In VitroResponses to Crude and Purified hCG in Human Thyroid Membranes*

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Abstract
Others have reported that hCG has intrinsic thyrotropic activity in the mouse. To obtain evidence of whether hCG has stimulatory activity in the human thyroid, we have studied the interactions of crude and pure hCG with receptors in human thyroid membranes by assessing the ability of these preparations to inhibit the binding of bovine [125I]TSH ([125I]-bTSH) and to activate adenylate cyclase activity therein. On the assumption that all activity in crude hCG was due to hCG itself, studies of the displacement of [125I]bTSH at 4 C in 20 DIM Tris-Cl-0.5% bovine serum albumin, pH 7.45, revealed differing preparations of crude hCG to be approximately 10- to 40- fold more potent than pure hCG on a molar basis. Gonadotropic activities of crude and pure hCG were then standardized in a radioreceptor assay for hCG in rat testis. No major discrepancy between the in vivo bioactivity and the activity determined in vitro radioreceptor assay of these preparations was revealed. Lineweaver-Burk analysis showed the inhibition of bTSH binding by crude and purified hCG preparations to be competitive in nature. In experiments conducted at 37 C in 20 mM Tris-Cl-0.5% bovine serum albumin containing 50 mM NaCl, pH 7.45, bTSH exhibited approximately 60-fold greater affinity for receptors than was noted in comparable experiments performed at 4 C. However, under these conditions the greater potency of crude than of pure hCG in inhibiting the binding of [125I]bTSH was retained, though some loss in the potency of crude hCG was noted. With one exception, five commercial preparations of crude hCG, as well as pure hCG itself, failed to exert any stimulatory effect on adenylate cyclase activity when tested at a concentration between 2,000–40,000 IU/ml, although a brisk stimulatory response to bTSH was noted. The one crude hCG preparation tested that appeared to stimulate adenylate cyclase was APL, but its activity can probably be explained by the phenol that it contains as a preservative, in view of the following evidence: 1) the stimulatory activity of APL was lost on dialysis and restored to dialyzed specimens by replacement of the original concentrations of phenol; 2) phenol itself, at concentrations comparable to those present in APL, was stimulatory to adenylate cyclase in human thyroid membranes. The results indicate that preparations of crude hCG contain a factor that inhibits the binding of [125I]bTSH to human thyroid membranes. Though not a demonstrable stimulator of adenylate cyclase in human thyroid particulate fractions, this or some other factor in crude hCG also inhibited the stimulation of adenylate cyclase in such membranes produced by bTSH. Pure hCG neither stimulated adenylate cyclase directly nor significantly inhibited its response to bTSH. The minor activity of pure hCG to displace [125I]bTSH from human thyroid membranes may reflect either a slight contamination with the same factor(s) that is active in crude hCG or a very weak activity intrinsic to pure hCG itself.