Crowding effects on EcoRV kinetics and binding
Crowding effects on EcoRV kinetics and binding
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DOI：
10.1016/s0006-3495(99)77154-7
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发表时间：
1999-12-01
影响因子：
3.4
通讯作者：
Bloomfield, VA
中科院分区：
文献类型：
--
作者：
Wenner, JR;Bloomfield, VA
细胞的胞质溶胶含有高浓度的小分子和大分子，但实验数据通常是在稀释溶液中获得的，不能反映体内条件。我们已经研究了拥挤效应，大分子EcoRV裂解加入高分子量的Ficoll 70的反应溶液。结果表明，Ficoll对整体EcoRV反应速度的影响令人惊讶地小，因为V-max和K-m的抵消性增加以及更强的非特异性结合。测量参数的变化可以在很大程度上归因于反应物活性的排除体积效应和蛋白质扩散的减缓。结合后的共体积减少似乎增强了非特异性结合强度，并且k（cat）似乎被更强的非特异性结合减慢，这减慢了产物释放。数据还表明，有效Ficoll颗粒体积随着其浓度增加超过几个重量百分比而减小，这可能是由于Ficoll相互渗透或压缩。
The cytosol of the cell contains high concentrations of small and large macromolecules, but experimental data are often obtained in dilute solutions that do not reflect in vivo conditions. We have studied the crowding effect that large macromolecules have on EcoRV cleavage by adding high-molecular-weight Ficoll 70 to reaction solutions. Results indicate that Ficoll has surprisingly little effect on overall EcoRV reaction velocity because of offsetting increases in V-max and K-m, and stronger nonspecific binding. The changes in measured parameters can largely be attributed to the excluded volume effects on reactant activities and the slowing of protein diffusion. Covolume reduction upon binding appears to reinforce nonspecific binding strength, and k(cat) appears to be slowed by stronger nonspecific binding, which slows product release. The data also suggest that effective Ficoll particle volume decreases as its concentration increases above a few weight percent, which may be due to Ficoll interpenetration or compression.