STRUCTURE, LOCATION AND FUNCTION OF BRAIN AMELIN

我们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)不同于突触素I (另一个4.1类似物)，并具有结合脑影蛋白的能力 固相分析法。在这项提案中描述的研究中， 小鼠脑内Amelin的含量将用一种 免疫斑点分析，并与脑影蛋白亚型进行比较 内容。Amelin在神经细胞中的亚细胞位置将是 由免疫电子显微镜利用 免疫金技术。氨基精氨酸与纯化脑的相互作用 溶液中的血影蛋白亚型将通过珠子过滤进行检测 测定及氨基精氨酸磷酸化对亲和力的影响 化学计量学已经确定。此外，我们还将本地化 旋转法测定Amelin与脑内血影蛋白分子的结合部位 复合体的阴影和电子显微镜。的能力 Amelin刺激脑内血影蛋白异构体-F-肌动蛋白的相互作用 将通过沉降法和低剪切法测定 粘度计测量。再一次阿美林的效果 这一功能上的磷酸化将被确定。终于 将确定Amelin的序列，并将其与 已发表的RBC 4.1和突触素I基因序列 代表Amelin基因编码区的基因将被分离 并通过双脱氧核苷酸链末端进行测序 方法。这些研究将使我们对 新发现的这个基因的结构、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.