Solubilization and characterization of the beta-adrenergic receptor binding sites of frog erythrocytes.

摘要: Specific beta-adrenergic receptors present in membrane preparations of frog erythrocytes were identified by binding (-)-[3H]dihydroalprenolol, a potent competitive antagonist. The (-)-[3H]dihydroalprenolol sites could be solubilized treatment purified erythrocyte fraction with the plant glycoside digitonin but not wide variety other detergents. appeared to soluble several independent experimental criteria including (a) failure sediment 105,000 X g for 2 hours; (b) passage through 0.22-mu Millipore filters; (c) chromatography on Sepharose 6B gels; and (d) electron microscopy. receptor retained all essential characteristics membrane-bound sites, namely rapid reversible agonists antagonists; strict stereospecificity toward both appropriate structure-activity relationships; saturability at low concentrations ligand; no affinity alpha-adrenergic drugs, nonphysiologically active catechol compounds, catecholamine metabolites. Based gel presence detergent, molecular weight is estimated greater than 130,000 150,000. Equilibrium studies indicated KD nM. Hill coefficients (nH) 0.77 curved Scatchard plots suggested negatively cooperative interactions among agreement previous findings sites. Kinetic an association rate constant K1 = 3.8 10(6) M-1 min-1 reverse k2 2.3 10(-3) 4 degrees. kinetically derived (k2/k1) 0.6 nM reasonable that determined equilibrium studies. labile temperature degrees stabilized high EDTA. Guanidine hydrochloride urea produced concentration-dependent losses activity which partially upon dialysis. Trypsin phospholipase A degraded proteases phospholipases as well DNase RNase without effect. Experiments group-specific reagents free lysine, tryptophan, serine, sulfhydryl groups may important binding. These suggest probably protein requires lipids functional integrity. Data obtained are consistent contention these represent physiologically relevant have been extracted from membranes full retention their properties.