PHOSPHOINOSITIDES IN NORMAL AND ABNORMAL HUMAN RED CELLS

存在于红细胞膜中的磷脂酰肌醇具有独特的 与许多其他类型的细胞相比，它具有更高的功能。在 红细胞中的肌醇磷脂起着重要作用 与更常见的观察信号相反的结构作用 转导功能。具体地说，这些脂质一直是 与控制原生双凹形状有关 红血球以及作为骨骼的组成部分 带4.1和血糖素之间的复合体。在这样的背景下， 本提案的重点是理解 肌醇磷脂的生理功能和酶学性质 红血球。第一个目标是提纯和表征 磷脂酰肌醇4-磷酸激酶。这种酶，它有 已被证明是红细胞骨架的一个组成部分，是一种 控制磷脂酰肌醇水平的关键因素 膜中含有4，5-二磷酸。因此，这种酶将是 纯化后，对其动力学性质进行了开和关的检测 骨架，该酶在骨架上的结合位置将 被指认出来。这项提案的第二个目标是研究 衰老红细胞的肌醇磷脂代谢。成熟 红细胞不能合成额外的磷脂酰肌醇 他们的这种水平下降的可能性 在它们的生命周期中，脂质尤其令人感兴趣 对老化的红细胞的活性的影响。一种新近开发的 分离衰老红细胞的程序将是 利用；这一过程涉及到兔的生物素化 红血球。衍生细胞的回输及其在体内的应用 在体内老化后，通过附着亲和素最终恢复 支持。该项目的第三个目标是检查 维持不同的肌醇磷脂池 红细胞膜。初步工作表明，有相当大的 部分红细胞磷脂酰肌醇与 框架，以及这个池和散装之间的交换 磷脂酰肌醇的速度很慢，很容易研究。这个 该项目的最终目标是检查患有不明原因疾病的患者 溶血性贫血的异常存在于 磷脂酰肌醇途径。

Phosphoinositides present in the erythrocyte membrane have a unique function when compared to many other cell types. In the erythrocyte the phosphoinositides play a structural role in contrast to the more commonly observed signal transduction function. Specifically, these lipids have been implicated in the control of the native biconcave shape of the erythrocyte as well as serving as a component in the skeletal complex between band 4.1 and glycophorin. With this background, the focus of the present proposal is to understand the physiological function and enzymology of the phosphoinositides in erythrocytes. The first goal is to purify and characterize phosphatidylinositol 4-phosphate kinase. This enzyme, which has been shown to be a component of the erythrocyte skeleton, is a critical element in controlling the level of phosphatidylinositol 4,5-bisphosphate in the membrane. Therefore, the enzyme will be purified, its kinetic properties examined both on and off the skeleton, and the binding site for this enzyme on the skeleton will be identified. The second goal of this proposal is to examine the phosphoinositide metabolism of senescent erythrocytes. Mature erythrocytes cannot synthesize additional phosphatidylinositol so that the possibility that they have a decreasing level of this lipid during their life span is of particular interest with regards to the viability of aged erythrocytes. A recently developed procedure for the isolation of senescent erythrocytes will be utilized; this procedure involves the biotinylation of rabbit erythrocytes. The reinfusion of the derivatized cells and their eventual recovery, after in vivo aging, by attachment to an avidin support. The third goal of the project is to examine the maintenance of distinct pools of phosphoinositides within the erythrocyte membrane. Preliminary work has shown that a sizable portion of the erythrocyte phosphoinositides are associated with the skeleton and that the exchange between this pool and the bulk of the phosphoinositides is slow enough to be readily studied. The final goal of the project is to examine patients with undefined hemolytic anemia for the presence of abnormalities in the phosphoinositide pathways.