Cayley S, Lewis B A, Guttman H J, Record M T
Program in Molecular Biology, University of Wisconsin-Madison 53706.
J Mol Biol. 1991 Nov 20;222(2):281-300. doi: 10.1016/0022-2836(91)90212-o.
The water-accessible volumes, the amounts of all significant osmolytes, and the protein concentration in the cytoplasm of aerobically grown Escherichia coli K-12 have been determined as a function of the osmolarity of the minimal growth medium. The volume of cytoplasmic water (Vcyto) decreases linearly with increasing osmolarity from 2.23(+/- 0.12) microliters/mg dry weight in cells grown at 0.10 OSM to 1.18(+/- 0.06) microliters/mg dry weight at 1.02 OSM. Above 0.28 OSM, growth rate decreases linearly with increasing osmolarity. The growth rate extrapolates to zero at an osmolarity of approximately 1.8, corresponding to an estimated Vcyto of 0.5(+/- 0.2) microliters/mg dry weight. Measurements of Vcyto in titrations of non-growing cells with the plasmolyzing agent NaCl were used to obtain volumes of "bound" water (presumably water of macromolecular hydration) and cytoplasmic osmotic coefficients for cells grown in medium of low (0.10 OSM) and moderate (0.28 OSM) osmolarity. The volume of bound water Vb is similar in the two osmotic conditions (Vb = 0.40(+/- 0.04) microliters/mg dry wt), and corresponds to approximately 0.5 g H2O/g cytoplasmic macromolecule. Since Vcyto decreases with increasing osmolarity, whereas Vb appears to be independent of osmolarity, water of hydration becomes a larger fraction of Vcyto as the osmolarity of the growth medium increases. Growth appears to cease at the osmolarity where Vcyto is approximately equal to Vb. K+ and glutamate (Glu-) are the only significant cytoplasmic osmolytes in cells grown in medium of low osmolarity. The amount of K+ greatly exceeds that of Glu-. Analysis of cytoplasmic electroneutrality indicates that the cytoplasm behaves like a concentrated solution of the K+ salt of cytoplasmic polyanions, in which the amount of additional electrolyte (K+ Glu-) increases with increasing osmolarity. As the osmolarity of the growth medium becomes very low, the cytoplasm approaches an electrolyte-free K+-polyanion solution. In vivo osmotic coefficients were determined from the variation of Vcyto with external osmolarity in plasmolysis titrations of non-growing cells. The values obtained (phi = 0.54(+/- 0.06) for cells grown at 0.10 OSM and phi = 0.71(+/- 0.11) at 0.28 OSM) indicate a high degree of non-ideality of intracellular ions arising from coulombic interactions between K+ and cytoplasmic polyanions. Analysis of these osmotic coefficients using polyelectrolyte theory indicates that the thermodynamic activity of cytoplasmic K+ increases from approximately 0.14 M in cells grown at an external osmolarity of 0.10 OSM to approximately 0.76 M at 1.02 OSM.(ABSTRACT TRUNCATED AT 400 WORDS)
已测定了需氧生长的大肠杆菌K - 12细胞质中的水可及体积、所有重要渗透溶质的含量以及蛋白质浓度，这些都是作为基本生长培养基渗透压的函数。细胞质水体积（Vcyto）随渗透压升高呈线性下降，从在0.10 OSM下生长的细胞中的2.23（±0.12）微升/毫克干重降至1.02 OSM时的1.18（±0.06）微升/毫克干重。高于0.28 OSM时，生长速率随渗透压升高呈线性下降。在渗透压约为1.8时生长速率外推至零，此时估计的Vcyto为0.5（±0.2）微升/毫克干重。用质壁分离剂NaCl对非生长细胞进行滴定来测量Vcyto，以获得低渗透压（0.10 OSM）和中等渗透压（0.28 OSM）培养基中生长的细胞的“结合”水体积（可能是大分子水合水）和细胞质渗透系数。在两种渗透条件下结合水体积Vb相似（Vb = 0.40（±0.04）微升/毫克干重），约相当于0.5克H₂O/克细胞质大分子。由于Vcyto随渗透压升高而降低，而Vb似乎与渗透压无关，随着生长培养基渗透压的增加，水合水在Vcyto中所占比例变得更大。在Vcyto约等于Vb的渗透压下生长似乎停止。K⁺和谷氨酸（Glu⁻）是在低渗透压培养基中生长的细胞中仅有的重要细胞质渗透溶质。K⁺的量大大超过Glu⁻的量。细胞质电中性分析表明，细胞质表现得像细胞质多阴离子的K⁺盐的浓溶液，其中额外电解质（K⁺ Glu⁻）的量随渗透压增加。随着生长培养基的渗透压变得非常低，细胞质接近无电解质的K⁺-多阴离子溶液。通过非生长细胞质壁分离滴定中Vcyto随外部渗透压的变化来测定体内渗透系数。得到的值（0.10 OSM下生长的细胞φ = 0.54（±0.06），0.28 OSM下φ = 0.71（±0.11））表明由于K⁺与细胞质多阴离子之间的库仑相互作用，细胞内离子具有高度非理想性。使用聚电解质理论分析这些渗透系数表明，细胞质K⁺的热力学活性从外部渗透压为0.10 OSM时生长的细胞中的约0.14 M增加到1.02 OSM时的约0.76 M。（摘要截断于400字）
