ELECTRIC FIELD SENSITIVITY OF YEAST H+

酵母 H 的电场敏感性

• 批准号: 2155420
• 负责人: RAYMOND Dean ASTUMIAN
• 金额: $ 10.83万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-28 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2155420
• 关键词: acidity /alkalinity; adenosinetriphosphatase; calcium transporting ATPase; cell membrane; electric field; electrical potential; electrodes; electromagnetic radiation; environmental radiation; enzyme activity; hydrogen; hydrolysis; ion transport; lipid bilayer membrane; membrane potentials; membrane transport proteins; radiation sensitivity; sodium potassium exchanging ATPase; tissue /cell culture; vesicle /vacuole; yeasts

The proposed research is targeted towards investigating the interactions between low frequency AC electric fields and membrane proteins. The basic approach will involve measuring the rate of reactions catalyzed by proton ATPase as a function of the frequency and amplitude of the field. We will use a very broad range of frequencies and amplitudes in order to establish a connection between effects seen at moderate fields (1-10 V/cm) and weak fields (1-100 mV/cm). Since our experiments will be carried out on vesicles rather than much larger mammalian cells, the corresponding fields anticipated to cause similar effects in human tissue are much smaller, so this work is directly relevant to understanding possible effects of powerline or household appliance fields (10(-6)-10(-3) V/m). The experiments will be carried out at many different conditions in order to test postulated mechanisms for the field-protein interactions. Some of these include electro-conformational coupling, effects of electric polarization of the double layer, and field inducted changes in ionic concentration in the double layer. We have derived a general expression for the field and amplitude dependence of the response to a weak oscillating field. This can be described as a sum of Lorenzian curves, where the amplitudes depend on the square of the applied field strength. The dependence of these amplitudes on the experimental conditions is different for different mechanisms. Thus, by measuring these amplitudes at different conditions we can distinguish between different mechanisms. The enzyme used in our study will be the H+ATPase from yeast because it is readily available in purified form from our collaborator, and because much evidence suggests a strong membrane potential dependence for this enzyme. Thus, there is a very good likelihood of experimental success. In initial experiments we will use enzyme in plasma membranes from fractions with very high specific activity. Later, we will use purified enzyme reconstituted in phospholipid bilayer vesicles.

拟议的研究旨在调查这些相互作用 低频交流电场和膜蛋白之间的相互作用。最基本的 该方法将包括测量质子催化的反应速度。 ATPase作为场的频率和幅度的函数。我们会 使用非常广泛的频率和幅度范围以确定 中场效应(1-10V/cm)和弱场效应之间的联系 场强(1-100 mV/cm)。因为我们的实验将在 囊泡而不是更大的哺乳动物细胞，相应的场 在人体组织中引起类似影响的可能性要小得多，因此 这项工作直接关系到理解 电力线或家用电器领域(10(-6)-10(-3)V/m)。 这些实验将按顺序在许多不同的条件下进行 以测试场-蛋白质相互作用的假设机制。其中一些 这些包括电子构象耦合，电子的影响 双电层的极化，以及场诱导的离子变化 在双层中的浓度。我们推导出了一个普遍的表达式 对于场和幅度的依赖关系，对弱响应 振荡场。这可以被描述为洛伦兹曲线的总和， 其中，幅度取决于外加磁场强度的平方。 这些幅度与实验条件的依赖关系为 对于不同的机制来说是不同的。因此，通过测量这些幅度在 不同的条件下，我们可以区分不同的机制。 我们研究中使用的酶将是酵母中的H+ATPase，因为它是 可随时从我们的合作者处获得纯净版，因为许多 有证据表明，这种酶对膜电位有很强的依赖性。 因此，实验成功的可能性很大。在首字母中 我们将在质膜中使用酶的实验 比活度非常高。稍后，我们将使用纯化的酶 在磷脂双层囊泡中重组。