Abstract
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.
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