Zimmerman S B, Trach S O
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.
J Mol Biol. 1991 Dec 5;222(3):599-620. doi: 10.1016/0022-2836(91)90499-v.
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 of Escherichia 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 intact E. coli. Macromolecule concentrations in the cytoplasm of E. coli in either exponential or stationary growth phase are estimated to be approximately 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 (lac repressor, RNA polymerase) are extremely sensitive to changes in salt concentration in studies in vitro, but are insensitive in studies in 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.
由于排阻体积效应，细胞内极高浓度的大分子可能会对细胞成分的热力学活性产生压倒性影响。为了估算此类效应的大小，我们对大肠杆菌的细胞质进行了实验研究。利用细胞和细胞提取物的参数来计算细胞质条件下的近似活度系数。这些计算需要了解提取物中大分子的大小、浓度和有效比容。大分子大小的表示方法，要么是通过应用两相分配测定法来定义相关的均相溶液，要么是利用凝胶过滤得到的大分子分子量分布。细胞质中大分子的浓度是通过对提取物进行分析，并对提取过程中发生的稀释进行校正而获得的。该系数是根据完整大肠杆菌中细胞质体积对蔗糖不渗透这一已知特性的实验确定的。指数生长期或稳定生长期大肠杆菌细胞质中大分子的浓度估计约为0.3至0.4 g/ml。大分子的比容是从聚乙二醇诱导的紧密堆积沉淀物的组成推断出来的。几种与DNA结合的特征明确的蛋白质（乳糖阻遏物、RNA聚合酶）在体外研究中对盐浓度变化极为敏感，但在体内研究中却不敏感。应用本研究中的活度系数表明，这种差异至少部分源于这些研究中排阻体积的不同。还讨论了活度系数在溶解度或缔合反应中的应用，以及与细胞生长阶段和渗透或其他效应相关的变化。讨论了使用模拟推断的细胞质拥挤条件的纯化大分子溶液的情况。
