Lang U, Vallotton M B
J Biol Chem. 1987 Jun 15;262(17):8047-50.
The distribution of calcium-activated, phospholipid-dependent protein kinase (protein kinase C) between cytosol and membrane fractions was examined in bovine adrenal glomerulosa cells treated with angiotensin II or potassium. Protein kinase C was isolated from cytosol and from detergent-solubilized particulate fractions by DEAE-cellulose chromatography. A major peak of activity for both the soluble and particulate forms of adrenal glomerulosa protein kinase C was eluted at 0.05-0.09 M NaCl. The soluble and particulate forms were found to constitute about 95 and 5%, respectively, of the total enzyme activity in unstimulated cells. A second peak of kinase activity was eluted with 0.15-0.19 M NaCl, which was not dependent on the presence of phospholipids. Exposure of isolated cells for 20 min to 10(-8) M angiotensin II resulted in a decrease in cytosolic activity to 30-40% of control values, and in a corresponding increase in protein kinase C activity associated with the particulate fraction. This hormone-induced redistribution was found to be dose-dependent with an ED50 of 2 nM for angiotensin II, and it occurred rapidly, reaching a plateau within 5-10 min. It was prevented by the specific antagonist [Sar1,Ala8]angiotensin II. By contrast, stimulation with 12 mM KCl did not change the subcellular distribution of protein kinase C activity. These results suggest that redistribution of protein kinase C represents an early step in the post-receptor activation cascade following angiotensin II, but not potassium stimulation of adrenal glomerulosa cells.
在用血管紧张素II或钾处理的牛肾上腺球状带细胞中,检测了钙激活的磷脂依赖性蛋白激酶(蛋白激酶C)在胞质溶胶和膜组分之间的分布。通过DEAE - 纤维素色谱法从胞质溶胶和去污剂溶解的颗粒组分中分离蛋白激酶C。肾上腺球状带蛋白激酶C的可溶性和颗粒性形式的主要活性峰在0.05 - 0.09M NaCl处洗脱。发现可溶性和颗粒性形式分别占未刺激细胞中总酶活性的约95%和5%。激酶活性的第二个峰在0.15 - 0.19M NaCl处洗脱,其不依赖于磷脂的存在。将分离的细胞暴露于10^(-8)M血管紧张素II 20分钟导致胞质活性降低至对照值的30 - 40%,并且与颗粒组分相关的蛋白激酶C活性相应增加。发现这种激素诱导的重新分布是剂量依赖性的,血管紧张素II的ED50为2nM,并且它迅速发生,在5 - 10分钟内达到平台期。它被特异性拮抗剂[Sar1,Ala8]血管紧张素II阻止。相比之下,用12mM KCl刺激不会改变蛋白激酶C活性的亚细胞分布。这些结果表明,蛋白激酶C的重新分布代表了血管紧张素II受体后激活级联反应的早期步骤,但不是钾刺激肾上腺球状带细胞的早期步骤。