title: Photoaffinity labeling of the alpha 1-adrenergic receptor using an 125I-labeled aryl azide analogue of prazosin.
abstract: alpha 1-Adrenergic receptor probes, which can be radioiodinated to yield high specific activity radioligands, have been synthesized and characterized. 2-[4-(4-Amino-benzoyl)piperazin-1-yl]-4-amino-6,7-dimethoxyquin azoline (CP63,155), an arylamine analogue of the selective alpha 1-adrenergic antagonist prazosin, and its iodinated derivative, 2-[4-(4-amino-3-[125I]iodobenzoyl)piperazin-1-yl]-4-amino-6, 7-dimethoxyquinazoline [( 125I]CP63,789), bind reversibly and with high affinity (KD = 1 nM and 0.6 nM, respectively) to rat hepatic membrane alpha 1-adrenergic receptors. Conversion of [125I]CP63,789 to the aryl azide yields a photolabile derivative, 2-[4-(4-azido-3-[125I]iodobenzoyl)piperazin-1-yl]-4-amino-6, 7-dimethoxyquinazoline [( 125I]CP65,526), which prior to photolysis binds competitively and with high affinity (KD = 0.3 nM). Binding of [125I]CP63,789 and [125I]CP65,526 (prior to photolysis) is rapid and saturable. Both ligands identify similar alpha 1-adrenergic receptor binding site concentrations as the parent probe, [3H]prazosin. Specific binding by these iodinated ligands is stereoselective and inhibited by a variety of adrenergic agents with a specificity typical of the alpha 1-adrenergic receptor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography of [125I]CP65,526-labeled rat hepatic membranes reveal major protein species with molecular weights of 77K, 68K and 59K. Each protein binds adrenergic ligands with stereoselectivity and with a specificity typical of the alpha 1-adrenergic receptor. Inclusion of multiple protease inhibitors during membrane preparation prior to SDS-PAGE does not alter the labeling of these peptides.(ABSTRACT TRUNCATED AT 250 WORDS)
mesh_terms: D000345:Affinity Labels / Q000138:chemical synthesis*; D000818:Animals; D001386:Azides / Q000138:chemical synthesis* / Q000378:metabolism; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D005260:Female; D007202:Indicators and Reagents; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008970:Molecular Weight; D011224:Prazosin / Q000031:analogs & derivatives / Q000378:metabolism; D011799:Quinazolines / Q000138:chemical synthesis* / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D011942:Receptors, Adrenergic, alpha / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D000345:Affinity Labels; D001386:Azides; D007202:Indicators and Reagents; D011799:Quinazolines; D011942:Receptors, Adrenergic, alpha; C041552:azidoprazosin; D011224:Prazosin