Biological activity of agarose-immobilized catecholamines.
Catecholamines substituted to agarose were synthesized in various ways. Norepinephrine and isoproterenol were linked to p-aminobenzamidohexyl agarose by an azo linkage to the catechol ring. Norepinephrine was also couple to hexyl agaros via the amino group, forming an amino, guanidino or amido bond. Biological activity of the immobilized catecholamines was determined by assessing their abilities to interact with adenylate cyclase in several membrane preparations and intact preparations of erythrocytes. In dog heart membranes, stimulation of adenylate cyclase by the catecholamine-gels could be accounted for by leached hormone which had been released from the gels. In frog erythrocyte membranes, leaching was minimal and no significant stimulation of adenylate cyclase was observed. Agarose-immobilized catecholamines, however, competitively inhibited isoproterenol stimulation of adenylate cyclase in these erythrocyte membranes indicating that catecholamines which are bound to agarose interact with the beta-adrenergic receptors as antagonists rather than agonists. When tested on intact frog erythrocytes, agarose immobilzed catecholamines did not increase the intracellular levels of cyclic AMP, although isoproterenol caused as 8-10 fold rise in these levels. Similarly, when tested for antagonist activity in the intact cells the agarose-catecholamines failed to inhibit the stimulation of cyclic AMP caused by isoproterenol. The difference observed in the beta-adrenergic antagonist activity of the agarose-bound catecholamines in membrane preparations and intact cells can be attributed to steric factors which could have prevented the access of the bead-bound ligands with the surface of the cell or to the possibility that receptors might be buried in the membrane matrix.
Biological activity of agarose-immobilized catecholamines. Catecholamines substituted to agarose were synthesized in various ways. Norepinephrine and isoproterenol were linked to p-aminobenzamidohexyl agarose by an azo linkage to the catechol ring. Norepinephrine was also couple to hexyl agaros via the amino group, forming an amino, guanidino or amido bond. Biological activity of the immobilized catecholamines was determined by assessing their abilities to interact with adenylate cyclase in several membrane preparations and intact preparations of erythrocytes. In dog heart membranes, stimulation of adenylate cyclase by the catecholamine-gels could be accounted for by leached hormone which had been released from the gels. In frog erythrocyte membranes, leaching was minimal and no significant stimulation of adenylate cyclase was observed. Agarose-immobilized catecholamines, however, competitively inhibited isoproterenol stimulation of adenylate cyclase in these erythrocyte membranes indicating that catecholamines which are bound to agarose interact with the beta-adrenergic receptors as antagonists rather than agonists. When tested on intact frog erythrocytes, agarose immobilzed catecholamines did not increase the intracellular levels of cyclic AMP, although isoproterenol caused as 8-10 fold rise in these levels. Similarly, when tested for antagonist activity in the intact cells the agarose-catecholamines failed to inhibit the stimulation of cyclic AMP caused by isoproterenol. The difference observed in the beta-adrenergic antagonist activity of the agarose-bound catecholamines in membrane preparations and intact cells can be attributed to steric factors which could have prevented the access of the bead-bound ligands with the surface of the cell or to the possibility that receptors might be buried in the membrane matrix.