Separation of solubilized A2 adenosine receptors of human platelets from non-receptor [3H]NECA binding sites by gel filtration

Abstract

Summary Human platelet membranes were solubilized with the zwitterionic detergent CHAPS (3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonate) and the solubilized extract subjected to gel filtration. Binding of the adenosine receptor agonist [3H]NECA (5′-N-ethylcarboxamidoadeno-sine) was measured to the eluted fractions. Two [³H]NECA binding peaks were eluted, the first of them with the void volume. This first peak represented between 10% and 25% of the [³H]NECA binding activity eluted from the column. It bound [³H]NECA in a reversible, saturable and GTP-dependent manner with an affinity of 46 nmol/1 and a binding capacity of 510 fmol/mg protein. Various adenosine receptor ligands competed for the binding of [³H]NECA to the first peak with a pharmacological profile characteristic for the A₂ adenosine receptor as determined from adenylate cyclase experiments. In contrast, most adenosine receptor ligands did not compete for [³H]NECA binding to the second, major peak. These results suggest that a solubilized A₂ receptor-GS protein complex of human platelets can be separated from other [³H]NECA binding sites by gel filtration. This allows reliable radioligand binding studies of the A₂ adenosine receptor of human platelets.