Nixon R A, Lewis S E, Marotta C A
J Neurosci. 1987 Apr;7(4):1145-58. doi: 10.1523/JNEUROSCI.07-04-01145.1987.
The progressive modification of newly synthesized neurofilament proteins (NFPs) during axoplasmic transport in mouse retinal ganglion cell (RGC) neurons was studied after RGC perikarya were pulse-labeled with 32P-orthophosphate or radiolabeled amino acids. The 3 NFP subunits, H(igh), M(iddle), and L(ow), were among a group of axonally transported proteins that incorporated high levels of 32P. Covalent addition of phosphate slowed the electrophoretic mobility of H and M on SDS polyacrylamide gels and shifted the charge of all 3 subunits toward more acidic pH values, thereby providing an index of the phosphorylation state of this radiolabeled population of NFPs. NFPs were extensively phosphorylated before they entered axons at the optic nerve level, and continued to be modified during transport along RGC axons at the optic nerve and tract level. H and M exhibited charge shifts of 0.2-0.6 units toward a more acidic pH during axoplasmic transport. The charge modifications became more prominent when NFPs reached distal axonal levels, which may indicate regional differences in the activity of this modification process along axons. By contrast, the L subunit became more basic in charge, consistent with decreases in the phosphorylation state during transport. Additional observations (Nixon and Lewis, 1986) that a considerable proportion of phosphate groups initially added to L and M were later removed as neurofilaments advanced along RGC axons support the notion that the changing phosphorylation state of NFP subunits during axoplasmic transport reflects a dynamic equilibrium between phosphorylation and dephosphorylation events. Topographical remodeling of phosphate groups on NFPs during axoplasmic transport is proposed as a possible mechanism for coordinating interactions between neurofilaments and other constituents, as these elements are transported and integrated into the axonal cytoskeleton.
在用32P-正磷酸盐或放射性标记氨基酸对小鼠视网膜神经节细胞（RGC）神经元的胞体进行脉冲标记后，研究了新合成的神经丝蛋白（NFP）在轴浆运输过程中的逐步修饰情况。3种NFP亚基，即高（H）、中（M）和低（L）亚基，是一组轴突运输蛋白，它们掺入了高水平的32P。磷酸盐的共价添加减缓了H和M在SDS聚丙烯酰胺凝胶上的电泳迁移率，并使所有3个亚基的电荷向更酸性的pH值移动，从而提供了该放射性标记的NFP群体磷酸化状态的指标。NFP在进入视神经水平的轴突之前就被广泛磷酸化，并在沿视神经和视束水平的RGC轴突运输过程中持续发生修饰。在轴浆运输过程中，H和M的电荷向更酸性的pH值移动了0.2-0.6个单位。当NFP到达轴突远端水平时，电荷修饰变得更加明显，这可能表明该修饰过程在轴突上的活性存在区域差异。相比之下，L亚基的电荷变得更碱性，这与运输过程中磷酸化状态的降低一致。其他观察结果（Nixon和Lewis，1986）表明，随着神经丝沿着RGC轴突前进，最初添加到L和M上的相当一部分磷酸基团后来被去除，这支持了轴浆运输过程中NFP亚基磷酸化状态的变化反映了磷酸化和去磷酸化事件之间动态平衡的观点。有人提出，轴浆运输过程中NFP上磷酸基团的拓扑重塑是协调神经丝与其他成分之间相互作用的一种可能机制，因为这些成分在运输过程中被整合到轴突细胞骨架中。
