MOLECULAR DEFECTS WITHIN THE SPECTRIN MEMBRANE SKELETON OF SICKLED CELLS

该项目的长期目标是确定分子 不可逆镰状细胞血影蛋白膜骨架内的缺陷 这些细胞使这些细胞锁定成镰刀形。在 初步研究表明， 只含有血影蛋白，蛋白质4.1和肌动蛋白保持镰刀形时， 分离自高密度镰状细胞。此外，我们有 表明血影蛋白的膜骨架。从正常的红细胞 当在37摄氏度下制备时， 密度镰状细胞仍然相关-事实上， 镰刀形核心骨架在37摄氏度下仍然存在， 共价键;因为这些骨架完全解离， 血影蛋白，蛋白4.1，和肌动蛋白在SDS的存在下。我们的假设 是血影蛋白、蛋白4.1或 肌动蛋白导致不可逆的镰状骨架， 是在37 ℃ 我们将用以下方法来检验这一假设 问题研究制备的正常和镰状细胞核心骨架的形态 在0 ℃和37 ℃下，将通过负染色进行研究， 电子显微镜功能上有缺陷的蛋白质将被 通过[1]研究血影蛋白-4.1-f-肌动蛋白相互作用鉴定， 当血影蛋白与对照分离时血影蛋白二聚体-二聚体相互作用 与镰状细胞相比。[2]血影蛋白-4.1-F-肌动蛋白相互作用的研究 当蛋白4.1从对照与镰状细胞中分离时。[3]第一章 确定镰状细胞相对于对照的聚合速率 肌动蛋白，以及镰状细胞与正常肌动蛋白原丝的能力 作为肌动蛋白聚合的种子。一旦异常蛋白质 已经确定了翻译后修饰导致 问题将通过反相HPLC分离来确定， 镰状细胞的蛋白水解酶与对照骨骼蛋白， 随后通过MS/MS质谱对改变的肽进行蛋白质测序 光谱法

The long term objective of this project is to identify the molecular defect(s) within the spectrin membrane skeleton of irreversibly sickled cells which cause these cells to lock into the sickled shape. In preliminary studies we have demonstrated that core skeletons which contain only spectrin, protein 4.1, and actin remain sickled when isolated from high density sickled cells. Furthermore, we have demonstrated that spectrin membrane skeletons. from normal red cells dissociate when prepared at 37 degrees C, while the skeletons from high density sickle cells remain associated- The fact that irreversibly sickled core skeletons remain associated at 37 degrees C is not due to covalent linkages; because these skeletons dissociate completely to spectrin, protein 4.1, and actin in the presence of SDS. Our hypothesis is that posttranslational modification(s) of spectrin, protein 4.1, or actin lead to an irreversibly sickled skeleton which resists dissociation at 37 degrees C. We will test this hypothesis with the following studies. The morphology of normal and sickle cell core skeletons prepared at 0 degrees C and 37 degrees C will he studied by negative staining and electronmicroscopy. The protein which is functionally defective will he identified by [1] studying the spectrin-4.1-f-actin interaction and spectrin dimer-dimer interaction when spectrin is isolated from control versus sickled cells. [2] Studying the spectrin-4.1-f-actin interaction when protein 4.1 is isolated from control versus sickled cells. [3] Determining the rate of polymerization of sickle cell versus control actin, and the ability of sickle cell versus normal actin protofilaments to serve as seeds for actin polymerization. Once the abnormal protein(s) have been identified the posttranslational modification(s) causing the problem will he identified by reverse phase HPLC separation of proteolytic digests of the sickle cell versus control skeletal protein, followed by protein sequencing of altered peptides by MS/MS mass spectrometry.