Isolation of adenylate cyclase-free, beta-adrenergic receptor from turkey erythrocyte membranes by affinity chromatography.

Abstract

The adenylate cyclase [ATP pyrophosphate-lyase (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 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 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%. 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. The .beta.-adrenergic receptor and the adenylate cyclase apparently are independent entities which may be separated on a functional basis.