Leid M, Schimerlik M I, Murray T F
College of Pharmacy, Oregon State University, Corvallis 97331.
Mol Pharmacol. 1989 Apr;35(4):450-7.
Porcine atrial adenosine receptors have been solubilized using a detergent system consisting of digitonin and sodium cholate and characterized with the agonist radioligand N6[125I]hydroxyphenylisopropyl) adenosine [125I]HPIA. 125IHPIA labeled an apparently homogeneous population of solubilized recognition sites with a Bmax of 88 +/- 4 fmol/mg of protein and a KD of 1.4 +/- 0.1 nM. Solubilization resulted in a 2.5-fold enrichment of adenosine receptor specific activity and an enhanced signal to noise ratio over that observed for porcine atrial membrane preparations. Solubilized cardiac adenosine receptors were relatively stable and exhibited many of the properties of membrane-bound receptors. The rank order potency of adenosine receptor agonists inhibiting the binding of [125I]HPIA was consistent with the labeling of a solubilized A1 adenosine receptor. Association rate experiments suggested that the interaction of [125I]HPIA with solubilized cardiac adenosine receptors was consistent with that of a simple bimolecular reaction. The dissociation constant calculated from kinetic data (0.73 nM) was in good agreement with that determined by equilibrium binding measurements (1.4 nM). The interaction of cardiac A1 adenosine receptors and guanine nucleotide binding protein(s) G protein(s) was retained in this detergent system. Addition of guanosine-5'-O-(3-thio)triphosphate to an equilibrated mixture of solubilized cardiac adenosine receptors and [125I]HPIA resulted in a rapid and complete dissociation of [125I]HPIA. This dissociation was resolved into two kinetic phases, which appear to arise from two populations of independent, noninterconvertible receptor-G protein complexes that display differing sensitivities to guanine nucleotides. The A1 adenosine receptor-G protein complex solubilized in digitonin/cholate appears to provide an excellent system by which agonist radioligand-receptor-G protein interactions can be further studied.
