Barkley M D, Riggs A D, Jobe A, Burgeois S
Biochemistry. 1975 Apr 22;14(8):1700-12. doi: 10.1021/bi00679a024.
The equilibrium association constants for the binding of a wide variety of effecting ligands of the lac repressor were measured by equilibrium dialysis. Also, detailed investigations of the apparent rate of dissociation of repressor-operator comples as a function of ligand concentration were carried out for several inducers and anti-inducers. The affinity of repressor-ligand comples for operator DNA was evaluated from the specific rate constants at saturating concentrations of effecting ligand. By fitting the experimental data depicting the functional dependence of the rate of dissociation upon ligand concentrations to calculated curves, assuming simple models of the induction mechanism, the equilibrium association constant for the binding of effecting ligand to repressor-operator comples was determined. Inducers reduce the affinity of lac repressor for operator DNA by a factor of approximately 1000 under standard conditions; the extent of destabilization depends on Mg2+ ion concentration. Anti-inducers increase the affinity of repressor for operator at most a factor of five. Only one neutral ligand, which binds to repressor without altering the stability of repressor-operator comples, was found. No homotropic or heterotropic interactions in the binding of effecting ligands either to repressor or to repressor-operator complex are evident.
通过平衡透析法测定了乳糖阻遏物多种效应配体结合的平衡缔合常数。此外，还针对几种诱导剂和抗诱导剂，详细研究了阻遏物 - 操纵基因复合物的表观解离速率与配体浓度的函数关系。根据效应配体饱和浓度下的特定速率常数，评估了阻遏物 - 配体复合物对操纵基因DNA的亲和力。通过将描述解离速率与配体浓度函数关系的实验数据拟合到计算曲线，假设诱导机制的简单模型，确定了效应配体与阻遏物 - 操纵基因复合物结合的平衡缔合常数。在标准条件下，诱导剂使乳糖阻遏物对操纵基因DNA的亲和力降低约1000倍；去稳定化程度取决于Mg2 +离子浓度。抗诱导剂最多使阻遏物对操纵基因的亲和力增加5倍。仅发现一种中性配体，它与阻遏物结合而不改变阻遏物 - 操纵基因复合物的稳定性。在效应配体与阻遏物或阻遏物 - 操纵基因复合物的结合中，均未发现同促或异促相互作用。
