Zachowski A, Favre E, Cribier S, Hervé P, Devaux P F
Biochemistry. 1986 May 6;25(9):2585-90. doi: 10.1021/bi00357a046.
When human erythrocytes are incubated with spin-labeled analogues of sphingomyelin, phosphatidylcholine, phosphatidylserine, or phosphatidylethanolamine, with a short beta chain (C5) bearing a doxyl group at the fourth carbon position, the labeled lipids incorporate readily in the outer monolayer. The incorporation is followed in fresh erythrocytes by a selective inward diffusion of the amino derivatives. This observation led us to postulate the existence of a selective ATP-dependent system that would flip aminophospholipids from the outer to the inner monolayer [Seigneuret, M., & Devaux, P. F. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3751-3755]. This study further examines the nature of this selective transport and demonstrates that it is mediated by a specific membrane protein. By measurement of the initial rate of transverse diffusion of spin-labeled lipids incorporated at various concentrations in the membrane outer leaflet of packed erythrocytes, apparent Km values were determined for the phosphatidylserine and phosphatidylethanolamine analogues. A ratio of approximately equal to 1/9.4 [corrected] was obtained (KmPS/KmPE). Using spin-labels bearing either a 14N or a 15N isotope, we have carried out competition experiments allowing us to measure simultaneously the transport of two different phospholipids. By this procedure, we show that phosphatidylserine and phosphatidylethanolamine compete for the same transport site but that phosphatidylserine has a higher affinity, in agreement with a lower apparent Km. On the other hand, the slow diffusion of the phosphatidylcholine or sphingomyelin analogues has no influence on the transport of phosphatidylserine or phosphatidylethanolamine. Experiments carried out in ghosts loaded with ATP enabled us to determine the activation energies for phosphatidylserine and phosphatidylcholine transverse diffusion.(ABSTRACT TRUNCATED AT 250 WORDS)
当人红细胞与鞘磷脂、磷脂酰胆碱、磷脂酰丝氨酸或磷脂酰乙醇胺的自旋标记类似物一起孵育时，这些类似物具有短的β链（C5），在第四个碳位置带有一个多昔基团，标记的脂质很容易掺入外层单分子层。在新鲜红细胞中，掺入后会发生氨基衍生物的选择性向内扩散。这一观察结果使我们推测存在一种选择性的ATP依赖系统，该系统会将氨基磷脂从外层单分子层翻转到内层单分子层[塞涅雷，M.，& 德沃，P. F.（1984年）美国国家科学院院刊81，3751 - 3755]。本研究进一步考察了这种选择性转运的性质，并证明它是由一种特定的膜蛋白介导的。通过测量在不同浓度下掺入密集红细胞膜外小叶的自旋标记脂质的横向扩散初始速率，确定了磷脂酰丝氨酸和磷脂酰乙醇胺类似物的表观Km值。得到了约等于1/9.4[校正后]的比值（KmPS/KmPE）。使用带有14N或15N同位素的自旋标记物，我们进行了竞争实验，从而能够同时测量两种不同磷脂的转运。通过这个过程，我们表明磷脂酰丝氨酸和磷脂酰乙醇胺竞争同一个转运位点，但磷脂酰丝氨酸具有更高的亲和力，这与较低的表观Km一致。另一方面，磷脂酰胆碱或鞘磷脂类似物的缓慢扩散对磷脂酰丝氨酸或磷脂酰乙醇胺的转运没有影响。在加载了ATP的血影中进行的实验使我们能够确定磷脂酰丝氨酸和磷脂酰胆碱横向扩散的活化能。（摘要截短于250字）
