Kinetics of binding of [3H]acetylcholine to Torpedo postsynaptic membranes: association and dissociation rate constants by rapid mixing and ultrafiltration.
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.[1]References
- Kinetics of binding of [3H]acetylcholine to Torpedo postsynaptic membranes: association and dissociation rate constants by rapid mixing and ultrafiltration. Boyd, N.D., Cohen, J.B. Biochemistry (1980) [Pubmed]
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