Molecular Cell Biology
Wiley InterScience Backfile Collection 1832-2000
Chemistry and Pharmacology
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 3H-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.
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