BIOCHEMICAL INVESTIGATION OF NEUROFILAMENTS

拟议研究的长期目标是在 分子水平，神经丝在组织中的作用 哺乳动物神经元的细胞骨架网络，并有助于 对该网络在轴突动力学中的作用的理解 运输。未来三年的研究目的是为了与 神经细丝的结构和生化对结构和功能的影响 在体外形成的一种缔合物的生物化学 神经丝和微管。四个相互关联的方法将是 有人了。(1)超速离心、电子显微镜和电泳法 将对神经细丝的解离进行调查 变成它们的组成原丝。其目的是定义蛋白质 作为第一步的原丝的组成和单位长度的质量 在确定它们的亚结构和自组装模式方面。(2)图案 两个大的神经丝蛋白的磷酸化将是 用蛋白质化学方法表征，找出大多数 磷酸盐位于蛋白质的一小部分。可能的 磷酸化在稳定神经细丝结构中的作用 将通过酶扰动的程度进行调查 磷酸化，然后通过以下方式评估结构的变化 超速离心法和电子显微镜。标识，通过使用 共价结合底物类似物，负责 磷酸化将会完成。(三)对新发案件的调查 已发现的微管-神经丝相关复合体将继续存在。 通过层析分离微管相关蛋白 通过络合物形成的粘度法测定， 将确定起交联剂作用的特定蛋白质(S)，以及 将确定磷酸化在确定其活性中的作用。 通过观察掺入的聚苯乙烯微珠的光散射 它、复合体的内部流动性以及它内部的几何形状 将对空间进行评估。将尝试在以下条件下形成该综合体 将在复杂中引起更高程度的各向异性的条件 比到目前为止所获得的更多。(4)进行初步研究 通过光散射和粘度计来确定肌动蛋白细丝是否形成 与神经丝相关的复合体。

The long-range goal of the proposed research is to understand, at the molecular level, the role of neurofilaments in the organization of the cytoskeletal network of mammalian neurons, and to contribute to understanding of the functioning of this network in the dynamics of axonal transport. The aim of the studies for the next three years is to relate the structure and biochemistry of neurofilaments to the structure and biochemistry of an associated complex that forms in vitro between neurofilaments and microtubules. Four interrelated approaches will be taken. (1) An ultracentrifugal, electron microscopic, and electrophoretic investigation will be carried out of the dissociation of neurofilaments into their component protofilaments. The intent is to define the protein composition and mass per unit length of the protofilaments as a first step in determining their substructure and mode of self-assembly. (2) Patterns of phosphorylation of the two large neurofilament proteins will be characterized by protein chemical methods to find out whether most of the phosphates are located in a small region of the proteins. The possible role of phosphorylation in stabilizing the structure of the neurofilaments will be investigated by enzymically perturbing the extent of phosphorylation, then assessing changes in the structure by ultracentrifugation and electron microscopy. Identification, by use of a covalently binding substrate analog, of the kinase responsible for the phosphorylation will be completed. (3) Investigation of the newly discovered microtubule-neurofilament associated complex will be continued. By chromatographic separation of the microtubule-associated proteins that mediate the complex and by viscometric assay of complex-formation, the specific protein(s) that act as cross-linkers will be identified, and the role of phosphorylation in determining their activity will be determined. By observation of light scattering from polystyrene beads incorporated into it, the internal mobility of the complex and the geometry of its internal spaces will be assessed. Attempts will be made to form the complex under conditions that will induce a higher degree of anisotropy in the complex than has so far been obtained. (4) A preliminary study will be carried out by light-scattering and viscometry to find out if actin filaments form an associated complex with neurofilaments.