Protein kinase activity in homogenates of control thyroid slices and those incubated with thyroid stimulating hormone (TSH) and prostaglandin E 1 was assayed and correlated with changes in cyclic adenosine 3':5' monophosphate (cAMP) concentrations and binding of [ 3H]cAMP. Both TSH and prostaglandin E 1 (25 μg/ml) increased protein kinase activity and the activity ratio (expressed as activity - CAMP to activity + cAMP). It is unlikely that such activation reflects effects of the increased cAMP liberated at the time of homogenization. Hormone induced activation of protein kinase persisted even after the homogenate had been diluted so that its cAMP concentration would be insufficient to achieve maximal activation of the enzyme. Homogenization of thyroid tissue in 0.5 M NaCl and chromatography using Sephadex G 100 did not seem to stabilize dissociation of protein kinase into its receptor and catalytic subunits. However, increasing amounts of NaCl in the homogenizing buffer were associated with an increase in the cAMP independence of enzyme activity. Dilution of the homogenate did not change the protein kinase activity ratio whether the homogenizing buffer contained NaCl or not. Increasing concentrations of NaF inhibited protein kinase activity. Within 1 to 3 min of incubation of thyroid slices with TSH, protein kinase activity and the activity ratio were increased significantly. This correlated quite well with increased cAMP concentrations in the slices and inhibition of [ 3H]cAMP binding to the homogenates. Maximal activation of the enzyme was achieved by 10 min which corresponds to the time of maximal effect on cAMP concentrations. Activation of protein kinase was achieved by 0.125 milliunit/ml of TSH and maximal effects with 0.5 to 1.25 milliunits/ml. These amounts agree well with those required for other effects of TSH. Although larger amounts of TSH produced even greater increases in cAMP concentrations this was not always associated with augmented inhibition of [ 3H]cAMP binding. These results are compatible with the concept that the TSH mediated increase in cAMP is associated with activation of protein kinase in the intact cell. They also suggest that not all of the intracellular cAMP is available for activation of protein kinase.
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|State||Published - 1 Dec 1975|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology