BIOCHEMICAL INVESTIGATION OF NEUROFILAMENTS

拟议研究的长期目标是了解，在 在分子水平上，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 了解这个网络在轴突动力学中的功能， 运输 未来三年的研究目标是 神经丝的结构和生物化学对结构和 生物化学：在体外形成的结合物， 神经丝和微管。 将采取四种相互关联的办法， 满员你 (1)一个超电泳，电子显微镜，和电泳 将对神经丝的解离进行研究 变成它们的原丝 目的是定义蛋白质 作为第一步， 来决定它们的结构和自我组装的模式。 (2)模式 这两个大的神经丝蛋白的磷酸化将是 通过蛋白质化学方法来确定是否大部分 磷酸盐位于蛋白质的小区域中。 可能的 磷酸化在稳定神经丝结构中的作用 将通过酶干扰的程度进行研究， 磷酸化，然后评估结构的变化， 超离心和电子显微镜。 通过使用 共价结合的底物类似物，激酶负责的 磷酸化将完成。 (3)调查新的 发现的微管-神经丝相关复合物将继续。 通过色谱分离微管相关蛋白， 介导复合物，并通过复合物形成的粘度测定， 将鉴定起交联剂作用的特定蛋白质， 将确定磷酸化在决定其活性中的作用。 通过观察聚苯乙烯珠粒的光散射， 它，复杂的内部流动性和其内部的几何形状， 空间将被评估。 将尝试在以下条件下形成复合体 在复合体中引起更高程度的各向异性的条件 比迄今为止所获得的。 (4)将进行初步研究 通过光散射和粘滞来确定肌动蛋白丝是否形成了一个 与神经丝相关的复合物。

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.