MOLECULAR AND RED CELL DETERMINANTS OF SICKLE CELL DISEASE

镰状细胞病的分子和红细胞决定因素

• 批准号: 6242831
• 负责人: ROBERT M BOOKCHIN
• 金额: $ 31.9万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-06 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6242831
• 关键词: cell water; cellular pathology; crosslink; erythrocytes; hemoglobin Ss; molecular pathology; protein structure function; recombinant proteins; sickle cell anemia; synthetic protein; vesicle /vacuole

The overall goal of this project is to characterize the structure and assembly mechanisms of the deoxy HbS polymer and gel in structure in within the red cells. Specific aims are: 1) Exploring the properties and functional consequences of the polymer water compartment (PWC). The idea is based on model studies predicting a volume shift associated with polymerization. Using C-70kD dextran the concentration of deoxy-HbS in the polymer, Cp, was calculated to be 54.7 g/dl. This value is lower than the concentration of Hb in the red cells (136 g/dl), and implies the presence of polymer associated water. It is proposed that this is associated with compartmentalization of red cell components. In particular, Dr. Bookchin wants to investigate the redistribution of organic phosphates, in view of their reported effect on enhancing gelation, and to assess whether changes in metabolic activity are associated with gelation of the deoxy HbS in the red cells. 2) Testing the variability of Cp in deoxy HbS alone and in mixture with other hemoglobins either chemically modified or variants produced by recombinant techniques. The idea is based on preliminary data in which 1:1 mixtures of HbS and HbA or HbC or HbF show an increase in Csat. 3) Implementing the Csat determination in the presence of dextran to microquantities and correlate it with the values obtained from the micro- kinetic assay of Dr. Ferrone. 4) Testing the role of membrane associated sites in the heterogenous nucleation of HbS. For these studies three types of RBC membranes will be prepared, inside out (IOVs) and right side out ghosts in which all the membrane components are preserved, cytoskeleton- free IOVs from which the cytoskeletal proteins have been released, and hybrid erythrocytes deoxygenation. 5) The identification of intra and inter contact points in the fibers, will be done focussing on sites which are susceptible to chemical modifications. A large variety of chemical modifications, either specific or introduced through a semisynthetic approach are proposed as a) amidation of specific Glu residue, b) construction of chimeric hemoglobins c) photoaffinity labeling of contact regions d) preparation of crosslinked asymmetric hybrids.

该项目的总体目标是表征结构和 脱氧 HbS 聚合物和凝胶结构的组装机制 红细胞内。 具体目标是： 1）探索属性和 聚合物水室（PWC）的功能后果。这个想法 基于模型研究预测与相关的体积变化 聚合。使用 C-70kD 葡聚糖测定脱氧 HbS 的浓度 聚合物 Cp 经计算为 54.7 g/dl。该值低于 红细胞中 Hb 的浓度 (136 g/dl)，意味着存在 聚合物缔合水。建议这与 红细胞成分的区室化。特别是布克钦博士 想要研究有机磷酸盐的重新分布，鉴于 他们报告的对增强凝胶作用的影响，并评估是否发生变化 代谢活性的变化与脱氧 HbS 的凝胶化有关 红细胞。 2) 测试单独脱氧 HbS 和脱氧 HbS 中 Cp 的变异性 与其他经过化学修饰或变体的血红蛋白的混合物 通过重组技术产生。这个想法是基于初步数据 其中 HbS 和 HbA 或 HbC 或 HbF 的 1:1 混合物显示出增加 Csat。 3) 在葡聚糖存在的情况下进行 Csat 测定 微量并将其与从微量获得的值相关联 Ferrone 博士的动力学分析。 4) 测试膜相关作用 HbS 的异质成核位点。对于这些研究三种类型 将制备红细胞膜，内朝外 (IOV) 和右朝外 保留所有膜成分的鬼影，细胞骨架- 已释放细胞骨架蛋白的游离 IOV，以及 杂交红细胞脱氧。 5) 内部和外部的识别 纤维中的相互接触点，将重点关注以下部位： 容易受到化学修饰。化学品种类繁多 修饰，无论是特定的还是通过半合成引入的 建议的方法为：a) 特定 Glu 残基的酰胺化，b) 嵌合血红蛋白的构建 c) 接触的光亲和标记 区域d)交联不对称杂化物的制备。