发生镰变过程，观察到的磷脂组织扰动反映了镰状细胞中预先存在的膜异常。加载ATP的空壳细胞给出相同的结果。除了在非常高的游离钙浓度（3 microM）下扩散几乎被消除外，改变细胞内钙浓度对脂质扩散没有影响。我们认为膜蛋白改变可能是观察到的异常现象的部分原因。
