INFLUENCES OF MACROMOLECULAR CROWDING ON BIOCHEMICAL SYSTEMS

大分子拥挤对生化系统的影响

• 批准号: 3917964
• 负责人: S B ZIMMERMAN
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3917964
• 关键词: DNA; DNA binding protein; DNA directed DNA polymerase; Escherichia coli; acidity /alkalinity; bacterial DNA; chemical binding; enzyme mechanism; enzyme substrate; genetic translation; intermolecular interaction; ionic strengths; oligonucleotides; polymers; ribosomes; serum albumin; temperature

The nick-translation reaction of E. coli DNA polymerase I (Pol I) was used as a model system to demonstrate the ability of macromolecular crowding to alter the response of an enzyme to a number of basic parameters, such as pH, temperature or inhibitors. In the presence of high concentrations of non-specific polymers, nick translation occurred under a variety of otherwise strongly inhibitory conditions. The conditions tested included a range of pH values or temperatures or inhibitory concentrations of urea, formamide or ethidium bromide. These crowding effects are accentuated at higher ionic strengths, suggesting their origin in increased binding between the polymerase and its DNA template- primer under crowded conditions. Kinetic measurements were consistent with such a mechanism. We have examined the effects of crowding on the association reactions of ribosomal particles. The equilibrium for the binding reaction between the 30 S and 50 S ribosomal subunits of E. coli is shifted towards formation of 70 S ribosomes in the presence of a variety of polymers. The polymers also increase a further interaction between 70 S particles to form the 100 S dimer. The requirement for relatively high concentrations of non-specific polymers indicates that the shifts in equilibria arise from excluded volume effects. Analysis using scaled particle theory is consistent with this mechanism. The effects of high concentrations of polymers in the interactions between ribosomal species may make important changes in the function of ribosomes under the crowded conditions which occur in vivo.

E.大肠杆菌DNA聚合酶I 被用作模型系统，以证明 大分子拥挤改变了酶的反应， 一些基本参数，如pH值、温度或抑制剂。 在高浓度的非特异性聚合物的存在下， 切口平移发生在各种其他强烈 抑制条件 测试的条件包括一系列 pH值或温度或尿素的抑制浓度， 甲酰胺或溴化乙锭。 这些拥挤效应是 在更高的离子强度下加剧，表明它们的起源是 聚合酶和它的DNA模板之间的结合增加- 在拥挤条件下的引物。 动力学测量 符合这样的机制。 我们已经研究了拥挤对协会的影响 核糖体颗粒的反应。 结合的平衡 30 S和50 S核糖体亚基之间的反应。杆菌 的存在下，向70 S核糖体的形成转移。 各种聚合物。 聚合物还进一步增加了 70 S颗粒之间的相互作用形成100 S二聚体。 的 要求相对高浓度的非特异性 聚合物表明，平衡的变化来自于 排除体积效应。 使用比例粒子理论分析是 与这一机制相一致。 高浓度的影响 核糖体物种之间相互作用中的聚合物可能使 拥挤环境下核糖体功能的重要变化 在体内发生的情况。