The beta-adrenergic receptor: rapid purification and covalent labeling by photoaffinity crosslinking.
New procedures for the rapid purification and covalent labeling of the beta-adrenergic receptors have been developed that should greatly accelerate progress in the study of these widely distributed adenylate cyclase-coupled receptors. Chromatography of solubilized receptor preparations on a Sepharose-alprenolol affinity gel followed by HPLC on steric exclusion columns lead to rapid (2 days) and high yield (approximately 30%) purification of the receptors from frog erythrocytes. The receptor obtained by these rapid procedures appears to be composed entirely of 58,000 Mr subunit(s) and to be identical to that previously purified by much lengthier procedures [Shorr, R. G. L., Lefkowitz, R. J. & Caron, M. G. (1981) J. Biol. Chem. 256, 5820-5826]. A novel, very high affinity, specific beta-adrenergic antagonist, p-aminobenzylcarazolol, has also been synthesized. It can be radioiodinated to theoretical specific radioactivity with 125I (2,200 Ci/mmol). This radioligand, which possesses an arylamine moiety, may then be covalently incorporated into the receptor binding subunit (58,000 Mr peptide) of the frog erythrocyte membranes by the use of the bifunctional photoactive crosslinker N-succinimidyl-6-(4'-azido-2'- nitrophenylamino)hexanoate (SANAH). Covalent incorporation is blocked by various drugs with a strict beta-adrenergic specificity. This suggests that the photoaffinity crosslinking approach may be useful for labeling a variety of small molecule and neurotransmitter receptors when appropriate ligands can be synthesized.
D000345:Affinity Labels; D000818:Animals; D001001:Anura; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003432:Cross-Linking Reagents; D004910:Erythrocyte Membrane / Q000032:analysis*; D004912:Erythrocytes / Q000032:analysis*; D010777:Photochemistry; D011941:Receptors, Adrenergic / Q000302:isolation & purification*; D011943:Receptors, Adrenergic, beta / Q000302:isolation & purification*
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D011941:Receptors, Adrenergic; D011943:Receptors, Adrenergic, beta