ATP alpha S is a ligand for P2Y receptors in synaptosomal membranes: solubilization of [S-35]ATP alpha S binding proteins associated with G-proteins

ATP alpha S was established as a P2Y receptor-specific ligand for assaying the solubilization of functional native P2Y receptors from synaptosomal mem branes. These receptors are not yet amenable to biochemical studies. High-a ffinity [S-35]ATPaS binding sites in synaptosomal membranes, solubilized wi th Brij58, retained the binding affinity and ligand specificity (ATP alpha S = ATP > 2-MeSATP > ADP, ADP beta S > AMP much greater than alpha,beta-MeA TP) corresponding to P2Y receptors. Mg2+ but not Ca2+, enhanced high-affini ty [S-35]ATPaS binding 30-fold, supporting specific recognition by P2Y rece ptors. ATP alpha S stimulated P2Y receptor-mediated [S-35]GTP gamma S bindi ng equipotently with ATP in synaptosomal membranes and in Brij58-solubilize d proteins demonstrating the association with G-proteins. Anion-exchange ch romatography of solubilized synaptosomal membrane proteins yielded two frac tions in which [S-35]ATP alpha S binding was regulated by GTP gamma S/Mg2+, thus possibly by heterotrimeric G-proteins. After a second chromatographic step (hydroxyapatite) the regulation of high-affinity [S-35]ATP alpha S bi nding by Mg2+ was still present, whereas the regulation by GTP gamma S/Mg2 was lost indicating the dissociation from G-proteins. Thus, conditions wer e found to stabilize ligand binding activity of solubilized P2Y receptors a nd to solubilize P2Y receptors associated with G-proteins. (C) 1999 Elsevie r Science Ltd. All rights reserved.