STUDIES OF SICKLING MECHANISMS AND RED CELL MEMBRANES

本研究的目的是：（1）进一步定义分子 脱氧HbS聚合的机制，通过研究非S 血红蛋白在聚合过程中，Hb解离平衡的影响 和不对称杂化聚合，并分析了具体的 通过关联溶液数据确定聚合物中的分子间接触位点 晶体学数据;（2）功能的进一步研究 含Hb-S的红细胞的性质;和（3） 血红蛋白病和其他疾病患者红细胞膜功能异常 异常红细胞 其中包括脱水机制的研究 SS细胞，通过定义其异常离子转运特性，包括 镰状化过程中钙敏感性K+通道的瞬时激活， 细胞异质性在K+通道Ca 2+敏感性中的可能作用， 扩散氯渗透性，和钙渗透性的改变， 泵活性;其他异常高钙红细胞的钙状态， 内吞囊泡的形成和滞留的作用和机制; 以及Hb与RBC膜的相互作用。 我们的方法将包括 研究了在各种条件下二元血红蛋白混合物的聚合物溶解度 条件下，使用示踪剂标记的白蛋白作为非聚合物的标记物， 通过停流技术测量配体动力学，以及 密度分级SS镰状化和红细胞氧平衡的滞后现象 在血浆和生理缓冲液中测定细胞和SA红细胞的张力; SS和其他红细胞的示踪剂通量研究，45 Ca，86 Rb和其他 同位素;红细胞密度分布变化监测;比较 生理条件下SS和其他RBC中阳离子和阴离子通量的研究 和实验模拟条件促进脱水;电子 内吞作用的显微照相研究和封闭囊泡的鉴定 脾切除者与脾完整者的RBC， 溶血状态;冷冻切片的电子探针X射线微量分析 红细胞体积、pH值和pH值变化动力学的计算机建模， 在特定条件下的离子组成，可以预测 对运输和体积调节假说的实验检验。

The proposed research is aimed at (1) further definition of the molecular mechanisms of deoxy-HbS polymerization, by studies of the role of non-S hemoglobins in polymerization, the effects of Hb dissociation equilibria and asymmetric hybrids in polymerization, and analysis of specific intermolecular contact sites in the polymer by correlating solution data with crystallographic data; (2) further studies of the functional properties of Hb-S containing red cells; and (3) expanded studies of the functional abnormalities of rbc membranes in hemoglobinopathic and other abnormal red cells. These include studies of the mechanism of dehydration of SS cells, by defining their abnormal ion transport properties, including transient activation of the Ca2+-sensitive K+-channel during sickling, the possible role of cell heterogeneity in Ca2+-sensitivity of K+ channels, diffusional chloride permeability, and alterations of Ca permeability and pump activity; the state of Ca in other abnormal rbc with high Ca contents, the role and mechanisms of formation and retention of endocytic vesicles; and interactions of Hb with the rbc membrane. Our methods will include studies of the polymer solubility of binary Hb mixtures under a variety of conditions, using tracer-labelled albumin as a marker of the nonpolymer phase; measurements of the ligand kinetics by stopped-flow techniques, and the hysteresis of sickling and rbc O2 equilibria of density-fractionated SS cells and SA red cells tonometered in plasma and physiological buffers; tracer flux studies of SS and other rbc with 45Ca, 86Rb, and other isotopes; monitoring of density distribution changes of rbc; comparative studies of cation and anion fluxes in SS and other rbc under physiological and experimental modeling conditions promoting dehydration; electron micrographic studies of endocytosis and identification of enclosed vesicles in rbc of splenectomized versus persons with spleen intact, in normal and hemolytic states; electron probe X-ray microanalyses of cryosections of rbc; and computer modeling of the kinetics of changes in rbc volume, pH and ionic composition under specified conditions, which can predict experimental testing of hypotheses about transport and volume regulation.