Isolation of adenylate cyclase-free, beta-adrenergic receptor from turkey erythrocyte membranes by affinity chromatography.
The adenylate cyclase [ATP pyrophosphatelyase (cyclizing); EC 4.6.1.1] and beta-adrenergic receptor of plasma membranes of turkey erythrocytes were solubilized in an active form by treatment with either NaF or guanylylimidodiphosphate and digitonin. The solubilized enzyme was no longer stimulated by catecholamines, NaF, or guanine nucleotides. The digitonin extract was chromatographed on an alprenolol-agarose derivative. While the bulk of protein and all the adenylate cyclase activity passed unretarded through the column, the receptor was retained. It eluted free of enzyme activity with an alprenolol solution containing 1 M NaCl; the yield was 25-30%. The protein content of the alprenolol eluates was too low to be estimated by the Lowry technique and was assessed by a more sensitive fluorometric method. Under these conditions, the beta-adrenergic receptor was purified approximately 2000-fold in a single step with retention of all its pharmacological properties. These experiments establish that the beta-adrenergic receptor and the adenylate cyclase are independent entities which may be separated on a functional basis.
D000262:Adenylyl Cyclases / Q000097:blood* / Q000302:isolation & purification; D000526:Alprenolol / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D002846:Chromatography, Affinity; D004910:Erythrocyte Membrane / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D007700:Kinetics; D011941:Receptors, Adrenergic / Q000302:isolation & purification; D011943:Receptors, Adrenergic, beta / Q000302:isolation & purification / Q000378:metabolism; D014422:Turkeys
D011941:Receptors, Adrenergic; D011943:Receptors, Adrenergic, beta; D000526:Alprenolol; D000262:Adenylyl Cyclases