Kinetics of [35S]dATPalphaS interaction with P2Y1 purinoceptor in rat brain membranes

Kinetics of [35S]dATPalphaS (2'deoxyadenosine-5'-[alpha-35S]-thiotriphosphate) interaction with rat brain membrane fragments was studied at 25 degrees C and at radioligand concentrations from 2 to 250 nM. At least two different ways of [35S]dATPalphaS interaction with the membranes were distinguished on the basis of radioligand on-rate. Firstly, the binding sites characterized by 'fast' on-rate can be observed. Secondly, the 'slow' binding sites were kinetically identified and quantified. As in both cases the bound radioligand could be displaced by excess of ATP, all these binding sites can be defined as 'specific sites'. In the 'slow' binding sites isomerization of the receptor-ligand complex was observed, as is typical for interaction of antagonists with G-protein coupled receptors, and the kinetic parameters for this interaction were similar with the appropriate data for the hP2Y1 receptors expressed in 1321N1 astrocytoma cells Therefore these sites could be assigned to the same receptor subtype in brain membranes while the 'fast' binding sites belong to other membrane-bound proteins, also interacting with ATP and its analogues. The kinetic properties of the latter sites were not analysed in detail.