Large-scale purification of [alpha]2-adrenergic receptor-enriched membranes from human platelets. Persistent association of guanine nucleotides with nonpurified membranes

Abstract

A simple large-scale purification of [alpha]2-adrenergic receptor-enriched membranes from human platelets is described. Binding of the antagonist [3H]yohimbine is enriched 3-5-fold compared to a crude membrane fraction. Binding of low concentrations of the partial agonist 3H-p-aminoclonidine is increased 15-20-fold due to a higher binding affinity for the purified membranes. A soluble inhibitor of 3H-p-aminoclonidine binding to purified membranes is found even in thrice-washed crude platelet membranes. The guanine nucleotides GDP and GTP are found to account for this inhibitory activity. Forskolin-stimulated adenylate cyclase activity is also enriched in the purified membrane fraction. Adenylate cyclase activity is inhibited by [alpha]2-agonist to a comparable extent in all membrane fractions. This membrane preparation should prove useful in studies of [alpha]2-adrenergic receptor mechanisms.