INFLUENCES OF MACROMOLECULAR CROWDING ON BIOCHEMICAL SYSTEMS

体外研究表明，排除性体积效应相对较大。 通过各种生化反应。考虑到高浓度的 活细胞内的大分子，预计这种排除 细胞中的体积效应将引起广泛的速率和 一系列细胞反应的平衡。我们设计了试验性的 帮助估计这些影响在体内的大小的程序。这个 目标是将体外参数修正为更适合于 细胞条件。 我们初步研究了大肠杆菌的细胞质隔膜。我们的 方法是测量黄连提取物中的排除体积参数 并对这些参数进行了校正，以使 萃取物中的大分子相对于细胞质的稀释 细胞。这种方法需要开发两个新的程序， 即适用于估计排除的体积参数的方法 在复杂的混合物中，例如基于两相分离的细胞提取物 系统，也是一个程序，以纠正萃取物浓度回到 到细胞质条件。 在一项不相关的研究中，这项研究源于早期对拥挤的测量 反应，我们已经开发了一种方法来量化 一组DNA限制片段中的特定成员。该方法应该 一般适用于涉及连接或特定部位的反应 切割特定的限制性片段。

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.