Abstract
We have recently described the affinity chromatography purification of the turkey erythrocyte β‐adrenergic receptor. The minute amounts obtained initially precluded extensive biochemical characterization. To improve the yield of the receptor, the erythrocyte membranes have been prepared by a new method. This procedure resulted in a 10‐fold higher receptor density in comparison with the membrane preparation used previously. The new membranes also contained a catecholamine‐sensitive guanine triphosphatase and an adenylate cyclase sensitive to Gpp(NH)p and l‐epinephrine. Solubilization by a double digitonin extraction resulted in a preparation containing 4–6 pmoles of ^3^H‐dihydroalprenolol binding sites per mg of membrane protein.
A single step of affinity chromatography on alprenolol‐sepharose of the soluble digitonin extract resulted in an additional 1,000‐fold purification of the receptor. The overall purification factor was 20,000 relative to the binding activity of the crude membrane preparations.
Electrophoresis in SDS‐polacrylamide of iodinated purified β‐receptors revealed, after autoradiography, the presence of four major components. Three of these, corresponding to molecular weights of 170,000, 33,000, and 30,000, respectively, were not affected by reduction with β‐mercaptoethanol and were not observed when the digitonin extracts were loaded on the affinity gel in the presence of an excess of l‐propranolol. A fourth 52,000‐dalton component (60,000 daltons after reduction with β‐mercaptoethanol) remained apparent even when affinity purification was prevented by addition of l‐propranolol.
Our results suggest that the β‐adrenergic receptor is composed of at least three subunits that interact by noncovalent bonds.