Boyd N D, Cohen J B
Biochemistry. 1980 Nov 11;19(23):5353-8. doi: 10.1021/bi00564a032.
An automated rapid-mixing ultrafiltration apparatus was constructed to measure at subsecond times the kinetics of binding of radiolabeled cholinergic ligands to nicotinic postsynaptic membranes isolated from Torpedo electric tissue. The dissociation of [3H]AcCh-receptor complexes at 7 and 23 degrees C was characterized by dissociation rate constants (kdissoc) of 0.05 s-1 and 0.15S-1, respectively. The association kinetics at low concentrations of [3H]AcCh were determined at 23 degrees C and found to be consistent with a model in which 20% of the AcCh binding sites preexist in a receptor conformation that binds AcCh with high affinity (KD = 3 nM) and with a bimolecular association constant, k+ = 5.7 x 10(7) M-1 SD-1. Initial studies of the association kinetics at higher AcCh concentrations revealed a transient low-affinity binding step that occurred relatively slowly. In the presence of 0.8 microM [3H]AcCh, this component of the association reaction was characterized by an experimental rate constant of 2 s-1. The observed binding kinetics are discussed with reference to the processes of channel activation and receptor desensitization.
构建了一种自动快速混合超滤装置，用于在亚秒级时间测量放射性标记的胆碱能配体与从电鳐电组织分离的烟碱型突触后膜结合的动力学。在7℃和23℃下，[3H]乙酰胆碱-受体复合物的解离分别以解离速率常数（kdissoc）为0.05 s-1和0.15 s-1为特征。在23℃下测定了低浓度[3H]乙酰胆碱时的结合动力学，发现其与一个模型一致，即20%的乙酰胆碱结合位点以高亲和力（KD = 3 nM）结合乙酰胆碱的受体构象预先存在，双分子结合常数k+ = 5.7×10(7) M-1 s-1。对较高乙酰胆碱浓度下结合动力学的初步研究揭示了一个相对缓慢发生的短暂低亲和力结合步骤。在0.8 microM [3H]乙酰胆碱存在下，该结合反应组分的实验速率常数为2 s-1。结合通道激活和受体脱敏过程对观察到的结合动力学进行了讨论。
