SPECTRIN-LIKE PROTEIN IN DEVELOPING BRAIN

我们的实验室首次鉴定出了一种免疫反应， 红细胞血影蛋白结构相关类似物 非红细胞 最近，我们鉴定并纯化了一种 小鼠红细胞血影蛋白免疫活性类似物 个脑袋 纯化的脑血影蛋白样分子是一个大的不对称 972，000 MW，10.5S，（AlphaBeta）2四聚体，由240，000道尔顿组成，和 235，000个道尔顿亚基。 而脑血影蛋白样蛋白和红细胞 血影蛋白具有共同抗原位点、形态、亚基组成， 功能区，二维胰蛋白酶肽分析表明，只有 小鼠脑和红细胞蛋白之间的有限同源性。 针对天然脑和RBC血影蛋白的抗体已被用于研究 免疫荧光法测定成年小鼠脑神经元胞体和细胞 过程特别富含血影蛋白。 在研究中， 这项建议，我们研究血影蛋白样蛋白的作用， 发育中的老鼠大脑 强化免疫生物化学和免疫荧光 对出生前15天、新生和出生后10天、21天、30天 产后90天将使我们能够关注神经元的活跃期 以及神经胶质细胞增殖、迁移和分化。 抗体 240 K红细胞、220 K红细胞、240 K脑血影蛋白和235 K脑血影蛋白亚基将被 用于间接免疫荧光研究， 脑组织以及神经细胞内;使用 相位显微镜、银染色和花青素染色切片将 包括. 用铁蛋白标记的抗体进行免疫电子显微镜检查， 用于血影蛋白亚基的超微结构定位。 的 血影蛋白样亚单位的类型和数量在神经个体发育将是 用免疫沉淀和免疫放射自显影技术测定 利用我们针对血影蛋白亚基的抗体 免疫荧光 因此图像将与子单元的识别相关 被可视化。 此外，还进行了一维和二维肽图谱分析。 放射性碘标记的多肽将用于确定 脑血影蛋白亚基的变化。 拟议的研究将 是第一次探究类幽灵蛋白的功能意义， 蛋白质在发育中的神经系统，并揭示了血影蛋白的作用 在神经细胞和组织中。 这项调查是正在进行的 在细胞和分子神经生物学的程序，旨在了解 正常和异常大脑发育的过程。

Our laboratory has made the first identification of an immunoreactive, structurally related analogue of red blood cell spectrin in various nonerythroid cells. Recently, we have identified and purified an immunoreactive analogue of rbc spectrin from a membrane fraction of mouse brain. The purified brain spectrin-like molecular is a large asymmetric 972,000 MW, 10.5S, (AlphaBeta)2 tetramer, consisting of 240,000 dalton and 235,000 dalton subunits. While brain spectrin-like protein and erythrocyte spectrin share common antigenic sites, morphology, subunit composition, and functional regions, two dimensional tryptic peptide analysis indicates only limited homology between the mouse brain and erythrocyte proteins. Antibodies against native brain and rbc spectrin have been used to study adult mouse brain by immunofluorescence, and neuronal perikarya and cell processes are particularly enriched in spectrin. In studies described in this proposal we examine the role of spectrin-like proteins in the developing mouse brain. Intensive immunobiochemical and immunofluorescent investigations of mice of ages 15 days prenatal, newborn, and 10, 21, 30, and 90 days postnatal will allow us to focus on active periods of neuronal and glial cell proliferation, migration, and differentiation. Antibodies to 240K rbc, 220K rbc, 240K brain and 235K brain spectrin subunits will be used in indirect immunofluorescent studies that will localize spectrin in brain tissue as well as within neural cells; structural correlates using phase microscopy, silver staining, and gallocyanin stained sections will be included. Immunoelectron microscopy with ferritin-labeled antibodies will be undertaken for ultrastructural localization of spectrin subunits. The type and quantity of spectrin-like subunits during neuro-ontogeny will be determined with immunoprecipitation and immunoautoradiographic techniques utilizing our antibodies to the spectrin subunits. Immunofluorescent images will thereby be correlated with the identification of the subunit being visualized. In addition, one and two dimensional peptide mapping of radioiodinated polypeptides will be used to ascertain structural changes in the brain spectrin subunits during development. The studies proposed will be the first inquiry into the functional significance of spectrin-like proteins in the developing nervous system and reveal the roles of spectrin in neural cells and tissues. This investigation is part of an ongoing program in cellular and molecular neurobiology which seeks to understand the processes of normal and abnormal brain development.