EFFECT OF HEMOGLOBIN MEMBRANE INTERACTIONS ON RED CELL FUNCTION

血红蛋白膜相互作用对红细胞功能的影响

• 批准号: 3736165
• 负责人: Mohandas Narla
• 金额: --
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3736165
• 关键词: antiport; erythrocyte membrane; erythrocytes; gel electrophoresis; glycophorin; hemoglobin; hemoglobin C; hemoglobin Ss; immunoprecipitation; membrane activity; membrane model; membrane structure; mutant; potassium chloride; sickle cell anemia; western blottings

The objective of this project is to test the hypothesis that interaction of mutant and unpaired Hb chains with specific membrane components affects KCI cotransport and deformability of red cells (RBC). Two Specific Aims will be pursued: 1) to determine the extent to which mutant unpaired Hb chains modify KCI co-transport, membrane deformability and mechanical strength, and 2) to identify membrane components that interact with Hb to mediate these effects. To define (IOV) and resealed membranes in the presence of normal Hb vs mutant Hb and excess alpha and beta globin chains. Differential extraction and readdition of selected membrane skeletal components will test for medication of Hb effects via the membrane skeleton, and maneuvers to block association of Hb chains with integral membrane proteins will test for medication by B3 and glycophrin. Differential effects of various type of Hb on membrane deformability and mechanical strength will be assessed by comparative assays of resealed membranes containing various concentrations and types of Hb, using the ektacytometer, a diffraction viscometer. To identify specific membrane components that interact with Hb to modulate these functions, RBC will be treated with variety of cross-linking agents at low concentrations to stabilize Hb/membrane associations in the intact cell and prevent their disruption during analysis. Hb cross-linking to specific membrane components will be detected by separating membrane proteins by polyacrylamide gel electrophoresis, preparing immunoblots and probing them with anti-Hb and antibodies against membrane components. Apparent complexes between Hb and membrane components will be confirmed by immunoprecipitation with antibody against the membrane component, and probing of immunoblots of the immunoprecipitate using anti-Hb. To probe for interaction of Hb with the protein that mediates KCI cotransport, rabbit polyclonal antibodies against the protein will be prepared, making use of the differential expression of the antigen in high K and low K sheep RBC. It is expected that information derived from these studies will provide new information about the effects of HbS and HbC on membrane function that may be useful in understanding the variable pathophysiologic manifestations in the sickling disorders.

该项目的目的是检验相互作用的假设 具有特定膜成分的突变体和未配对的HB链 影响KCI共转运和红细胞（RBC）的可变形性。 二 将追求具体目标：1）确定多大程度上 突变的未配对HB链修改KCI共同传输，膜变形。 和机械强度，以及2）确定膜成分 与HB相互作用以调解这些效果。 定义（IOV）并重新密封 在正常Hb与突变体Hb和过量α的存在下膜 Beta Globin链。 选定的差分提取和读取 膜骨骼成分将通过 膜骨架和操纵以阻止HB链的关联 伴有整体膜蛋白将通过B3和 甘氨酸。 各种HB对膜的差异影响 可变形性和机械强度将通过比较评估 含有各种浓度和类型的重新密封膜的测定 HB的of of ektacyTometer，衍射粘度计。 识别 与HB相互作用以调节这些的特定膜成分 功能，RBC将用各种交联剂处理 低浓度以稳定完整的HB/膜关联 细胞并防止分析过程中的破坏。 HB交联 通过分离膜检测特定的膜成分 通过聚丙烯酰胺凝胶电泳的蛋白质，准备免疫印迹和 用抗HB和针对膜成分的抗体探测它们。 HB和膜组件之间的明显复合物将得到确认 通过用抗膜成分的抗体免疫沉淀，并 使用抗Hb探测免疫沉淀的免疫印迹。 探测 HB与介导KCI共晶的蛋白质相互作用， 将制备针对蛋白质的兔多克隆抗体，使 在高k和低k中使用抗原的差分表达 绵羊RBC。 预计从这些研究中得出的信息 将提供有关HBS和HBC对膜的影响的新信息 可能有助于理解变量有用的功能 病态疾病中的病理生理表现。