Identification of a receptor for ADP on blood platelets by photoaffinity labelling.
The synthesis of a new analogue of ADP, 2-(p-azidophenyl)-ethythioadenosine 5'-diphosphate (AzPET-ADP), is described. This compound contains a photolabile phenylazide group attached to the ADP molecule by a thioether link at the purine 2 position. It has been prepared in radioactive form with 32P in the beta-phosphate at a specific radioactivity of 100 mCi/mumol. The reagent activated platelets, causing shape change and aggregation, with somewhat lower affinity than ADP. On photolysis the affinity was increased. The reagent also inhibited platelet adenylate cyclase stimulation by prostaglandin E1, with considerably higher affinity than ADP. On photolysis the affinity was decreased. AzPET-ADP competitively inhibited the binding of 2-methylthio[beta-32P]ADP, a ligand for the receptor by which ADP causes inhibition of adenylate cyclase. In the dark, AzPET-[beta-32P]ADP bound reversibly and with high affinity to a single population of sites similar in number to the sites that bind 2-methylthio[beta-32P]ADP. Binding was inhibited by ADP and by ATP and by p-chloromercuribenzenesulphonic acid (pCMBS). On exposure to u.v. light in the presence of platelets, AzPET-[beta-32P]ADP was incorporated covalently but non-specifically into several platelet proteins, although prominent intracellular proteins were not labelled. Specific labelling was confined to a single region of SDS/polyacrylamide gels, overlying but not comigrating with actin. Incorporation of radioactivity into this region was inhibited by ADP and by ATP as well as by ADP beta S, ATP alpha S and pCMBS, but not by adenosine, GDP or AMP. Inhibition of AzPET-[beta-32P]ADP incorporation was closely correlated with inhibition of equilibrium binding of 2-methylthio[beta-32P]ADP. These results suggests that the labelled protein, which migrates with an apparent molecular mass of 43 kDa in reduced gels, is the receptor through which ADP inhibits adenylate cyclase.
Cristalli
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Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA 19140.
Mills
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DC
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000345:Affinity Labels; D001386:Azides / Q000138:chemical synthesis / Q000378:metabolism*; D001667:Binding, Competitive; D001792:Blood Platelets / Q000378:metabolism*; D005260:Female; D006801:Humans; D008297:Male; D010782:Photolysis; D011983:Receptors, Purinergic / Q000378:metabolism*; D014466:Ultraviolet Rays
C081089:2-(4-azidophenyl)ethylthioadenosine 5'-diphosphate; D000345:Affinity Labels; D001386:Azides; D011983:Receptors, Purinergic; D002731:4-Chloromercuribenzenesulfonate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate