EFFECT OF HEMOGLOBIN MEMBRANE INTERACTIONS ON RED CELL FUNCTION

这个项目的目标是检验相互作用的假设 具有特定膜组分的突变和未配对的Hb链 影响KCI共转运和红细胞(RBC)的变形能力。二 将追求具体目标：1)确定 突变的未配对Hb链改变KCI共转运，膜变形性 和机械强度，以及2)识别膜组件 与HB互动以调节这些影响。定义(IOV)并重新密封 存在正常Hb与突变Hb和过量α-Hb的膜 贝塔珠蛋白链。精选的差示提取和重现 膜骨架成分将通过以下途径测试乙肝的药物效果 膜骨架，以及阻断Hb链结合的操作 带有完整膜蛋白的B3和B3将检测药物 血糖素。不同类型的Hb对膜的不同影响 变形性和机械强度将通过比较法进行评估 含有不同浓度和类型的再密封膜的检测 对于Hb，使用的是衍射粘度计--EKTACYTOMMENTER。要确定 与Hb相互作用的特定的膜组件来调节这些 功能，红细胞将被各种交联剂处理在 低浓度以稳定完整的Hb/膜结合 细胞，并防止它们在分析过程中被破坏。Hb交叉链接到 用分离膜检测特定的膜组分 蛋白通过聚丙烯酰胺凝胶电泳法，制备免疫印迹和 用抗-Hb和抗膜成分抗体来探测它们。 Hb和膜组分之间的表观络合物将得到确认 通过与抗膜成分的抗体进行免疫沉淀，以及 用抗-Hb检测免疫沉淀物的免疫印迹。探查 对于Hb与介导KCI共转运的蛋白质的相互作用， 将制备兔抗该蛋白的多克隆抗体，使 抗原差异表达在高钾和低钾中的应用 绵羊红细胞。预计从这些研究中获得的信息 将提供有关HBS和HBC对膜的影响的新信息 可能对理解变量有用的函数 镰状病症的病理生理表现。

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.