Proteins from morphologically differentiated neuroblastoma cells promote tubulin polymerization
Proteins from morphologically differentiated neuroblastoma cells promote tubulin polymerization
复制标题
来自形态分化的神经母细胞瘤细胞的蛋白质促进微管蛋白聚合
作者：
N. Seeds;R. Maccioni
小鼠神经母细胞瘤C-1300的克隆细胞（N18）可被诱导经历形态分化，其特征在于含有许多微管的非常长的神经突（> 150微米）的生长。由于微管的数量和长度的显着增加显然不是由于微管蛋白亚基的浓度增加，额外的大分子在N18细胞轴突形成过程中微管蛋白聚合的调节中的可能作用进行了检查。使用其中低浓度（< 4 mg/ml）纯化的脑微管蛋白的聚合需要微管相关蛋白（MAP）的体外系统，测定来自神经母细胞瘤和神经胶质瘤细胞的高速上清液（250，000 g）在脑微管蛋白的聚合中替代MAP的能力。只有来自“分化的”N18细胞的上清液具有聚合能力。这些上清液的电子显微镜观察未能证明成核结构（环或盘）的存在。活性因子在蔗糖梯度离心中在约7S时沉淀，并在170，000 molwt蛋白质区域中从4 B琼脂糖凝胶洗脱。此外，当在限制性MAP存在下测试时，来自其他细胞的无活性上清液不抑制聚合。因此，微管的形成伴随着神经细胞瘤细胞中的神经突生长，似乎是由额外的大分子因子的存在下，可能是功能等同于与脑微管发现的MAP的调节。
Clonal cells (N18) of the mouse neuroblastoma C-1300 can be induced to undergo a morphological differentiation characterized by the outgrowth of very long neurites (> 150 microns) that contain many microtubules. Because the marked increase in the number and length of microtubules is apparently not due to an increase in the concentration of tubulin subunits, the possible role of additional macromolecules in the regulation of tubulin polymerization during neurite formation by N18 cells was examined. Using an in vitro system where the polymerization of low concentrations (< 4 mg/ml) of purified brain tubulin requires microtubule-associated proteins (MAPs), high-speed supernates (250,000 g) from neuroblastoma and glioma cells were assayed for their ability to replace MAPs in the polymerization of brain tubulin. Only the supernates from "differentiated" N18 cells were polymerization competent. Electron microscope observations of these supernates failed to demonstrate the presence of nucleation structures (rings or disks). The active factor(s) sedimented at approximately 7S on sucrose gradient centrifugation and eluted from 4B Sepharose in the region of 170,000 mol wt proteins. Furthermore, the inactive supernates from other cells did not inhibit polymerization when tested in the presence of limiting MAPs. Thus, microtubule formation accompanying neurite outgrowth in neuroblastoma cells appears to be regulated by the presence of additional macromolecular factor(s) that may be functionally equivalent to the MAPs found with brain microtubules.