| Abstract: | The goal of this study was to synthesize biotinylated derivatives of alprenolol, a beta-adrenergic antagonist, and to determine whether these ligands could bind simultaneously to both avidin (a biotin-binding protein) and to the beta-adrenergic receptor. Such ligands would be useful for beta-adrenergic receptor localization and purification, since avidin can be covalently labelled with fluorescent or electron-dense markers or can be linked to solid supports for affinity chromatography. Three biotinyl derivatives of alprenolol were synthesized and characterized. Each derivative bound to avidin and also possessed high affinity for the duck erythrocyte beta-adrenergic receptor. Two of the compounds, biotinyl-caproyl-cysteaminyl-alprenolol (BCCA) and biotinyl-dodecanoyl-cysteaminyl-alprenolol (BDCA) had the same affinities for the duck erythrocyte beta-adrenergic receptor (membrane-bound or digitonin-solubilized) in the absence and presence of avidin. This indicated that high affinity complexes could be formed between the beta-adrenergic receptor and avidin using these bifunctional biotinyl-alprenolol ligands. In contrast, biotinyl-cysteaminyl-alprenolol (BCA), in which the distance between the biotin and alprenolol moieties was shorter, had greatly reduced affinity for the duck erythrocyte beta-adrenergic receptor in the presence of avidin. Additional studies showed that BDCA, avidin-BDCA, and ferritin-avidin-BDCA were equally potent in inhibiting the isoproterenol stimulation of cAMP accumulation in intact HeLa cells. The data reported in this paper demonstrate the importance of an appropriate spacer sequence to allow correct apposition of the receptor and avidin molecules, and suggest that BDCA may be a useful probe for beta-adrenergic receptor localization and purification. |
