Visualization of the turkey erythrocyte beta-adrenergic receptor.
We have recently described the affinity chromatography purification of the turkey erythrocyte beta-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 is SDS-polyacrylamide of iodinated purified beta-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 beta-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 beta-mercaptoethanol) remained apparent even when affinity purification was prevented by addition of l-propranolol. Our results suggest that the beta-adrenergic receptor is composed of at least three subunits that interact by noncovalent bonds.
D000262:Adenylyl Cyclases / Q000097:blood; D000818:Animals; D002846:Chromatography, Affinity; D004082:Dihydroalprenolol / Q000097:blood; D004910:Erythrocyte Membrane / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000097:blood; D007545:Isoproterenol / Q000097:blood; D008970:Molecular Weight; D011941:Receptors, Adrenergic / Q000378:metabolism*; D011943:Receptors, Adrenergic, beta / Q000302:isolation & purification / Q000378:metabolism*; D014422:Turkeys
D011941:Receptors, Adrenergic; D011943:Receptors, Adrenergic, beta; D004082:Dihydroalprenolol; D020558:GTP Phosphohydrolases; D000262:Adenylyl Cyclases; D007545:Isoproterenol