MOLECULAR INTERACTIONS IN ERYTHROID CELL MEMB

跨膜蛋白的物理性质，包括侧链 流动性，旋转流动性，空间相互作用，以及高和低 亲和力结合作用，是细胞的重要决定因素 膜的结构和功能。红系细胞提供了一个理想的系统 来研究调节这种物理行为的分子机制 属性。最近对人类红细胞的研究导致了一项工作 控制平均侧向和侧向运动的分子机制模型 红血球阴离子交换剂带3的转动迁移率。立体的 基于血影蛋白的膜骨架提供的阻碍相互作用 似乎调节带3的横向扩散速度；低亲和力 与膜骨架蛋白ankyrin和Anyrin的结合作用 蛋白质4.2调节带3的旋转扩散速度；高 与锚定蛋白的亲和结合作用确定其组分 可旋转固定的带3；以及带3齐聚以控制 横向不动频带的分数3。该模型在 目前的提议，在单个带3分子的水平上，在3 相关红系细胞系统：慢性粒细胞白血病患者的成熟红细胞 遗传性溶血性贫血和血影蛋白，锚蛋白，带3缺陷， 蛋白4.1和蛋白4.2；小鼠品系的成熟红细胞 遗传性溶血性贫血和血影蛋白和锚蛋白缺陷，以及 来自在Ankyrin，Band中存在工程缺陷的转基因小鼠 3和蛋白4.2；人和小鼠红系细胞的分化 在文化上。主要的红细胞唾液酸糖蛋白的物理性质， 血糖素A也在成熟的红细胞中检测，在 分化的红系细胞。单粒子跟踪用于 辨析个体平移运动的四种模式 蛋白质分子，包括不受限制的扩散，受限的 扩散、定向移动和固定。激光光镊子 用于量化高亲和力结合强度和低亲和力结合强度 天然中涉及单个蛋白质分子的相互作用 膜环境。荧光光漂白的恢复和偏振 使用荧光耗竭来测量平均侧方和 分别是跨膜蛋白的旋转迁移率。膜 生化和超微结构与跨膜蛋白的关系 单个分子水平上的物理性质。这些研究 将第一次引导人们从分子水平上理解 物理作用力限制了跨膜蛋白的流动性。澄清 这些力量的性质和大小将有助于解释 遗传性球形红细胞增多症等膜疾病的病理生理学 和遗传性椭圆形红细胞增多症。

Physical properties of transmembrane proteins, including lateral mobility, rotational mobility, steric interactions, and high and low affinity binding interactions, are important determinants of cell membrane structure and function. Erythroid cells provide an ideal system for study of the molecular mechanisms that regulate such physical properties. Recent studies in human red blood cells have led to a working model of the molecular mechanisms controlling the average lateral and rotational mobility of band 3, the red cell anion exchanger. Steric hindrance interactions provided by the spectrin based membrane skeleton appear to regulate the rate of band 3 lateral diffusion; low affinity binding interactions with the membrane skeletal proteins ankyrin and protein 4.2 to regulate the rate of band 3 rotational diffusion; high affinity binding interactions with ankyrin to determine the fraction of rotationally immobile band 3; and band 3 oligomerization to control the fraction of laterally immobile band 3. This model is tested in the present proposal, at the level of individual band 3 molecules, in three related erythroid cell systems: mature red cells from patients with hereditary hemolytic anemias and defects in spectrin, ankyrin, band 3, protein 4.1, and protein 4.2; mature red cells from mouse strains with hereditary hemolytic anemias and defects in spectrin and ankyrin, and from transgenic mouse strains with engineered defects in ankyrin, band 3, and protein 4.2; and differentiating human and murine erythroid cells in culture. The physical properties of the major RBC sialoglycoprotein, glycophorin A, are also examined in mature red cells and in differentiating erythroid cells. Single particle tracking is used to distinguish among four modes of translational motion of individual protein molecules, including unrestricted diffusion, constrained diffusion, directed movement, and immobilization. Laser optical tweezers are employed to quantify the strength of high and low affinity binding interactions involving individual protein molecules in the native membrane environment. Fluorescence photobleaching recovery and polarized fluorescence depletion are used to measure the average lateral and rotational mobility, respectively, of transmembrane proteins. Membrane biochemistry and ultrastructure are correlated with transmembrane protein physical properties at the level of individual molecules. These studies will lead, for the first time, to a molecular understanding of the physical forces constraining transmembrane protein mobility. Elucidation of the nature and magnitude of such forces will help to explain the pathophysiology of membrane disorders including hereditary spherocytosis and hereditary elliptocytosis.