Characterization of [3H]Ro 5-4864 binding to calmodulin using a rapid filtration technique.
The benzodiazepine [3H]Ro 5-4864 bound specifically and saturably to an apparently homogenous, univalent species of binding site on the calmodulin molecule with an associated equilibrium dissociation rate constant (Kd) of 644 +/- 121 nM. The rates of association (K1) and dissociation (K-1) governing this interaction were estimated to be 7.66 x 10(3) M-1 sec-1 and 2.9 x 10(-3) sec-1, respectively, yielding a non-equilibrium determination of the Kd to be 379 nM. Such binding of [3H]Ro 5-4864 was protein-, pH-, and temperature-dependent and demonstrated pharmacological selectivity. Only benzodiazepine compounds (chlordiazepoxide, diazepam and Ro 5-4864) inhibited [3H]Ro 5-4864 binding to calmodulin with inhibitory equilibrium dissociation constants (Ki) less than 10 microM. The benzodiazepine compounds Ro 15-1788 and flunitrazepam did not displace [3H]Ro 5-4864 binding to calmodulin nor did a number of pharmacologically active non-benzodiazepine compounds (Ki values greater than 10 microM). Consideration of the stoichiometry yielded an approximate mole ratio of 0.90:1.0 (Ro 5-4864: calmodulin), suggesting that there is one binding site for Ro 5-4864 per molecule of calmodulin. The data reveal that the binding of [3H]Ro 5-4864 to calmodulin fulfills the major criteria of a ligand binding to a receptor. Such an interaction may underlie some of the pharmacological actions of Ro 5-4864-like compounds.
Morgan
P F
PF
Biological Psychiatry Branch, National Institute of Mental Health, Bethesda, MD 20892.
Patel
J
J
Marangos
P J
PJ
D001570:Benzodiazepinones / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008024:Ligands; D011485:Protein Binding; D013329:Structure-Activity Relationship; D013696:Temperature
D001570:Benzodiazepinones; D002147:Calmodulin; D008024:Ligands; C028513:4'-chlorodiazepam; D002118:Calcium