STRUCTURE, LOCATION AND FUNCTION OF BRAIN AMELIN

我们的实验室发现了一种结构和免疫学上的 哺乳动物脑组织中RBC蛋白4.1的类似物。 这个大脑 蛋白4.1类似物（称为amelin）不同于突触蛋白I （另一个4.1类似物），并具有结合脑血影蛋白的能力， 固相分析。 在本提案所述的研究中， 将用一种新的方法定量小鼠脑中的釉蛋白含量， 免疫斑点试验，并与脑血影蛋白亚型进行比较 内容 amelin在神经细胞中的亚细胞定位将是 通过免疫电子显微镜测定， 免疫金技术 Amelin与纯化脑的相互作用 溶液中血影蛋白亚型将通过珠过滤进行检测 测定，和釉蛋白磷酸化对亲和力的影响 和化学计量确定。 此外，我们会将 通过旋转法测定脑血影蛋白分子中amelin的结合位点 阴影和电子显微镜的复杂。 的能力 amelin刺激脑血影蛋白亚型-F-肌动蛋白相互作用 将通过沉降试验和低剪切试验确定 粘度测量 同样，amelin的作用 将确定磷酸化对该功能的影响。 最后 将确定amelin的序列，并与 先前公布的RBC 4.1和突触蛋白I的序列。 cDNA 将分离代表amelin基因编码区的 并通过双脱氧核苷酸链终止进行测序 法 这些研究将使我们全面了解 这种新发现的结构、位置和功能 神经膜骨架蛋白

Our laboratory has discovered a structural and immunological analogue of rbc protein 4.1 in mammalian brain tissue. This brain protein 4.1 analogue (termed amelin) is distinct from synapsin I (another 4.1 analogue), and has the ability to bind brain spectrin in a solid phase assay. In the studies described in this proposal, the content of amelin in mouse brain will be quantitated with an immunodot assay, and compared to brain spectrin isoform content. The subcellular location of amelin in neural cells will be determined by immunoelectron microscopy utilizing the immunogold technology. Amelin's interaction with purified brain spectrin isoforms in solution will be tested by a bead filtration assay, and the effect of amelin phosphorylation upon the affinity and stoichiometry ascertained. In addition, we will localize the binding site for amelin along the brain spectrin molecule by rotary shadowing and electron microscopy of the complex. The ability of amelin to stimulate the brain spectrin isoform-F-actin interaction will be determined by sedimentation assays and low shear viscometry measurements. Again the effect of amelin phosphorylation upon this function will be ascertained. Finally the sequence of amelin will be determined, and compared to previously published sequences for rbc 4.1 and synapsin I. cDNA representing the coding region of the amelin gene will be isolated and sequenced by the dideoxy nucleotide chain termination method. These studies will give us a comprehensive knowledge of the structure, location, and function of this newly discovered neural membrane skeletal protein.