Photoaffinity labeling of the beta-adrenergic receptor.
A new photoactive beta-adrenergic antagonist, p-azidobenzylcarazolol (pABC) has been synthesized by combining a carbazole moiety with a p-azido-benzyl substituent. The compound has been labeled with tritium to a specific activity of 26 Ci/mmol. In frog erythrocyte membranes, [3H]p-azido-benzylcarazolol binds to the beta-adrenergic receptor with the expected beta 2 specificity and with high affinity (KD congruent to 100 +/- 10 pM). Unlabeled p-azido-benzylcarazolol can irreversibly inactivate the [3H]dihydroalprenolol-binding activity of frog erythrocyte membranes in a photodependent manner which can be prevented by beta-adrenergic agents. Incubation of frog erythrocyte membranes or digitonin-solubilized preparations of these membranes or digitonin-solubilized preparations of these membranes which had been enriched in beta-adrenergic receptors by a Sepharose-alprenolol chromatography step led to covalent incorporation of radioactivity into a Mr = 58,000 peptide. Specific incorporation of [3H]pABC into the Mr = 58,000 peptide could be prevented by both beta-adrenergic agonists and antagonists. This peptide has previously been purified and shown to contain the beta-adrenergic receptor-binding site (Shorr, R. G. L., Lefkowitz, R. J., and Caron, M. G. (1981) J. Biol. Chem. 256, 5820-5826). Thus, photoaffinity labeling of the beta-adrenergic receptor protein directly identifies the same hormone-binding subunit as has been isolated by conventional purification techniques.
D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D001386:Azides; D001667:Binding, Competitive; D002227:Carbazoles / Q000138:chemical synthesis / Q000378:metabolism*; D004910:Erythrocyte Membrane / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D011412:Propanolamines; D011898:Ranidae; D011941:Receptors, Adrenergic / Q000378:metabolism*; D011943:Receptors, Adrenergic, beta / Q000302:isolation & purification / Q000378:metabolism*
D000345:Affinity Labels; D001386:Azides; D002227:Carbazoles; D011412:Propanolamines; D011941:Receptors, Adrenergic; D011943:Receptors, Adrenergic, beta; C032509:4-azidobenzylcarazolol