Cyclic AMP-stimulated protein kinase prepared from bovine thyroid glands

Abstract

A protein kinase that was stimulated three- to fourfold by cyclic AMP (5 × 10 −6 M) was partially purified from bovine thyroid glands. Histones and protamine were better substrates for the enzyme than casein or bovine serum albumin, and stimulation induced by cyclic AMP was greater using the former two substances. The concentration of cyclic AMP that gave half-maximal activation (K m) was 9.1 × 10 −8 M, and 5 × 10 −6 M cyclic AMP produced maximal stimulation. The K m for ATP was 1.3 × 10 −5 M both in the presence and absence of cyclic AMP. However, cyclic AMP (5 × 10 −6 M) increased the V max of the enzyme 3.5-fold. Other cyclic nucleotides (cyclic IMP, cyclic GMP, cyclic UMP, cyclic CMP, and dibutyryl-cyclic AMP) also activated the enzyme but only at concentrations considerably higher than that required for cyclic AMP. Substitution of 2.5 m M Co ++ for 2.5 m M Mg ++ increased basal protein kinase activity and the response induced by cyclic AMP. Replacement of Mg ++ by Mn ++ caused reduction in basal and cyclic AMP mediated activities. Basal enzyme activity was very low when Ca ++ was used instead of Mg ++, and cyclic AMP produced very little stimulation. Adenosine and ADP (5 × 10 −5 M) markedly inhibited basal and cyclic nucleotide (cyclic AMP, cyclic GMP, and cyclic IMP) activated protein kinase activity. Similar concentrations of FMN, FAD, GDP, adenine, and AMP were either inactive or much less effective. Although these data provide some properties of a cyclic AMP-dependent protein kinase in the thyroid, its role in thyroid gland function still remains to be elucidated.