EFFECT OF MICROTUBULAR PROTEINS ON CELL SURFACES

这一建议是基于微管中的两项新发现。 生物化学：1)在生理缓冲液的存在下， HCO3-/CO2，在微管蛋白上形成碳氨基，这是 与微管的pH依赖性改变有关 组件；以及2)疏水性(可能是膜)微管蛋白 容易从微管中分离出来的蛋白质可以从 哺乳动物的大脑和培养的细胞。 微管蛋白在HCO3-/CO2中的性质表明 微管组装调控的重要机制 我们建议详细探讨；善意的存在 疏水性微管蛋白为微管提供了新的途径 影响细胞功能。 我们的总体目标是确定 CO2/HCO3-效应，以定义氨基甲酸酯的后果 微管功能的形成，并确定 疏水性微管与微管的分子差异 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 生理pH和二氧化碳。氨基甲酸酯是用质子来测量的。 二氧化碳和蛋白质溶液快速混合后释放： 碳氨基的形成和解离常数 反应胺可以计算出来。CO2/HCO3-缓冲液似乎 在碱性pH条件下促进微管组装，我们将测量 氨基甲酸酯的形成与组装和映射的关系 (微管相关蛋白)结合。氨基甲酸酯的位置 形成可能局限于羧基末端区域 以及在这一区域的功能，如暴君和 将会检查堕落的情况。二价阳离子结合作用的研究 二氧化碳的存在将通过EPR(电子顺磁)进行测量 共振)。 我们将尝试分析疏水性的结构基础 膜微管蛋白的性质及其研究进展 功能。将寻求与可溶性微管蛋白的区别 双向凝胶电泳法和多肽图谱。这个 存在脂肪酸和肌醇的共价掺入 将会被检查。与微管蛋白的共聚反应 接受测试。疏水微管蛋白的量将为 作为细胞-细胞和细胞-基质接触的函数进行测量。

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.