Abstract
We propose a method for obtaining kinetic parameters of unlabeled ligands which is based on analyzing the effect of their competition on the binding kinetics of a labeled ligand. In this method (competition kinetics), the binding kinetics of a labeled ligand are measured in the absence and in the presence of given concentrations of a competing unlabeled ligand. The rate equations appropriate to the particular kinetic model may be solved employing linear homogeneous differential equations. The rate constants appearing in the final integrated expressions can be evaluated by nonlinear regression. We have demonstrated the validity and applicability of the method to study receptor systems, using the muscarinic receptors in rat brain cortex as an experimental system. We have determined the various kinetic parameters of two muscarinic antagonists (N-methyl-4-[3H]piperidyl benzilate and (-)-N-[3H]methylscopolamine) by following directly their binding kinetics. These parameters were compared with those obtained for the same unlabeled ligands in homogeneous and heterogeneous competition studies. In all cases, the parameters obtained by the competition kinetics method were close to those extracted from direct binding kinetics, and in cases where both types of studies were performed on the same preparations, the parameters obtained by the two methods were essentially identical.
Highlights
Of a labeled ligandI.n this method (competitionkinet- In thismethod, the binding kinetics of ics), the bindingkinetics of a labeled ligand are mea- a labeled ligand are measured in the absence andinthe suredintheabsenceand in thepresenceof given presence of given concentrations of a competing unlabeled concentrations of a competing unlabeled ligand
The parameters obtainebdy the competition to employ ligands whose binding kinetics can be investigated kinetics method were close to those extracted from direct bindingkinetics, and incases where both types of studies were performed on the same preparations, the parameters obtained by thetwo methods were essentially identical
As in the case ofthe homogeneous competi- experimentally determined valuoefs the equilibrium dissociation studies, the rate constants extracted from the heteroge- tion constants, lending further supportto the validity of the neouscompetitioncurves didnot depend on the concentration competition kinetics method
Summary
We propose a method for obtainingkinetic parame- two ligands competing on one receptor site. Ters of unlabeled ligands whichis based on analyzing We propose here a method based on analyzing the effect of the effect of their competitionon the bindingkinetics ligand competition on the binding kinetics of a labeled ligand. The parameters obtainebdy the competition to employ ligands whose binding kinetics can be investigated kinetics method were close to those extracted from direct bindingkinetics, and incases where both types of studies were performed on the same preparations, the parameters obtained by thetwo methods were essentially identical. A direct test of the validity of the method is whether the kinetic parameters extracted by competition kinetics agree with those obtained independently by direct binding experiments. The binding of antagonists to the muscarinic receptors in rat brain cortex fulfills the requirements for such a study
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