Boyd N D, Cohen J B
Biochemistry. 1980 Nov 11;19(23):5344-53. doi: 10.1021/bi00564a031.
The kinetics of binding of [3H]acetylcholine (AcCh) and [3H]carbamoylcholine (Carb) to membrane-bound nicotinic receptor from Torpedo electric tissue have been measured on the second time scale by rapid mixing and ultrafiltration. The concentration dependence of the association kinetics of agonist binding and the kinetics of ligand dissociation and receptor reisomerization following the removal of agonist are analyzed in terms of a model in which the observed binding is by a single population of receptors that exists in the absence of agonist in two interconvertible conformations, one binding agonist weakly (R1) and the other binding with high affinity (R2). A computer simulation has been used to determine values of rate and equilibrium constants characterizing the ligand interactions with the two conformations and for the conformational equilibrium in the presence and absence of agonist. At 4 degrees C, R1/R2 - 4.5, and the half-time for isomerization for low to high affinity of unliganded receptor is equal to 200 s, while for receptors occupied by either AcCh of Carb the half-time is reduced to approximately 4 s. For AcCh the apparent dissociation constants of the low- and high-affinity conformations re 800 nM and 2 nM, respectively (Keq = 8 nM), and for Carb the values are 30 microM and 25 nM (Keq = 100 nM). The dissociation rate constant of [3H]AcCh from R2 is equal to 0.04 s-1. The results are further discussed in terms of alternate less satisfactory reaction models and are compared with the receptor conformational equilibria deduced by the use of other kinetic techniques.
通过快速混合和超滤技术，在第二个时间尺度上测量了[3H]乙酰胆碱（AcCh）和[3H]氨甲酰胆碱（Carb）与电鳐电组织膜结合烟碱受体的结合动力学。根据一个模型分析了激动剂结合的缔合动力学的浓度依赖性以及去除激动剂后配体解离和受体再异构化的动力学。在该模型中，观察到的结合是由单一群体的受体进行的，该受体在没有激动剂的情况下以两种可相互转化的构象存在，一种与激动剂弱结合（R1），另一种与激动剂高亲和力结合（R2）。已使用计算机模拟来确定表征配体与两种构象相互作用以及存在和不存在激动剂时构象平衡的速率和平衡常数的值。在4℃时，R1/R2 = 4.5，未结合配体的受体从低亲和力异构化为高亲和力的半衰期等于200秒，而对于被AcCh或Carb占据的受体，半衰期缩短至约4秒。对于AcCh，低亲和力和高亲和力构象的表观解离常数分别为800 nM和2 nM（Keq = 8 nM），对于Carb，值分别为30 μM和25 nM（Keq = 100 nM）。[3H]AcCh从R2的解离速率常数等于0.04 s-1。将根据其他不太令人满意的反应模型进一步讨论结果，并与使用其他动力学技术推导的受体构象平衡进行比较。
