Maguire M E, Wiklund R A, Anderson H J, Gilman A G
J Biol Chem. 1976 Mar 10;251(5):1221-31.
[125I]Iodohydroxybenzylpindolol, an extremely potent beta-adrenergic antagonist, has been purified to theoretical specific activity (2200 Ci/mmol) and used as a ligand to characterize the beta-adrenergic receptors of cultured rat glioma cells and other cultured cell clones. Appropriate receptor sites were identified by stereoselective competition for binding by a number of adrenergic agonists and antagonists, by correlation between the potency of these compounds to inhibit binding and to affect adenylate cyclase activity, and by correlation of binding with the presence or absence of response to catecholamines (stimulation of adenylate cyclase) in various cell clones. In equilibrium experiments, the dissociation constant for binding of the iodinated ligand to the beta-adrenergic receptor of a clone of rat glioma cells (C6TG1A) was 250 pM; the corresponding value for a clone of human fibroblasts (VA2) was 15 pM. For C6TG1A, KD was verified by analysis of the kinetics of binding: k1 = 10(8) 1/mol/min: k-1 = 0.017/min. This rate of dissociation of ligand from the receptor was also established by study of the rate of activation of adenylate cyclase by isoproterenol after prior equilibration with iodohydroxybenzylpindolol. For VA2 cells, where affinity was higher, the rate of reversal of binding was only 0.0035/min. C6TG1A contained approximately 4000 receptor sites/cell (75 fmol/mg of protein), and these sites appeared to be coupled to adenylate cyclase in a stoichiometric manner. A second site with equal affinity for iodohydroxybenzylpindolol (KD = 250 pM) was also identified in C6TG1A by both kinetic analysis and equilibrium binding studies. While most compounds that interacted with the beta-adrenergic receptor also influenced binding to the second site, the latter did not distinguish between stereoisomers of propranolol, and its affinity for the other compounds tested was poorer.
[125I]碘羟基苄基吲哚洛尔是一种极其强效的β-肾上腺素能拮抗剂，已被纯化至理论比活度（2200居里/毫摩尔），并用作配体来表征培养的大鼠胶质瘤细胞和其他培养细胞克隆的β-肾上腺素能受体。通过多种肾上腺素能激动剂和拮抗剂对结合的立体选择性竞争、这些化合物抑制结合和影响腺苷酸环化酶活性的效力之间的相关性，以及结合与各种细胞克隆中对儿茶酚胺（腺苷酸环化酶的刺激）反应的有无之间的相关性，确定了合适的受体位点。在平衡实验中，碘化配体与大鼠胶质瘤细胞克隆（C6TG1A）的β-肾上腺素能受体结合的解离常数为250皮摩尔；人成纤维细胞克隆（VA2）的相应值为15皮摩尔。对于C6TG1A，通过结合动力学分析验证了KD：k1 = 10(8) 1/摩尔/分钟：k-1 = 0.017/分钟。在与碘羟基苄基吲哚洛尔预先平衡后，通过异丙肾上腺素激活腺苷酸环化酶的速率研究，也确定了配体从受体上解离的速率。对于亲和力较高的VA2细胞，结合逆转的速率仅为0.0035/分钟。C6TG1A每个细胞含有约4000个受体位点（75飞摩尔/毫克蛋白质），这些位点似乎以化学计量方式与腺苷酸环化酶偶联。通过动力学分析和平衡结合研究，在C6TG1A中还鉴定出了对碘羟基苄基吲哚洛尔具有同等亲和力的第二个位点（KD = 250皮摩尔）。虽然大多数与β-肾上腺素能受体相互作用的化合物也影响与第二个位点的结合，但后者不能区分普萘洛尔的立体异构体，并且其对其他测试化合物的亲和力较差。
