MEMBRANE TRANSPORT PROCESSES IN RED BLOOD CELLS

我们的主要目标是深入了解分子机制 负责红细胞中电解质的膜运输 以及这些机制在调节细胞体积中所起的作用。 这项工作的目的是建立 Na：K泵复合体的磷酸化及其作用的侧向性 不同的配体参与，与运输的离子的转移。 研究还将针对定义能量关系和 运输和细胞代谢之间的耦合机制。我们计划 钠与钾的泵化学计量比与细胞钠的关系研究 浓度，尤其是在人的红细胞中。这些研究将是 进行了钠、钾净移动量的测量和泵的生电 用荧光法估算膜电势的贡献 染色技术。因为细胞体积和新陈代谢(乳酸产生) 在这些情况下，这些参数也会受到干扰 随之而来的是膜的可能参与 磷酸甘油酸激酶和Na，K-ATPase定义了ATP的膜池。 膜池的功能后果将一起进行评估 尝试标记组成池的蛋白质。我们建议 进一步刻画联合发生的~(32)P-inorg的外流 钠在非偶联的钠外流中。我们还将努力了解 已知Na/Na交换与Na-ATPase活性的关系 以非耦合模式发生。此外，我们还将进一步研究 ADP+促进泵进行钠外排的新模式 被ATP抑制的P-inorg，在其中P-inorg被运输 和娜娜一起。我们还建议研究不同的 膜电位对Na/K不同部分反应或模式的影响 泵试图确定泵反应中的潜在敏感步骤 周而复始。有待研究的变化包括磷酰化的变化 Na和K在细胞内的电位和不同的梯度 薄膜。

Our primary aim is to gain insight into the molecular mechanisms responsible for the membrane transport of electrolytes in red blood cells and the function these mechanisms have in the regulation of cell volume. The work is oriented toward establishing the relationship between phosphorylation of the Na:K pump complex, and the sidedness of action of various ligands involved, with the translocation of the transported ions. Studies will also be directed toward defining the energy relationships and coupling mechanisms between transport and cell metabolism. We plan to study the pump stoichiometry of Na to K as a function of cell Na concentration, especially in human red cells. These studies will be carried out measuring net Na and K movements and the pump's electrogenic contribution to the membrane potential estimated by means of a fluorescence dye technique. Since cell volume and metabolism (lactate production) are perturbed under many of these situations, these parameters will also be followed together with the possible involvement of membrane phosphoglycerate kinase and Na,K-ATPase defining a membrane pool of ATP. Functional consequences of the membrane pool will be evaluated together with an attempt to label the proteins comprising the pool. We propose to characterize further the efflux of 32P-inorg that occurs in conjunction with Na in Uncoupled Na efflux. We will also try to understand the relationship between Na/Na exchange and Na-ATPase activities that are known to take place in the Uncoupled mode. In addition, we will study further a New mode of Na efflux carried out by the pump that is promoted by ADP + P-inorg that is inhibited by ATP and in which P-inorg is transported together with Na. We also propose to study the effects of varying the membrane potential on the different partial reactions or modes of the Na/K pump trying to identify potential-sensitive steps in the pump's reaction cycle. Variations to be studied include alterations in the phosphoryl potential and different sets of the gradients of Na and K across the cell's membrane.