EFFECT OF MICROTUBULAR PROTEINS ON CELL SURFACES

这项研究涉及两个基本但鲜为人知的领域 微管和微管蛋白的生物学和生物化学 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 微管的蛋白质亚单位，微管蛋白。 在以前的微管组装/拆卸的研究中，我们证明了一个 PH的增加提高了组装的临界浓度，并增加了 微管体外药敏试验。我们现在的目标是确定 PH在体内微管调节中的作用。使用新的光谱技术 方法：建立微管解体与微管解体的关系。 在生理条件下测定J774小鼠巨噬细胞的pH值 Na~+-H~+交换在碱化中的作用，并扩展分析 一种神经母细胞瘤系，在该瘤系中自发的微管 在单个细胞中，拆解/组装可以与pH相关。 我们采用了一种从牛膜微管蛋白分离的策略。 脑突触体揭示了疏水性微管蛋白是 在化学上与可溶性微管蛋白不同，但它与 微管蛋白单抗及其与可溶性牛脑的共聚 微管蛋白。这一过程还发现了几个潜在的疏水症 微管相关蛋白。简而言之，突触体膜是 从蔗糖梯度中取出，用1%Triton X-100提取 磷酸-谷氨酸缓冲液。提取物在105g的温度下离心60次。 Min和小球被丢弃。加入GTP-MG，上清液 在5微克分子紫杉醇存在下孵育30分钟。这种混合物 然后通过15%的蔗糖垫在105g下重新加热30分钟。 然后用Triton X-114提取得到的颗粒，并 疏水蛋白按Bordier进行分离。在十二烷基硫酸钠凝胶上， 约50%的二聚体约55千道尔顿的物种存在于 疏水萃取物。这种蛋白质与一种单克隆体交叉反应。 免疫印迹上的微管蛋白抗体，但在多肽上表现出明显的差异 N-氯代丁二酰亚胺消化后的MAP。至少高出两个，低出一个 分子量蛋白质在提取物中是独一无二的。这些 推测的图谱以及所描述的疏水性微管蛋白表明了新的方法 其中膜功能可能受到微管蛋白的影响。(L)

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)