INFLUENCES OF MACROMOLECULAR CROWDING ON BIOCHEMICAL SYSTEMS

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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 these 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 from two-phase partition and gel filtration assays. Macromolecule concentrations in the cytoplasm of E.coli are obtained from experiments using the known impermeability of the cytoplasmic volume to sucrose. 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 in vitro studies, but are insensitive in in vivo studies. 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.