[32P]3’-0-(4-Benzoyl)benzoyl ATP as a Photoaffinity Label for a Phospholipase C-coupled Pay-Purinergic Receptor*

A 32P-labeled ATP analog, 3’-0-(4-benzoyl)benzoyl ATP (BzATP) previously shown to be an agonist at P,,-purinergic receptors (Boyer J. L., and Harden T. K. (1989) Mol. Pharmacol. 36, 831~835), has been used as a probe for the Pzy-purinergic receptor on turkey erythrocyte plasma membranes. In the absence of light, [32P]BzATP bound to membranes with high affinity (Kn = 5 nM), and in a saturable and reversible manner. The binding of [32P]BzATP was competitively inhibited by ATP and ADP analogs (a-methylthioadenosine 5’-triphosphate > adenosine 5’-0-(2-thiodiphosphate) > BzATP > ATP > /3,-y-methyleneadenosine 5’-triphosphate > 5’-adenylylimidodiphosphate) with pharmacological specificity consistent with that of a Pzy-purinergic receptor. Guanine nucleotides (guanosine 5’-0-(3-thiotriphosphate) > GTP > guanosine 5’0-(2-thiodiphosphate) > GMP) noncompetitively inhibited the binding of radioligand. Photolysis of [32PJ BzATP-prelabeled membranes resulted in incorporation of radiolabel into a protein of approximately 53,000 Da. Photolabeling was inhibited in a concentration-dependent manner by ATP and ADP analogs with a potency order characteristic for a Pzu-purinergic receptor and was modulated by guanine nucleotides. A protein of approximately 53,000 daltons was also labeled by [32P]BzATP in membranes from several other tissues known to express the PzY-purinergic receptor. These results suggest that [32P]BzATP can be used to label covalently the P&purinergic receptor and that this radioprobe will be a useful reagent for further characterization and purification of the PZYpurinergic receptor.