INFLUENCES OF MACROMOLECULAR CROWDING ON BIOCHEMICAL SYSTEMS

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In vitro studies have demonstrated relatively large excluded volume effects upon a variety of biochemical reactions. Given the high concentrations of macromolecules within living cells, it is anticipated that such excluded volume effects in cells will cause wide-spread changes in rates and equilibria of a host of cellular reactions. We have devised experimental procedures to help estimate the magnitudes of these effects in vivo. The goal is the correction of in vitro parameters to values more appropriate to cellular conditions. We have initially studied the cytoplasmic compartment of E.coli. Our approach is to measure excluded volume parameters in extracts of spheroplasts and cells of E.coli and to correct these parameters for the dilution of macromolecules in the extract relative to the cytoplasm of the cells. This approach has required the development of two new procedures, namely an assay suitable for the estimation of excluded volume parameters in complex mixtures such as cell extracts based upon a two-phase partition system, and also a procedure for correction of extract concentrations back to cytoplasmic conditions. In an unrelated study which arose from earlier measurements of crowding reactions, we have developed a method for quantitating reactions between specific members of a set of DNA restriction fragments. The method should be generally applicable to reactions involving ligation or site-specific cleavage of specific restriction fragments.