Toews M L, Harden T K, Perkins J P
Proc Natl Acad Sci U S A. 1983 Jun;80(12):3553-7. doi: 10.1073/pnas.80.12.3553.
The interactions of agonists and antagonists with beta-adrenergic receptors on intact 1321N1 human astrocytoma and C62B rat glioma cells were studied by using the radioligand (-)-[125I]iodopindolol. Competition binding assays were performed at 37 degrees C under equilibrium conditions and in short-time nonequilibrium assays that approximated initial velocity conditions for binding of the radioligand. The theoretical basis and experimental validation of the initial velocity approach for determining binding affinities of rapidly equilibrating ligands are presented. For the agonists isoproterenol and epinephrine, high binding affinities that approximated their apparent affinities for binding in membranes and for increase of cyclic AMP concentrations in intact cells could be demonstrated only in short-time assays; in contrast, much lower affinities were observed with equilibrium (60-min) assays as reported previously for various cell lines. High-affinity binding of isoproterenol to 1321N1 cells also was observed in equilibrium (6-hr) binding assays carried out on ice. These results indicate that in the native state the intact cell beta-adrenergic receptor has a high binding affinity for agonists and suggest that incubation at 37 degrees C in the presence of an agonist converts the receptors to a form with low affinity for agonists.
利用放射性配体（-）-[¹²⁵I]碘吲哚洛尔，研究了激动剂和拮抗剂与完整的1321N1人星形细胞瘤细胞和C62B大鼠胶质瘤细胞上β-肾上腺素能受体的相互作用。在37℃下，于平衡条件下以及在近似放射性配体结合初始速度条件的短时间非平衡试验中进行竞争结合试验。介绍了确定快速平衡配体结合亲和力的初始速度方法的理论基础和实验验证。对于激动剂异丙肾上腺素和肾上腺素，只有在短时间试验中才能证明其具有高结合亲和力，这种高亲和力接近它们在膜中结合以及在完整细胞中增加环磷酸腺苷浓度时的表观亲和力；相反，如先前针对各种细胞系所报道的，在平衡（60分钟）试验中观察到的亲和力要低得多。在冰上进行的平衡（6小时）结合试验中，也观察到异丙肾上腺素与1321N1细胞的高亲和力结合。这些结果表明，在天然状态下，完整细胞的β-肾上腺素能受体对激动剂具有高结合亲和力，并表明在37℃下于激动剂存在的情况下孵育会使受体转变为对激动剂具有低亲和力的形式。
