Boney C, Fink D, Schlichter D, Carr K, Wicks W D
J Biol Chem. 1983 Apr 25;258(8):4911-8.
The effect of purified beef heart cAMP-dependent protein kinase catalytic subunit on tyrosine aminotransferase activity in intact cultured rat H35 hepatoma cells was directly tested by micro-injection using human red blood cell ghosts as vehicles. Although the micro-injection procedure itself produced temporary fluctuations in protein synthesis and in tyrosine aminotransferase activity in H35 cells, after a recovery period of 8-12 h, these parameters returned to normal in parallel with restoration of full inducibility of the aminotransferase by both 8-Br-cAMP and dexamethasone. Eight to sixteen hours after fusion of H35 cells with unloaded ghosts, ghosts loaded with bovine serum albumin or mock-loaded with the partially purified protein kinase catalytic subunit, no significant change in the activity of the aminotransferase was detected. In contrast, fusion with ghosts loaded with the catalytic subunit at concentrations between 0.1-2 mg/ml caused reproducible 2-3-fold increases in enzyme activity. Homogeneous preparations of the catalytic subunit exhibited even greater potency as an inducer. The effect was both time- and concentration-dependent and was abolished by inactivation of the catalytic subunit with N-ethylmaleimide prior to loading. The partially purified inhibitor of protein kinase from beef heart, while not affecting basal tyrosine aminotransferase activity, selectively inhibited the ability of 8-Br-cAMP but not that of dexamethasone to stimulate the activity of this enzyme. In addition, micro-injection of the pure regulatory subunit of the kinase blocked the response of the aminotransferase to low concentrations of 8-Br-cAMP. These results provide strong support for the proposition that the catalytic subunit of protein kinase mediates the effects of cAMP on the synthesis of tyrosine aminotransferase.
以人红细胞空壳为载体，通过显微注射直接检测纯化的牛心cAMP依赖性蛋白激酶催化亚基对完整培养的大鼠H35肝癌细胞中酪氨酸转氨酶活性的影响。尽管显微注射操作本身会使H35细胞中的蛋白质合成和酪氨酸转氨酶活性产生暂时波动，但在8 - 12小时的恢复期后，这些参数会恢复正常，同时转氨酶对8 - Br - cAMP和地塞米松的完全诱导性也会恢复。在H35细胞与未装载物质的空壳、装载牛血清白蛋白的空壳或模拟装载部分纯化的蛋白激酶催化亚基的空壳融合8至16小时后，未检测到转氨酶活性有显著变化。相比之下，与装载浓度为0.1 - 2 mg/ml催化亚基的空壳融合会使酶活性可重复地增加2 - 3倍。催化亚基的均一制剂作为诱导剂表现出更强的效力。这种作用具有时间和浓度依赖性，并且在装载前用N - 乙基马来酰亚胺使催化亚基失活后该作用消失。来自牛心的部分纯化的蛋白激酶抑制剂，虽然不影响基础酪氨酸转氨酶活性，但选择性地抑制了8 - Br - cAMP而非地塞米松刺激该酶活性的能力。此外，显微注射该激酶的纯调节亚基可阻断转氨酶对低浓度8 - Br - cAMP的反应。这些结果为蛋白激酶催化亚基介导cAMP对酪氨酸转氨酶合成的影响这一观点提供了有力支持。
