[32P]3'-O-(4-benzoyl)benzoyl ATP as a photoaffinity label for a phospholipase C-coupled P2Y-purinergic receptor.
A 32P-labelled ATP analog, 3'-O-(4-benzoyl)benzoyl ATP (BzATP) previously shown to be an agonist at P2Y-purinergic receptors (Boyer J. L., and Harden T. K. (1989) Mol. Pharmacol. 36, 831-835), has been used as a probe for the P2Y-purinergic receptor on turkey erythrocyte plasma membranes. In the absence of light, [32P]BzATP bound to membranes with high affinity (KD approximately 5 nM), and in a saturable and reversible manner. The binding of [32P]BzATP was competitively inhibited by ATP and ADP analogs (2-methylthioadenosine 5'-triphosphate greater than adenosine 5'-O-(2-thiodiphosphate) greater than BzATP greater than ATP greater than beta,gamma-methyleneadenosine 5'-triphosphate greater than 5'-adenylylimidodiphosphate) with pharmacological specificity consistent with that of a P2Y-purinergic receptor. Guanine nucleotides (guanosine 5'-O-(3-thiotriphosphate) greater than GTP greater than guanosine 5'-O-(2-thiodiphosphate) greater than GMP) noncompetitively inhibited the binding of radioligand. Photolysis of [32P] 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 P2Y-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 P2Y-purinergic receptor. These results suggest that [32P]BzATP can be used to label covalently the P2Y-purinergic receptor and that this radioprobe will be a useful reagent for further characterization and purification of the P2Y-purinergic receptor.
Department of Pharmacology, University of North Carolina, School of Medicine, Chapel Hill 27599.
J L
JL
Boyer
C L
CL
Cooper
T K
TK
Harden
D000227:Adenine Nucleotides / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D007700:Kinetics; D008027:Light; D010761:Phosphorus Radioisotopes; D011983:Receptors, Purinergic / Q000378:metabolism*; D013873:Thionucleotides / Q000494:pharmacology; D014422:Turkeys; D010738:Type C Phospholipases / Q000097:blood*
D000227:Adenine Nucleotides; D000345:Affinity Labels; D006150:Guanine Nucleotides; D010761:Phosphorus Radioisotopes; D011983:Receptors, Purinergic; D013873:Thionucleotides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D010738:Type C Phospholipases