REGULATION OF ERYTHROPOIESIS BY CELL SURFACE COMPONENTS

我们已经发现红细胞爆发促进活性（BPA）由以下表达： 淋巴细胞质膜 我们现在建议利用生物化学方法 明确的培养体系： 1.纯化膜相关BPA。 我们将利用各种 生物化学方法，包括免疫亲和法、凝集素亲和法和凝胶法 过滤高效液相色谱法（HPLC）和反相 用高效液相色谱法从淋巴细胞质膜和脱落细胞提取物中分离纯化BPA 囊泡 将通过等电聚焦对纯化组分进行表征 和蔗糖梯度分级。 最终，纯化的BPA将被 进行氨基酸分析。 2.确定可溶性和泡状BPA的相关性。 我们会委聘 抗血清和单克隆抗体与BPA反应，以检测是否 从膜和溶液中纯化的BPA是抗原相关的。 将进行研究，以确定BPA的两种物理形式是否 通过类似的机制从类似的细胞类型中释放，如果它们作用于 相似的骨髓亚群。 每种生长因子的靶细胞将被 组织化学和形态学特征。 3.定义囊泡相关BPA释放的动力学。 累积量 以及产生BPA的细胞群的囊泡形成和BPA释放速率 将被确定。 我们的研究结果将与旨在 测量培养物中所选膜组分的生物合成。 4.检查BPA的作用模式。 我们将确定BPA脱落是否 免疫反应的组成过程或一部分。 研究将 进行检查是否囊泡涉及细胞骨架成分 以及膜脂和磷脂成分是否对细胞的生长和分化重要。 脱落过程 我们将确定囊泡是否与 如果是的话，这是什么性质的？ 联合可能是使用电子显微镜的方法。 最终我们 将定位和纯化BPA受体，其长期目标是 研究受体激活的细胞内机制。

We have found that erythroid burst promoting activity (BPA) is expressed by lymphocyte plasma membranes. We now propose to utilize a biochemically defined culture system to: 1. Purify membrane-associated BPA. We will utilize a variety of biochemical methods, including immunoaffinity, lectin affinity and gel filtration high-performance liquid chromatography (HPLC) and reverse-phase HPLC, to purify BPA from extracts of lymphocyte plasma membranes and shed vesicles. Purified fractions will be characterized by isoelectric focusing and sucrose gradient fractionation. Ultimately, purified BPA will be subjected to amino acid analysis. 2. Determine relatedness of soluble and vesicular BPA. We will employ antisera and monoclonal antibodies that react with BPA to detect whether purified BPA from membranes and in solution are antigenically related. Studies will be performed to determine if both physical forms of BPA are released by similar mechanisms from similar cell types, and if they act on similar marrow subpopulations. Target cells for each growth factor will be characterized histochemically and morphologically. 3. Define kinetics of vesicle-associated BPA release. Cumulative amounts and rates of vesiculation and BPA release by BPA-producing cell populations will be determined. Our findings will be correlated with studies aimed at measuring the biosynthesis of selected membrane components in culture. 4. Examine modes of BPA action. We will determine whether BPA shedding is a constitutive process or part of an immune reaction. Studies will be performed to examine whether vesiculation involves cytoskeletal components and whether membrane lipid and phospholipid composition is important to the shedding process. We will determine whether vesicles associate preferentially with marrow cells and, if so, what the nature of this association might be using electronmicroscopic methods. Ultimately, we will locate and purify BPA receptors with the projected long-range goal to examine intracellular mechanisms for receptor activation.