Thyroid-stimulating Hormone Binding to Beef Thyroid Membranes: RELATION TO ADENYLATE CYCLASE ACTIVITY

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Binding of [3H]thyroid-stimulating hormone (TSH) and 125I-TSH to beef thyroid membranes was studied under conditions to give optimum binding. Binding was maximal at pH 5.5, was complete within 5 min, and was stable for at least 90 min at 0 and 22° for low and high TSH concentrations, respectively. At pH 7.5, two binding sites were found with apparent affinity constants of 2.2 to 3.1 x 108 m -1 and 0.7 to 1.1 x 106 m -1. Because of lower specific activity, [3H]TSH was used primarily in the concentration range of the low affinity constant while 125I-TSH was used at concentrations corresponding to both constants. Binding of [3H]TSH and 125I-TSH was equivalent when measured in the same concentration range. TSH binding was inhibited by divalent cations (Ca2+, Mg2+, Sr2+, Mn2+, and Ba2+) at concentrations above 0.1 m m by reducing the number of binding sites. Monovalent cations inhibited binding at concentrations above 10 to 15 m m . The order of potency for inhibition of binding by monovalent cations was Na+ ≃ K+ g Li+. Inhibition of binding by cations was independent of TSH concentration. When binding was determined under adenylate cyclase assay conditions, TSH binding was reduced to approximately one-third of the control levels and the higher apparent affinity constant was reduced to 1.6 x 107 m -1 whereas the second constant was unchanged. These constants coincide roughly with the midpoints of the concentration curve of TSH activation of adenylate cyclase. TSH was not displaced from the membranes by ITP and GTP at concentrations which enhance cyclase but only at concentrations above 1 m m ; the effect of ITP and GTP on binding was independent of medium conditions, TSH concentration or the presence of a regenerating system. TSH binding studied in the presence of activators and inhibitors of adenylate cyclase permitted classification of the compounds into four groups: (a) agents which affect cyclase and TSH binding in a parallel fashion (Ca2+, Na+, Li+, pronase, and cobramine B); (b) agents which enhance cyclase but decrease TSH binding (K+ and Mg2+); (c) agents which inhibit cyclase but enhance binding (phospholipase A and filipin); (d) agents which alter cyclase but not binding (ITP, GTP, F-, chlorpromazine, gramicidin S, valinomycin, and cetyl pyridinium chloride). It is concluded that: (a) TSH binding is relatively stable and major structural alterations are required to affect binding; (b) the effect of activators and inhibitors on TSH binding is independent of TSH concentration and medium conditions; (c) most modulators act in the cyclase system at a stage beyond hormone binding; (d) the response of TSH binding to nucleoside triphosphates indicates that the thyroid membrane system is different from other membrane systems; and (e) the relationship between hormone binding and cyclase activation is complex and direct correlation between binding and activation is not presently possible.