Isolation of an erythrocyte membrane protein that mediates Ca2+-dependent transbilayer movement of phospholipid
Isolation of an erythrocyte membrane protein that mediates Ca2+-dependent transbilayer movement of phospholipid
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DOI：
10.1074/jbc.271.29.17205
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发表时间：
1996-07-19
影响因子：
4.8
通讯作者：
Wiedmer, T
中科院分区：
文献类型：
--
作者：
Basse, F;Stout, JG;Wiedmer, T
红细胞、血小板和其他细胞中细胞内Ca 2+的升高会引发内外小叶之间质膜磷脂（PL）的快速重新分布，破坏正常的不对称分布。因此，磷脂酰丝氨酸和其他脂质通常被隔离到内小叶变得暴露在细胞表面。这种Ca 2+诱导的磷脂酰丝氨酸向活化的、损伤的或凋亡的细胞的表面的移动赋予质膜促凝血性质，其促进纤维蛋白凝固并提供通过网状内皮系统去除细胞的信号。为了鉴定介导这种Ca 2+依赖性“PL乱序酶”活性的膜的组分，我们从细胞骨架耗尽的红细胞血影的去污剂提取物中进行了具有这种活性的膜组分的纯化和重建。活性组分通过其介导7-硝基苯-2-氧杂-1，3-二氮唑-4-基标记的PL在重构的脂蛋白体小叶之间的Ca 2+依赖性再分布的能力来鉴定。该PL乱序酶活性通过多个色谱步骤与类似于37 kDa的单一多肽共洗脱，所述多肽被纯化至通过银染色分辨的表观均一性。与该蛋白带相关的活性被胰蛋白酶灭活，分离的蛋白质在蛋白脂质体中重构，介导7-硝基苯-2-氧杂-1，3-二氮唑-4-基-PL在膜小叶之间的非选择性双向转运，在20和60 μ M Ca 2+之间具有半最大活化（饱和度>100 μ M），模拟红细胞膜固有的Ca 2+依赖性跨双层脂质运动。
Elevation of intracellular Ca2+ in erythrocytes, platelets, and other cells initiates rapid redistribution of plasma membrane phospholipids (PL) between inner and outer leaflets, collapsing the normal asymmetric distribution. Consequently, phosphatidylserine and other lipids normally sequestered to the inner leaflet become exposed at the cell surface. This Ca2+-induced mobilization of phosphatidylserine to the surface of activated, injured, or apoptotic cells confers a procoagulant property to the plasma membrane, which promotes fibrin clotting and provides a signal for cell removal by the reticuloendothelial system, To identify the constituent of the membrane that mediates this Ca2+-dependent ''PL scramblase'' activity, we undertook purification and reconstitution of membrane component(s) with this activity from detergent extracts of erythrocyte ghosts depleted of cytoskeleton. Active fractions were identified by their capacity to mediate the Ca2+-dependent redistribution of 7-nitrobenz-2-oxa-1,3-diazol-4-yl-labeled PL between leaflets of reconstituted proteoliposomes. This PL scramblase activity co-eluted through multiple chromatographic steps with a single polypeptide of similar to 37 kDa, which was purified to apparent homogeneity as resolved by silver staining. The activity associated with this protein band was inactivated by trypsin, The isolated protein reconstituted in proteoliposomes mediated nonselective, bidirectional transport of 7-nitrobenz-2-oxa-1,3-diazol-4-yl-PL between membrane leaflets, with half-maximal activation between 20 and 60 mu M Ca2+ (saturation >100 mu M), mimicking the Ca2+-dependent transbilayer lipid movement intrinsic to the erythrocyte membrane.