Chuang L F, Zhao F K, Chuang R Y
Department of Pharmacology, School of Medicine, University of California, Davis 95616.
Biochim Biophys Acta. 1989 Jul 21;992(1):87-95. doi: 10.1016/0304-4165(89)90054-8.
Protein kinase C (PKC) was purified to near homogeneity from human leukemia ML-1 cells. The purified enzyme showed a single polypeptide band of 80 kDa on SDS-polyacrylamide gel after electrophoresis, and was totally dependent on Ca2+/phospholipid for activity. Diacylglycerol and the tumor-promoting on Ca2/phospholipid for activity. Diacylglycerol and the tumor-promoting phorbol esters stimulated the enzyme activity. Autophosphorylation of PKC purified from phenyl-Sepharose column showed both 80- and 37 kDa polypeptides. Further fractionation of PKC on a hydroxyapatite column revealed two peaks of enzyme activity, indicating that there may be two different forms of protein kinase C present in human leukemia cells. The purified PKC was used to phosphorylate RNA polymerase II of human leukemia cells in vitro and the autoradiogram showed that RNA polymerase II large subunits (240, 220 and 150 kDa) were phosphorylated in a time-dependent manner.
蛋白激酶C（PKC）从人白血病ML-1细胞中纯化至接近均一。纯化后的酶在SDS-聚丙烯酰胺凝胶电泳后呈现出一条80 kDa的单一条带，其活性完全依赖于Ca2+/磷脂。二酰基甘油和促肿瘤的佛波酯可刺激该酶的活性。从苯基琼脂糖柱纯化的PKC的自磷酸化显示出80 kDa和37 kDa的两种多肽。在羟基磷灰石柱上对PKC进一步分级分离显示出两个酶活性峰，表明人白血病细胞中可能存在两种不同形式的蛋白激酶C。纯化的PKC用于体外磷酸化人白血病细胞的RNA聚合酶II，放射自显影片显示RNA聚合酶II的大亚基（240、220和150 kDa）以时间依赖性方式被磷酸化。
