Kinetic analysis of [ 35S]dATPαS interaction with P2y 1 nucleotide receptor

Abstract

The kinetics of 2′-deoxyadenosine-5′-O-(1-thiotriphosphate) ([ 35S]dATPαS) interaction with membrane fragments of transfected astrocytoma 1321N1 cells, expressing human P2Y 1 receptors, and the same wild-type cells, not expressing P2Y receptors were studied. Binding of this radioligand was observed with both types of membranes, but sites showing slow on-rate were found only on the transfected cells. These “slow” binding sites behaved as a kinetically homogeneous population and their interaction with the radioligand was shown to occur in two steps, R+A ⇄ K A RA ⇄ k −i k i (RA) , including the relatively slow isomerization of the complex RA into ( RA). Evidence was obtained to assign the isomerized (“slow”) binding sites on the transfected cells as P2Y 1 receptor sites, differentiated from other binding sites of non-receptor origin by kinetic analysis, and characterised by the kinetic parameters K A =59±19 nM, k i =(9.0±0.8)10 −3 s −1 and k − i =(3.9±0.7)10 −3 s −1. [ 35S]dATPαS binding, with kinetic criteria, can be of value for differentiation of the receptor sites from non-receptor sites and thus provides solid basis for radioligand assay of P2Y 1 receptors.