Robles-Flores M, Alcántara-Hernández R, García-Sáinz J A
Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, D.F.
Biochim Biophys Acta. 1991 Aug 13;1094(1):77-84. doi: 10.1016/0167-4889(91)90028-v.
Two main forms of protein kinase C (PKC) activity were found in rat hepatocytes using DEAE-cellulose chromatography: PKC 1 and PKC 2. Treatment of cells with 1 microM 12-O-tetradecanoylphorbol 13-acetate (TPA) for 15 min caused a marked loss of PKC 1 activity and only a small loss of PKC 2 activity. Hydroxyapatite column chromatography resolved PKC 1 into three distinct peaks 1a, 1b and 1c, and PKC 2 into four peaks 2a, 2b, 2c and 2d. Immunoblot analysis with isozyme-specific monoclonal antibodies identified peak 1a as PKC-beta and peak 1b as PKC-alpha; the other peaks of activity were not identified. Treatment with TPA provoked a loss of activity of peaks 1b (PKC-alpha) and 1c, whereas peak 1a (PKC-beta) activity was not affected. The peaks of activity corresponding to PCK 2 did not show any major change due to TPA treatment except peak 2d that decreased. The apparent disappearance of PKC histone-kinase activity induced by TPA was also observed using other substrates (protamine or vinculin). The TPA-induced decrease in activity occurs in a time-dependent and dose-dependent fashion. However, the time-courses, the extent of depletion and the potency order of phorbol esters in induction of an activity decrease in the two groups of isoforms exhibited substantial differences.
利用二乙氨基乙基纤维素色谱法在大鼠肝细胞中发现了蛋白激酶C（PKC）的两种主要活性形式：PKC 1和PKC 2。用1微摩尔12 - O - 十四酰佛波醇13 - 乙酸酯（TPA）处理细胞15分钟，导致PKC 1活性显著丧失，而PKC 2活性仅轻微丧失。羟基磷灰石柱色谱法将PKC 1分离为三个不同的峰1a、1b和1c，将PKC 2分离为四个峰2a、2b、2c和2d。用同工酶特异性单克隆抗体进行免疫印迹分析，确定峰1a为PKC -β，峰1b为PKC -α；其他活性峰未得到鉴定。用TPA处理导致峰1b（PKC -α）和1c的活性丧失，而峰1a（PKC -β）的活性未受影响。与PKC 2相对应的活性峰，除峰2d降低外，经TPA处理未显示出任何主要变化。使用其他底物（鱼精蛋白或纽蛋白）也观察到TPA诱导的PKC组蛋白激酶活性明显消失。TPA诱导的活性降低呈时间依赖性和剂量依赖性。然而，两组同工型中佛波酯诱导活性降低的时间进程、消耗程度和效力顺序存在显著差异。
