ESTIMATION OF MACROMOLECULE CONCENTRATIONS AND EXCLUDED VOLUME EFFECTS FOR THE CYTOPLASM OF ESCHERICHIA-COLI
ESTIMATION OF MACROMOLECULE CONCENTRATIONS AND EXCLUDED VOLUME EFFECTS FOR THE CYTOPLASM OF ESCHERICHIA-COLI
复制标题
DOI：
10.1016/0022-2836(91)90499-v
复制
发表时间：
1991-12-05
影响因子：
5.6
通讯作者：
TRACH, SO
中科院分区：
文献类型：
--
作者：
ZIMMERMAN, SB;TRACH, SO
由于排除了体积效应，细胞内非常高浓度的大分子可能会对细胞成分的热力学活动产生压倒性的影响。为了估计这种影响的程度，我们对大肠杆菌的细胞质进行了实验研究。细胞和细胞提取物的参数用于计算细胞质条件的近似活性系数。这些计算需要提取物中大分子的大小、浓度和有效比体积的表示。通过应用两相分布测定来定义相关的均匀溶液或通过使用来自凝胶过滤的大分子的分子质量分布来获得大分子尺寸表示。通过对提取过程中发生的稀释进行校正，从提取物分析中获得细胞质中的大分子浓度。该因子是根据完整大肠杆菌细胞质体积对蔗糖的已知不渗透性的实验确定的。大肠杆菌。 E.细胞质中的大分子浓度。指数生长期或稳定生长期的大肠菌素估计约为 0.3 至 0.4 g/ml。大分子比体积是从聚乙二醇诱导的密堆积沉淀物的组成中推断出来的。几种与 DNA 结合的特征明确的蛋白质（lacrepressor、RNA 聚合酶）在体外研究中对盐浓度的变化极其敏感，但在体内研究中则不敏感。应用当前工作中的活度系数表明，这种差异至少部分是由这些研究中排除体积的差异引起的。还讨论了活度系数在溶解度或缔合反应中的应用，以及与细胞生长期和渗透或其他效应相关的变化。讨论了模拟细胞质拥挤条件的纯化大分子溶液的使用。
The very high concentration of macromolecules within cells can potentially have an overwhelming effect on the thermodynamic activity of cellular components because of excluded volume effects. To estimate the magnitudes of such effects, we have made an experimental study of the cytoplasm ofEscherichia coli. Parameters from cells and cell extracts are used to calculate approximate activity coefficients for cytoplasmic conditions. These calculations require a representation of the sizes, concentrations and effective specific volumes of the macromolecules in the extracts. Macromolecule size representations are obtained either by applying a two-phase distribution assay to define a related homogeneous solution or by using the molecular mass distribution of macromolecules from gel filtration. Macromolecule concentrations in cytoplasm are obtained from analyses of extracts by applying a correction for the dilution that occurs during extraction. That factor is determined from experiments based upon the known impermeability of the cytoplasmic volume to sucrose in intactE. coli. Macromolecule concentrations in the cytoplasm ofE. coliin either exponential or stationary growth phase are estimated to be ≈0.3 to 0.4 g/ml. Macromolecule specific volumes are inferred from the composition of close-packed precipitates induced by polyethylene glycol.Several well-characterized proteins which bind to DNA (lacrepressor, RNA polymerase) are extremely sensitive to changes in salt concentration in studiesin vitro, but are insensitive in studiesin vivo. Application of the activity coefficients from the present work indicates that at least part of this discrepancy arises from the difference in excluded volumes in these studies. Applications of the activity coefficients to solubility or to association reactions are also discussed, as are changes associated with cell growth phase and osmotic or other effects. The use of solutions of purified macromolecules that emulate the crowding conditions inferred for cytoplasm is discussed.