Sandnes D, Jacobsen F W, Jacobsen S
Department of Pharmacology, University of Oslo, Norway.
Pharmacol Toxicol. 1987 Oct;61(4):265-70. doi: 10.1111/j.1600-0773.1987.tb01816.x.
Inhibition of total 125I-ICYP binding to intact human mononuclear leucocytes at 32 degrees by propranolol and (+/-) CGP-12177 was biphasic. The high affinity component of 125I-ICYP binding, representing approximately 30% of total, was stereospecific, while the low affinity binding site was inhibited without stereospecificity. (-) Isoproterenol inhibited the high affinity component of 125I-ICYP binding only, with low affinity. By performing binding studies in intact cells at 4 degrees or in broken cell preparations at 37 degrees, the fraction of total 125I-ICYP binding representing specific binding was increased, and agonist affinity was high. Inhibition of 3H-CGP-12177 binding to intact cells at 32 degrees demonstrated a high fraction of specific binding and high agonist affinity. Computer-assisted analysis of total radioligand binding determined over a broad concentration range revealed two populations of saturable 125I-ICYP binding sites in intact cells as well as in broken cell preparations, while 3H-CGP-12177 binding demonstrated only one saturable binding site. The number of high affinity 125I-ICYP binding sites was comparable to the number of saturable 3H-CGP-12177 binding sites. Receptor numbers determined by analysis of total radioligand binding were comparable to receptor numbers determined by subtraction of non-specific binding, determined in the presence of a high concentration of competing ligand. Analysis of total radioligand binding was found to be a better procedure because it eliminates the use of an arbitrary concentration of unlabelled ligand and improves the accuracy of the assay.
普萘洛尔和（±）CGP - 12177在32℃时对完整人单核白细胞上125I - ICYP总结合的抑制呈双相性。125I - ICYP结合的高亲和力成分约占总量的30%，具有立体特异性，而低亲和力结合位点的抑制无立体特异性。（-）异丙肾上腺素仅以低亲和力抑制125I - ICYP结合的高亲和力成分。通过在4℃的完整细胞中或37℃的破碎细胞制剂中进行结合研究，125I - ICYP总结合中代表特异性结合的部分增加，且激动剂亲和力较高。在32℃时对完整细胞上3H - CGP - 12177结合的抑制显示出高比例的特异性结合和高激动剂亲和力。在较宽浓度范围内对总放射性配体结合进行计算机辅助分析表明，完整细胞以及破碎细胞制剂中存在两类可饱和的125I - ICYP结合位点，而3H - CGP - 12177结合仅显示一个可饱和结合位点。125I - ICYP高亲和力结合位点的数量与可饱和的3H - CGP - 12177结合位点数量相当。通过分析总放射性配体结合确定的受体数量与通过减去在高浓度竞争配体存在下测定的非特异性结合所确定的受体数量相当。发现分析总放射性配体结合是一种更好的方法，因为它无需使用任意浓度的未标记配体，提高了测定的准确性。
