PATHOPHYSIOLOGY OF RED CELL MEMBRANE DISEASES

这项建议主要涉及 某些遗传性红细胞膜疾病中的膜骨架。它是 分为4个部分：首先，我们将描述三者之间的相互作用 主要骨骼蛋白(血影蛋白、肌动蛋白和4.1)之间的相互作用 正常的红细胞。我们将测量这些化合物的化学计量比和亲和力 交互作用时，使用新的 光亲和试剂，并测定其物理性能和 蛋白质4.1的自结合。第二，我们将确定新的膜 骨骼缺陷通过系统分析膜蛋白组成， 膜的磷酸化，以及每一种骨骼蛋白的相互作用 使用已有的技术。第三，我们将重新刻画3个 发现各种形式的遗传性球形红细胞增多症(HS)。在类型I HS中 (血影蛋白缺乏)我们将重点研究血影蛋白的影响 赤字对磷脂不对称的影响，我们将比较 用磷酸化和去磷酸化的血影蛋白进行重组 膜的稳定性和整体蛋白质的迁移率。第II类HS(缺陷在 SPECTIN的4.1结合位点)我们将分离并鉴定2 缺陷者体内缺失的含硫醇类胰多肽 并定位这些肽与4.1结合位点的关系。我们 还将研究这种缺陷的细胞后果，特别是 膜表面损失的机理。在III型HS中(不稳定的血影蛋白 结合位点)，我们将看到是否由于 不稳定的锚蛋白，并试图确定不稳定的原因。 第四，我们将分析储存的血液中骨骼蛋白的相互作用， 聚焦于幽灵蛋白肌动蛋白结合部位的缺陷 在存储的早期，并通过减少来修正。我们将比较 这一缺陷的发展是由于形态、表面积和 分析异常血影蛋白中的硫醇基团； 并确定缺陷及其细胞后果是否可以 通过在还原条件下储存血液来防止的。

This proposal is primarily concerned with the structure and function of the membrane skeleton in certain inherited red cell membrane disorders. It is divided into 4 parts: First, we will characterize interactions of the 3 major skeletal proteins (spectrin, actin and 4.1) with each other in the normal red cell. We will measure the stoichiometry and affinity of these interactions, localize the respective binding domains with a new photoaffinity reagent, and determine the physical properties and self-association of protein 4.1. Second, we will identify new membrane skeletal defects by systematically analyzing membrane protein composition, membrane phosphorylation, and each of the skeletal protein interactions using established techniques. Third, we will characterize 3 newly discovered forms of hereditary spherocytosis (HS). In type I HS (deficiency of spectrin) we will focus on the effects of the spectrin deficit on phospholipid asymmetry and we will compare the effects of reconstitution with phosphorylated and dephosphorylated spectrin on membrane stability and integral protein mobility. In type II HS (defect in the 4.1 binding site of spectrin) we will isolate and characterize 2 thiol-containing tryptic peptides that are missing in the defective spectrin and locate these peptides in relation to the 4.1 binding site. We will also study the cellular consequences of this defect, particularly the mechanism of membrane surface loss. In type III HS (unstable spectrin binding site) we will see if the spectrin binding defect is due to an unstable ankyrin and attempt to identify the cause of the instability. Fourth we will analyze skeletal protein interactions in stored blood, focusing on a defect in the actin binding site of spectrin that develops early in storage and is corrected by reduction. We will compare the development of this defect to changes in the morphology, surface area, and metabolism of stored cells; analyze thiol groups in the abnormal spectrin; and determine whether the defect and its cellular consequences can be prevented by storage of blood under reducing conditions.