RED CELL CYTOSKELETON AND MEMBRANE COMPARTMENTED ATP

描述：这一新应用程序的长期目标是 对ATP的膜轨迹进行详细的表征 为红细胞的Na/K和Ca++泵提供燃料以及这一池在 调节膜功能。申请者之前已经表明 在红细胞的细胞骨架/膜复合体中存在一个 三磷酸腺苷隔间，这是两个最接近的能源 钠钾泵和钙泵。拟议研究的目标是 要找出这个ATP的位置，请识别 细胞骨架蛋白参与了细胞间隔化过程。至 实现这一目标有以下四个具体目标：1. 在人类红血球幽灵候选蛋白中鉴定 通过光标记这些蛋白质直接参与到ATP池中 带有[a-32P]ATP衍生物，该衍生物被特别掺入 池；2.确定在幽灵中的ATP池的重组 通过预处理，细胞骨架/膜蛋白已经被 去除时伴随着形成膜池的能力的丧失。 红细胞膜三磷酸腺苷池特性的研究 膜/细胞骨架相互作用有缺陷的变体；3.确定 应用激光共聚焦显微镜观察三磷酸腺苷池的膜形态 用ATP的荧光衍生物填充池；以及4. 用自内向外的囊泡测定膜ATP的性质 钙和钠/钾泵的池驱动钙和钠吸收。这些研究将 提供有关这方面的生理意义的信息 钙、钠转运中的ATP库。据预计， 这些目标的成功实现将为 膜ATP池在能量储备中的作用 在瞬变应力期内维持离子泵的作用 作为一种假定的转导机制，细胞的细胞骨架可以 感知蜂窝能量存储。

DESCRIPTION: The long term objective of this new application is to perform detailed characterization of the membrane locus of ATP that fuels red cell's Na/K and Ca++ pumps and the role of this pool in regulating membrane function. The applicant has previously shown that within the cytoskeletal/membrane complex of red cell exists a compartment of ATP that is the proximate energy source for both the Na/K pumps and Ca pumps. The objective of the proposed studies is to find out where this ATP is located by identifying the cytoskeletal proteins involved in the compartmentalization process. To achieve this goal the following four specific aims are outlined: 1. Identify in human red blood cell ghosts candidate proteins that are directly involved with the ATP pool by photolabelling these proteins with an [a-32P] ATP derivative that is specifically incorporated into the pool; 2. Determine the reassemblage of the ATP pool in ghosts in which, by pretreatment, cytoskeletal/membrane proteins have been removed with concomitant loss of the ability to form the membrane pool. Study the ATP pool characteristics of ghosts prepared from red cell variants with defective membrane/cytoskeletal interactions;3. Determine the membrane topography of ATP pools by confocal microscopy after filling the pools with a fluorescent derivative of ATP; and 4. Determine, using inside-out vesicles, the properties of the membrane ATP pool driven Ca and Na uptake by Ca and Na/K pumps. These studies will provide information regarding the physiologic significance of this ATP pool in Ca and Na transport. It is anticipated that the successful accomplishment of these goals will provide new insights into the role of membrane ATP pool serving both as an energy reserve capable of sustaining ion pumping during periods of transient stress, as well as a putative transduction mechanism by which cell's cytoskeleton may sense cellular energy stores.