Kinetic analysis of [35S]dATP alpha S interaction with P2y(1) nucleotide receptor

The kinetics of 2'-deoxyadenosine-5'-O-(1-thiotriphosphate) ([(35)S]dATP alpha S) 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). [(35)S]dATP alpha S 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.