ATPalphaS is a ligand for P2Y receptors in synaptosomal membranes: solubilization of [35S]ATPalphaS binding proteins associated with G-proteins.
ATPalphaS was established as a P2Y receptor-specific ligand for assaying the solubilization of functional native P2Y receptors from synaptosomal membranes. These receptors are not yet amenable to biochemical studies. High-affinity [35S]ATPalphaS binding sites in synaptosomal membranes, solubilized with Brij58, retained the binding affinity and ligand specificity (ATPalphaS = ATP > 2-MeSATP > ADP, ADPbetaS > AMP >>> alpha,beta-MeATP) corresponding to P2Y receptors. Mg2+ but not Ca2+, enhanced high-affinity [35S]ATPalphaS binding 30-fold, supporting specific recognition by P2Y receptors. ATPalphaS stimulated P2Y receptor-mediated [35S]GTPgammaS binding equipotently with ATP in synaptosomal membranes and in Brij58-solubilized proteins demonstrating the association with G-proteins. Anion-exchange chromatography of solubilized synaptosomal membrane proteins yielded two fractions in which [35S]ATPalphaS binding was regulated by GTPgammaS/Mg2+, thus possibly by heterotrimeric G-proteins. After a second chromatographic step (hydroxyapatite) the regulation of high-affinity [35S]ATPalphaS binding by Mg2+ was still present, whereas the regulation by GTPgammaS/Mg2+ was lost indicating the dissociation from G-proteins. Thus, conditions were found to stabilize ligand binding activity of solubilized P2Y receptors and to solubilize P2Y receptors associated with G-proteins.
Schäfer
R
R
Institut für Neurobiochemie, Medizinische Fakultät, Otto-von-Guericke-Universität Magdeburg, Germany.
Reiser
G
G
D000818:Animals; D002413:Cations, Divalent; D002462:Cell Membrane / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D008297:Male; D019920:Photoaffinity Labels; D011869:Radioligand Assay; D051381:Rats; D017208:Rats, Wistar; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D012995:Solubility; D013462:Sulfur Radioisotopes / Q000378:metabolism; D013574:Synaptosomes / Q000378:metabolism*; D013873:Thionucleotides / Q000378:metabolism*
D002413:Cations, Divalent; D019920:Photoaffinity Labels; D018048:Receptors, Purinergic P2; D013462:Sulfur Radioisotopes; D013873:Thionucleotides; C017817:adenosine 5'-(1-thio)triphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins