Dekkers D W, Comfurius P, Schroit A J, Bevers E M, Zwaal R F
Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, The Netherlands.
Biochemistry. 1998 Oct 20;37(42):14833-7. doi: 10.1021/bi981011w.
The outward movement (flop) of fluorescently labeled analogues of phosphatidylserine (PS) and phosphatidylcholine (PC) in human and murine red blood cells (RBC) was examined. 1-Oleoyl-2-[6(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl (C6-NBD) analogues of PS and PC were incorporated in the inner leaflet of the plasma membrane through the action of aminophospholipid translocase or through equilibration upon prolonged incubation, respectively. After removal of noninternalized probe, externalization of C6-NBD-PS or C6-NBD-PC from the inner to outer leaflet was monitored by continuous incubation of the cells in the presence of bovine serum albumin. Flop rates for both probes in intact human RBC were virtually identical (t1/2 approximately 1.5 h), confirming earlier findings by Bitbol et al. [Bitbol, M., et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 6783-6787] and Connor et al. [Connor, J., et al. (1992) J. Biol. Chem. 267, 19412-19417]. Flop activity in resealed RBC ghosts could only be found upon coinclusion of both ATP and oxidized glutathione (GSSG). Furthermore, flop in intact cells was sensitive to verapamil (IC50 = 5-7 microM), vincristine (IC50 = 20 microM), and indomethacin (IC50 = 50 microM), suggesting the involvement of proteins conferring multidrug resistance (MDR). Experiments with RBC from knock-out mice for multidrug resistance P-glycoproteins (Mdr1a/1b-/- and Mdr2-/-) and multidrug resistance protein 1 (Mrp1-/-) revealed that Mrp1 is responsible for the observed flop of the fluorescent lipid analogues. We found no indications for outward transport of endogenous PS by any of these drug-transporting proteins as measured by a sensitive prothrombinase assay. Neither aminophospholipid translocase nor Ca2+-induced lipid scramblase activities were affected in RBC of these knock-out mice. We conclude that lipid floppase activity, as detected with lipid probes, reflects the activity of MRP1 recognizing the modified lipid analogues as xenobiotics to be expelled from the cell.
对人源和鼠源红细胞（RBC）中磷脂酰丝氨酸（PS）和磷脂酰胆碱（PC）的荧光标记类似物的外向运动（翻转）进行了检测。PS和PC的1-油酰基-2-[6（7-硝基苯并-2-恶唑-1,3-二氮杂环丁烷-4-基）氨基]己酰（C6-NBD）类似物分别通过氨基磷脂转位酶的作用或长时间孵育后的平衡作用，掺入质膜的内小叶中。去除未内化的探针后，通过在牛血清白蛋白存在下连续孵育细胞，监测C6-NBD-PS或C6-NBD-PC从内小叶向外小叶的外化过程。完整人RBC中两种探针的翻转速率几乎相同（t1/2约为1.5小时），证实了Bitbol等人[Bitbol, M.,等人（1988年）美国国家科学院院刊85, 6783 - 6787]和Connor等人[Connor, J.,等人（1992年）生物化学杂志267, 19412 - 19417]早期的发现。只有在同时包含ATP和氧化型谷胱甘肽（GSSG）时，才能在重封的RBC空泡中检测到翻转活性。此外，完整细胞中的翻转对维拉帕米（IC50 = 5 - 7 microM）、长春新碱（IC50 = 20 microM）和吲哚美辛（IC50 = 50 microM）敏感，表明赋予多药耐药性（MDR）的蛋白质参与其中。对多药耐药P-糖蛋白（Mdr1a/1b-/-和Mdr2-/-）和多药耐药蛋白1（Mrp1-/-）基因敲除小鼠的RBC进行的实验表明，Mrp1负责观察到的荧光脂质类似物的翻转。通过灵敏的凝血酶原酶测定法测量，我们没有发现这些药物转运蛋白中有任何一种介导内源性PS外向转运的迹象。这些基因敲除小鼠的RBC中，氨基磷脂转位酶和Ca2+诱导的脂质翻转酶活性均未受到影响。我们得出结论，用脂质探针检测到的脂质翻转酶活性反映了MRP1将修饰的脂质类似物识别为要从细胞中排出的外源性物质的活性。
