Comfurius P, Williamson P, Smeets E F, Schlegel R A, Bevers E M, Zwaal R F
University of Limburg, Cardiovascular Research Institute Maastricht. Department of Biochemistry. The Netherlands.
Biochemistry. 1996 Jun 18;35(24):7631-4. doi: 10.1021/bi9606859.
Cellular activation, accompanied by elevation of cytoplasmic Ca2+ levels, can induce a progressive loss of plasma membrane phospholipid asymmetry, resulting from increased transbilayer movement (flip-flop) of phospholipids. While this process has been demonstrated in a variety of different cells, it is most active in blood platelets. In order to test whether this lipid scrambling process is mediated by a membrane protein, platelet membranes were solubilized in cholate and fractionated by anion exchange chromatography, and fractions were reconstituted into phospholipid vesicles by detergent dialysis in the presence of small amounts of fluorescent (NBD) phospholipids. Using dithionite reduction to monitor the transbilayer location of NBD phospholipids, it was shown that addition of Ca2+ and ionomycin to vesicles reconstituted with a particular fraction results in transbilayer movement of the fluorescent phospholipid analogs from the vesicle's inner to outer leaflet. Lipid vesicles reconstituted in the absence of membrane protein, or reconstituted with another platelet membrane protein fraction, were devoid of this activity. Heating the active fraction or incubating it with pronase or the SH reagent pyridyldithioethylamine markedly diminished the ability of the vesicles to translocate fluorescent phospholipid analogs across the bilayer in response to Ca2+ and ionophore. These results argue that a membrane protein (or proteins) from blood platelets is required to catalyze Ca2+-induced transbilayer movement of phospholipids, suggesting its (or their) involvement in the loss of lipid asymmetry that can occur during cellular activation.
细胞活化伴随着细胞质Ca2+水平升高，可导致质膜磷脂不对称性逐渐丧失，这是由于磷脂跨双层运动（翻转）增加所致。虽然这一过程已在多种不同细胞中得到证实，但在血小板中最为活跃。为了测试这种脂质紊乱过程是否由膜蛋白介导，将血小板膜用胆酸盐溶解并通过阴离子交换色谱法分级分离，然后在少量荧光（NBD）磷脂存在下通过去污剂透析将各组分重构成磷脂囊泡。利用连二亚硫酸盐还原法监测NBD磷脂的跨双层位置，结果表明，向用特定组分重构的囊泡中添加Ca2+和离子霉素会导致荧光磷脂类似物从囊泡内叶向外叶的跨双层运动。在没有膜蛋白的情况下重构的脂质囊泡，或用另一种血小板膜蛋白组分重构的脂质囊泡，都没有这种活性。加热活性组分或用链霉蛋白酶或SH试剂吡啶二硫代乙胺孵育，会显著降低囊泡响应Ca2+和离子载体使荧光磷脂类似物跨双层转运的能力。这些结果表明，血小板中的一种（或多种）膜蛋白是催化Ca2+诱导的磷脂跨双层运动所必需的，这表明其（或它们）参与了细胞活化过程中可能发生的脂质不对称性丧失。
