Caron M G, Lefkowitz R J
Biochim Biophys Acta. 1976 Sep 24;444(2):472-86. doi: 10.1016/0304-4165(76)90391-3.
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.
以多种方式合成了取代到琼脂糖上的儿茶酚胺。去甲肾上腺素和异丙肾上腺素通过与儿茶酚环的偶氮键连接到对氨基苯甲酰己基琼脂糖上。去甲肾上腺素还通过氨基与己基琼脂糖偶联，形成氨基、胍基或酰胺键。通过评估固定化儿茶酚胺与几种膜制剂和完整红细胞制剂中腺苷酸环化酶相互作用的能力，来测定其生物活性。在犬心膜中，儿茶酚胺凝胶对腺苷酸环化酶的刺激可归因于从凝胶中释放出来的浸出激素。在蛙红细胞膜中，浸出极少，未观察到对腺苷酸环化酶的显著刺激。然而，琼脂糖固定化的儿茶酚胺竞争性抑制这些红细胞膜中异丙肾上腺素对腺苷酸环化酶的刺激，这表明与琼脂糖结合的儿茶酚胺作为拮抗剂而非激动剂与β-肾上腺素能受体相互作用。当在完整的蛙红细胞上进行测试时，琼脂糖固定化的儿茶酚胺并未增加细胞内环磷酸腺苷的水平，而异丙肾上腺素可使这些水平升高8 - 10倍。同样，当在完整细胞中测试拮抗剂活性时，琼脂糖 - 儿茶酚胺未能抑制异丙肾上腺素引起的环磷酸腺苷刺激。在膜制剂和完整细胞中观察到的琼脂糖结合儿茶酚胺的β-肾上腺素能拮抗剂活性差异，可归因于空间因素，其可能阻止了珠结合配体与细胞表面的接触，或者归因于受体可能埋藏在膜基质中的可能性。
