Ca2+ sensitivity of phospholipid scrambling in human red cell ghosts
Ca2+ sensitivity of phospholipid scrambling in human red cell ghosts
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DOI：
10.1054/ceca.1999.0029
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发表时间：
1999-04-01
期刊：
影响因子：
4
通讯作者：
Roufogalis, BD
中科院分区：
文献类型：
--
作者：
Woon, LA;Holland, JW;Roufogalis, BD
红细胞以及血小板、淋巴细胞和其他细胞的质膜中的磷脂不对称分布，鞘磷脂和磷脂酰胆碱主要位于双层的外叶中，磷脂酰丝氨酸和磷脂酰乙醇胺位于内叶中。已知Ca2+可以通过激活称为磷脂乱序酶的蛋白质来破坏磷脂不对称性，所述磷脂乱序酶以很大程度上非选择性的方式影响双向磷脂运动。由于Ca2+也抑制氨基磷脂移位酶，其Mg2 +-ATP酶活性负责氨基磷脂从外叶主动移位到内叶，因此准确测定scramblase对细胞内游离Ca2+的敏感性是重要的。在本研究中，我们已经利用了有利的Kd，Mag-fura-2的钙在高微摩尔范围内，以确定游离钙2+水平与重新密封的人红细胞血影脂质混乱。的Ca2+的敏感性进行了测量，在平行的易位的荧光标记的脂质纳入鬼双层。磷脂的混乱被认为是半最大限度地激活在63 - 88 μ M的细胞内游离Ca 2+。更广泛的适用性的方法和钙敏感性测定的生理意义进行了讨论。
The phospholipids in plasma membranes of erythrocytes, as well as platelets, lymphocytes and other cells are asymmetrically distributed, with sphingomyelin and phosphatidylcholine residing predominantly in the outer leaflet of the bilayer, and phosphatidylserine and phosphatidylethanolamine in the inner leaflet. It is known that Ca2+ can disrupt the phospholipid asymmetry by activation of a protein known as phospholipid scramblase, which affects bidirectional phospholipid movement in a largely non-selective manner. As Ca2+ also inhibits aminophospholipid translocase, whose Mg2+-ATPase activity is responsible for active translocation of aminophospholipids from the outer to the inner leaflet, it is important to accurately determine the sensitivity of scramblase to intracellular free Ca2+. In the present study we have utilized the favourable K-d, of Mag-fura-2 for calcium in the high micromolar range to determine free Ca2+ levels associated with lipid scrambling in resealed human red cell ghosts. The Ca2+ sensitivity was measured in parallel to the translocation of a fluorescent-labelled lipid incorporated into the ghost bilayer. The phospholipid scrambling was found to be half-maximally activated at 63-88 mu M free intracellular Ca2+. The wider applicability of the method and the physiological implications of the calcium sensitivity determined is discussed.