Adenosine 3′,5′-Monophosphate-Dependent Protein Kinase Activity in Soluble Thyrotropin Receptor Complex*

Abstract

CAMP-dependent protein kinase activity was present in a soluble TSH receptor fraction. The K_m of this enzyme was 2.2 × 10^-6 M for casein substrate in the absence or presence of 10^-5 M CAMP. A [³H]cAMP-binding protein was also found in this fraction. The Ka for [³H]cAMP binding was 0.11 × 10⁶ M^-1 with a total binding capacity of 3 nmol/mg protein. After fractionation using a continuous sucrose density gradient, one of the several [125I]iodobovine TSH-binding peaks corresponded to a [³H]cAMP-binding peak. After fractionation on a sucrose density gradient containing 0.4 M NaCl at pH 6.5, a major peak of protein kinase activity was shown. This protein kinase activity was stimulated by adding 10^-5 M cAMP. A peak of [3H]cAMP-binding activity corresponded to the same peak. Protein kinase activity in the receptor fraction was stimulated by adding 6 mg/ml bovine TSH. The soluble TSH receptor fraction also has an adenylate cyclase activity stimulated by TSH.