EFFECT OF MICROTUBULAR PROTEINS ON CELL SURFACES

这项提议是基于微管的两个新发现 生物化学：1）在生理缓冲液存在下， HCO 3-/CO2时，微管蛋白上形成氨基甲酰， 与微管的pH依赖性改变有关 组装;和2）疏水性（推测为膜）微管蛋白 可容易地与微管蛋白分离， 哺乳动物脑和培养细胞。 HCO 3-/CO2中微管蛋白的性质表明， 调节微管组装的重要机制 我们建议详细探讨;是否存在善意 疏水微管蛋白提出了新的方式，其中“微管”可以 影响细胞功能。 我们的总体目标是确定 CO2/HCO 3-效应，以确定氨基甲酸盐的后果 形成微管功能，并确定 疏水和微管之间的分子差异 微管蛋白及其在培养细胞中的表达。 微管蛋白氨基甲酸酯的形成将在24小时内测量。 生理pH和CO2。 氨基甲酸酯通过质子测量 CO2和蛋白质溶液快速混合后的释放： 形成的氨基甲酰基和 可以计算反应性胺。 CO2/HCO 3缓冲液似乎 促进微管组装在碱性pH值，我们将测量 作为组装和MAP的函数的氨基甲酸酯形成 （微管相关蛋白）结合。 氨基甲酸酯位点 形成可能局限于羧基末端区域 并在该区域起作用，例如酪氨酸化， 将检查去酪氨酸作用。 二价阳离子结合在 将通过EPR（电子顺磁共振）测量CO2的存在 共振）。 我们将尝试分析疏水性的结构基础， 膜微管蛋白的性质，并开始研究其 功能 与可溶性微管蛋白的差异将利用 2-双向电泳和肽图谱。 的 存在脂肪酸和肌醇的共价结合 将被审查。 与微管蛋白的共聚将 得到考验 疏水微管蛋白的量将是 作为细胞-细胞和细胞-基质接触的函数测量。

This proposal is based on two novel findings in microtubule biochemistry: 1) in the presence of the physiological buffer, HCO3-/CO2, carbamino groups are formed on tubulin, and this is associated with an altered pH-dependency of microtubule assembly; and 2) a hydrophobic (presumably membrane) tubulin readily separable from microtubule protein can be obtained from mammalian brain and cultured cells. The properties of microtubule protein in HCO3-/CO2 suggest important mechanisms for the regulation of microtubule assembly we propose to explore in detail; the existence of a bona fide hydrophobic tubulin suggests new ways in which 'microtubules' can affect cell function. Our overall objectives are to determine the mechanism of the CO2/HCO3- effects, to define the consequences of carbamate formation for microtubule function, and to determine the molecular differences between the hydrophobic and microtubule tubulins and their expression in cultured cells. Tubulin carbamate formation will be measured over the physiological pH and CO2. Carbamate is measured by proton release following the rapid mixing of CO2 and protein solutions: carbamino groups formed and the dissociation constants of the reactive amines can be calculated. CO2/HCO3- buffers appear to promote microtubule assembly at alkaline pH and we will measure carbamate formation as a function of assembly and MAP (microtubule associated protein) binding. Sites of carbamate formation are probably localized to the carboxy-terminal region and functions at this region such as tyrosination and detyrosination will be examined. Divalent cation binding in the presence of CO2 will be measured by EPR (electron paramagnetic resonance). We will attempt to analyze the structural basis of the hydrophobic properties of the membrane tubulin and begin to study its function. Differences from soluble tubulin will be sought utilizing 2-dimensional electrophoresis, and peptide mapping. The presence of covalent incorporation of fatty acid and myoinositol will be examined. Copolymerization with microtubule protein will be tested. The quantity of the hydrophobic tubulin will be measured as a function of cell-cell and cell-substrate contact.