MOLECULAR AND RED CELL DETERMINANTS OF SICKLE CELL DISEASE

描述(由申请人提供)： 这个项目带来了这个实验室在分子和细胞方面的工作和专业知识。 镰状细胞病的病理生理学与其他四个实验室合作，每个实验室 不同领域的专家，共同的目标是对结构进行详细描述， 脱氧-Hb S聚合物、纤维和凝胶的相互作用和组装动力学 血红蛋白S在红细胞中的行为。我们强调这些目标：1)描述 脱氧-HB S聚合物、纤维和凝胶的相互作用部位，特别注意 易受特定修饰的重要部位，通过半合成方法和直接 化学修饰；对不同结构上特定残基的特定顺式或反式作用 聚合物的水平；以及非S HBs如何参与聚合；2)关联我们的微观 排除容积法测定聚合物的溶解度，将右旋糖苷与铁试剂进行微动力学聚合？S新的微动力学分析方法，使热力学分析可以用这种方法；3) 进一步考察聚合物水室(PWC)在2，3-二氯苯酚再分配中的作用 DPG、ATP、RBC酶和可能影响聚合的小分子 过程及其对细胞代谢的影响；4)检测脱氧血红蛋白S的构象变化 在不同条件下聚合，以解释观察到的DPG的排斥作用，以及IHP的溶解性降低效应4)研究产生半色素和氧化膜损伤的Hb S细胞内不稳定的机制5)评估红细胞膜的正常或异常(SS修饰)组分在动力学和组装中的作用 胞内聚合物。每个重组、修饰和/或交联的杂化四聚体Hb 专门设计用来测试不同水平的聚合物和凝胶中各个位置的作用 结构(Manning，Acharya)，将通过我们的平衡溶解度微量分析来检验 (葡聚糖-Csat)，并与微量动力学测定法(Ferrone)相关。选定的物种将是 用约瑟夫的电子显微镜方法和Briehl的直接方法检查 纤维生长和组装的可视化。我们共同的目标是详细描述 脱氧-Hb S聚合物和凝胶、溶液和红色的结构和组装机理 细胞，临床目的是促进设计针对镰状细胞的特定治疗剂 疾病，以及提出完整的机制和模型描述的基本目标 生物系统中蛋白质聚合和解聚的渗透效应。

DESCRIPTION (provided by applicant): This project brings the work and expertise of this laboratory on the molecular and cellular pathophysiology of sickle cell disease into collaboration with four other laboratories, each expert in different areas, with the common goal of a detailed description of the structure, interactions and kinetics of assembly of the deoxy-HB S polymers, fibers and gel and the behavior of Hb S in the red cell. We emphasize these objectives: 1) To characterize the interaction sites of the deoxy-Hb S polymers, fibers and gels, with particular attention to important sites susceptible to specific modification, by semisynthetic approaches and direct chemical modification; to the specific cis or trans role of specific residues at different structural levels of the polymer; and how non-S Hbs participate in polymerization; 2) to correlate our micro excluded volume assay of the polymer solubility, the dextran-Csat with Ferrone?s new micro-kinetic assay of polymerization, to permit thermodynamic analyses with this method; 3) to examine further the role of the polymer water compartment (PWC) on the redistribution of 2,3- DPG, ATP, RBC enzymes and small molecules which may affect both the polymerization process and its effects on cell metabolism; 4) to test for conformational changes in deoxy-Hb S polymerized in different conditions, to explain the observed exclusion of DPG, and solubility-lowering effect if IHP 4)to investigate the mechanisms of intracellular instability of Hb S, which generates hemichromes and oxidative membrane damage 5) to assess the role of normal or abnormal (SS-modified) components of the RBC membrane on the kinetics and assembly of intracellular polymer. Each recombinant, modified, and/or cross-linked hybrid tetramer Hb designed specifically to test the roles of individual sites in different levels of polymer and gel structure (Manning, Acharya), will be examined by our micro-assay of equilibrium solubility (dextran-Csat) and correlated with the micro-kinetic assay (Ferrone). Selected species will be examined by Joseph's electron microscopic methods, and by Briehl's methods of direct visualization of fiber growth and assembly. We share the goal of a detailed characterization of the structure and assembly mechanisms of the deoxy-Hb S polymer and gel, in solution and red cells, with the clinical aim of facilitating the design of specific therapeutic agents for sickle cell disease, and the basic aim of advancing a full model description of the mechanism and osmotic effects of protein polymerization and depolymerization in biological systems.