Phospholipid flippase activity of the reconstituted P-glycoprotein multidrug transporter
Phospholipid flippase activity of the reconstituted P-glycoprotein multidrug transporter
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DOI：
10.1021/bi0024456
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发表时间：
2001-06-12
期刊：
影响因子：
2.9
通讯作者：
Sharom, FJ
中科院分区：
文献类型：
--
作者：
Romsicki, Y;Sharom, FJ
P-糖蛋白多药转运蛋白作为一种ATP动力的外排泵，用于多种疏水性药物、天然产物和肽类。该蛋白质被提议在膜的疏水内部与其底物相互作用。有间接证据表明，P-糖蛋白也可以运输，或“翻转”，短链荧光脂质小叶之间的膜。在这项研究中，我们使用的荧光猝灭技术直接表明，P-糖蛋白重组成蛋白脂质体易位各种NBD脂质从外到内小叶的双层。翻转酶的活性依赖于ATP水解的蛋白脂质体的外表面，并抑制钒酸盐。P-糖蛋白对磷脂、移位磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰丝氨酸和鞘磷脂表现出广泛的特异性。翻转的脂质衍生物包括具有长、短、不饱和和饱和酰基链的分子，以及具有共价连接至酰基链或头基的NBD基团的物质。直接估计在37 ℃下20分钟内从外叶到内叶的脂质移位程度，其范围为0.36-1.83 nmol/mg蛋白质。磷脂翻转被抑制在浓度依赖性，饱和的方式由各种底物和调制器，包括长春碱，维拉帕米，和环孢素A，和抑制的效率相关以及与Pgp结合的亲和力。综上所述，这些结果表明，P-糖蛋白进行脂质转运和药物转运通过相同的path. The转运蛋白可能是一个通用的翻转酶的疏水分子与正确的空间属性，是存在于膜内部。
The P-glycoprotein multidrug transporter acts as an ATP-powered efflux pump for a large variety of hydrophobic drugs, natural products, and peptides. The protein is proposed to interact with its substrates within the hydrophobic interior of the membrane. There is indirect evidence to suggest that P-glycoprotein can also transport, or "flip", short chain fluorescent lipids between leaflets of the membrane. In this study, we use a fluorescence quenching technique to directly show that P-glycoprotein reconstituted into proteoliposomes translocates a wide variety of NBD lipids from the outer to the inner leaflet of the bilayer. Flippase activity depended on ATP hydrolysis at the outer surface of the proteoliposome, and was inhibited by vanadate. P-Glycoprotein exhibited a broad specificity for phospholipids, and translocated phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin. Lipid derivatives that were flipped included molecules with long, short, unsaturated, and saturated acyl chains and species with the NBD group covalently linked to either acyl chains or the headgroup. The extent of lipid translocation from the outer to the inner leaflet in a 20 min period at 37 degreesC was directly estimated, and fell in the range of 0.36-1.83 nmol/mg of protein. Phospholipid flipping was inhibited in a concentration-dependent, saturable fashion by various substrates and modulators, including vinblastine, verapamil, and cyclosporin A, and the efficiency of inhibition correlated well with the affinity of binding to Pgp. Taken together, these results suggest that P-glycoprotein carries out both lipid translocation and drug transport by the same path. The transporter may be a generic flippase for hydrophobic molecules with the correct steric attributes that are present within the membrane interior.