Field J B, Bloom G, Kerins M E, Chayoth R, Zor U
J Biol Chem. 1975 Jul 10;250(13):4903-10.
Protein kinase activity in homogenates of control thyroid slices and those incubated with thyroid-stimulating hormone (TSH) and prostaglandin EI was assayed and correlated with changes in cyclic adenosine 3':5'-monophosphate (cAMP) concentrations and binding of [3H]cAMP. Both TSH and prostaglandin E1 (25 mug/ml) increased protein kinase activity and the activity ratio (expressed as activity - cAMP to activity plus 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. In contrast to the previous results of J. D. Corbin, T. R. Soderling, and C. R. Park ((1973 J. Biol. Chem. 248, 1813) using adipose tissue, 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 NcCl 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.
对对照甲状腺切片以及与促甲状腺激素（TSH）和前列腺素E1一起孵育的甲状腺切片匀浆中的蛋白激酶活性进行了测定，并将其与环磷腺苷（cAMP）浓度变化以及[3H]cAMP结合情况相关联。TSH和前列腺素E1（25μg/ml）均增加了蛋白激酶活性以及活性比率（以活性 - cAMP与活性 + cAMP表示）。这种激活不太可能反映匀浆时释放的cAMP增加所产生的影响。即使匀浆被稀释，使得其cAMP浓度不足以实现酶的最大激活，激素诱导的蛋白激酶激活仍持续存在。与J.D.科尔宾、T.R.索德林和C.R.帕克（(1973)《生物化学杂志》248, 1813）先前使用脂肪组织的结果相反，在0.5M NaCl中对甲状腺组织进行匀浆以及使用葡聚糖G - 100进行色谱分析似乎并未稳定蛋白激酶解离为其受体和催化亚基。然而，匀浆缓冲液中NaCl量的增加与酶活性对cAMP的独立性增加相关。无论匀浆缓冲液是否含有NaCl，匀浆的稀释都不会改变蛋白激酶活性比率。NaF浓度的增加会抑制蛋白激酶活性。甲状腺切片与TSH孵育1至3分钟内，蛋白激酶活性和活性比率显著增加。这与切片中cAMP浓度的增加以及[3H]cAMP与匀浆结合的抑制情况相当吻合。酶的最大激活在10分钟时达到，这与对cAMP浓度的最大效应时间相对应。0.125毫单位/ml的TSH可实现蛋白激酶的激活，0.5至1.25毫单位/ml时达到最大效应。这些量与TSH其他效应所需的量非常吻合。尽管大量的TSH会使cAMP浓度有更大的增加，但这并不总是与[3H]cAMP结合的增强抑制相关。这些结果与TSH介导的cAMP增加与完整细胞中蛋白激酶激活相关的概念相符。它们还表明并非所有细胞内的cAMP都可用于激活蛋白激酶。
