EFFECT OF MICROTUBULAR PROTEINS ON CELL SURFACES

这项研究解决了两个基本但知之甚少的领域， 微管与微管蛋白生物学与生物化学 微管组装的生理调节，以及微管的存在和 膜与主体之间直接相互作用的结果 微管蛋白亚基，微管蛋白。 在以前的微管组装/拆卸的研究中，我们表明， pH的增加提高了组装的临界浓度， 体外微管药物敏感性研究。 我们现在的目标是确定 pH在体内微管调节中的作用。 使用新的光谱 方法，我们将建立微管解体与 pH在生理条件下在J774小鼠巨噬细胞中，测定 Na的作用+-H型+ 碱化交换，并扩展分析 神经母细胞瘤细胞系中， 拆卸/组装可以与单细胞中的pH相关。 我们采用了一种从牛血清中分离膜微管蛋白的方法， 大脑突触体，揭示了疏水微管蛋白， 在化学上不同于可溶性微管蛋白，但与 微管蛋白单克隆抗体与可溶性牛脑共聚 微管蛋白 该程序还揭示了几种潜在的疏水性 微管相关蛋白。简言之，突触体膜是 从蔗糖梯度中取出，用1%Triton X-100在 磷酸-谷氨酸缓冲液。 将提取物在10 ℃下离心5g为60 min，弃去沉淀。 加入GTP-Mg，并将上清液 在5微摩尔紫杉醇存在下孵育30分钟。 然后重新集中在105g，通过15%蔗糖缓冲液30 min。 然后用Triton X-114萃取所得沉淀，并将其浓缩。 根据Bordier分配的疏水蛋白。 在SDS凝胶上， 大约50%的约55千道尔顿的二聚体物质存在于 疏水性提取物。 这种蛋白质与单克隆抗体交叉反应， 微管蛋白抗体的免疫印迹，但显示出明显的差异， N-氯代琥珀酰亚胺消化后的图谱。 至少两个高一个低 分子量蛋白质是唯一存在于提取物中的。 这些 假定的MAPs以及描述的疏水微管蛋白提示了新的途径， 其中膜功能可能受到微管蛋白的影响。 （左）

This research addresses two fundamental but poorly understood areas of the biology and biochemistry of microtubules and microtubule proteins: the physiological regulation of microtubule assembly, and the existence and consequence of direct interaction between membranes and the principal protein subunit of microtubules, tubulin. In previous studies of microtubule assembly/disassembly, we showed that an increase in pH raises the critical concentration for assembly and increases the drug sensitivity of microtubule in vitro. Our goal now is to determine the role of pH in in vivo microtubule regulation. Using new spectroscopic methods, we will establish the relationship of microtubule disassembly and pH under physiological conditions in the J774 mouse macrophage, determine the role of Na+-H+ exchange in alkalinization, and extend the analysis to a neuroblastoma line in which spontaneous microtubule disassembly/assembly can be correlated with pH in single cells. We have employed a strategy for isolation of membrane tubulin from bovine brain synaptosomes that has revealed hydrophobic tubulins that are chemically distinct from soluble tubulins but which cross-react with a monoclonal tubulin antibody and copolymerize with soluble bovine brain tubulin. The procedure has also uncovered several potential hydrophobic microtubule-associated proteins. Briefly, synaptosomal membranes are removed from a sucrose gradient and extracted with 1% Triton X-100 in phosphate-glutamate buffer. The extract is centrifuged at 105g for 60 min and the pellet discarded. GTP-Mg is added and the supernatant incubated in the presence of 5 micromolar Taxol for 30 min. This mixture is then recentrifuged at 105g for 30 min through a 15% sucrose cushion. The resulting pellet is then extracted with Triton X-114 and the hydrophobic proteins partitioned according to Bordier. On SDS gels, roughly 50% of the dimeric approximately 55 kilodalton species is found in the hydrophobic extract. This protein cross-reacts wih a monoclonal tubulin antibody on immunoblot but shows distinct differences in peptide map after N-chlorosuccinimide digestion. At least two higher and one lower molecular weight proteins are uniquely present in the extract. These putative MAPs as well as the hydrophobic tubulin described suggest new ways in which membrane functions may be affected by microtubule proteins. (L)