Bennett C F, Crooke S T
Department of Molecular Pharmacology, Smith Kline and French Laboratories, Swedeland, Pennsylvania 19479.
J Biol Chem. 1987 Oct 5;262(28):13789-97.
Two peaks of phosphoinositide-specific phospholipase C (PI-PLC) activity were resolved when guinea pig uterus cytosolic proteins were chromatographed on a DEAE-Sepharose column. The first peak of enzyme activity eluting from the DEAE-Sepharose column (PI-PLC I) was further purified to homogeneity, whereas the second peak of enzyme activity was enriched 300-fold. PI-PLC I migrated as a 62-kDa protein on sodium dodecyl sulfate-polyacrylamide gels. Antibodies prepared against PI-PLC I failed to react with PI-PLC II. PI-PLC I hydrolyzed all three phosphoinositides, exhibiting a greater Vmax for phosphatidylinositol 4,5-bisphosphate greater than phosphatidylinositol 4-phosphate greater than phosphatidylinositol. Hydrolysis of phosphatidylinositol was calcium-dependent, whereas significant hydrolysis of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate occurred in the presence of 2.5 mM EGTA. At physiological concentrations of calcium, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate were the preferred substrates. Antibodies specific for PI-PLC I reacted with a 62-kDa protein in both the cytosol and membrane fractions from guinea pig uterus. Quantitation of the immunoblots revealed that 25% of the 62-kDa protein was membrane-associated, whereas only 5% of the total enzyme activity was membrane-associated. Approximately 20% of the membrane-bound phospholipase C activity and immunoreactive material were loosely bound, whereas the remainder required detergent extraction for complete solubilization. The 62-kDa protein associated with the membrane fractions did not bind lectin affinity columns, suggesting that it was not glycosylated. PI-PLC I was identified as a phosphoprotein in [32P]orthophosphate-labeled rat basophilic leukemia (RBL-1) cells by two-dimensional gel electrophoresis followed by immunoblotting. In untreated cells, 32P-labeled PI-PLC I was found in the cytosolic fraction. Treatment of RBL-1 cells with those phorbol esters which are known to activate the Ca2+/phospholipid-dependent enzyme protein kinase C, resulted in a time-dependent increase in the phosphorylation of both membrane-bound and cytosolic PI-PLC I. Thus, in RBL-1 cells, protein kinase C may play an important role in the regulation of phospholipase C through protein phosphorylation.
当豚鼠子宫胞质蛋白在DEAE - 琼脂糖柱上进行层析时，可分辨出磷酸肌醇特异性磷脂酶C（PI - PLC）活性的两个峰。从DEAE - 琼脂糖柱上洗脱下来的第一个酶活性峰（PI - PLC I）被进一步纯化至均一，而第二个酶活性峰则被富集了300倍。PI - PLC I在十二烷基硫酸钠 - 聚丙烯酰胺凝胶上迁移时表现为一种62 kDa的蛋白质。针对PI - PLC I制备的抗体与PI - PLC II不发生反应。PI - PLC I能水解所有三种磷酸肌醇，对磷脂酰肌醇4,5 - 二磷酸的最大反应速度大于磷脂酰肌醇4 - 磷酸大于磷脂酰肌醇。磷脂酰肌醇的水解依赖于钙，而在2.5 mM乙二醇双四乙酸（EGTA）存在的情况下，磷脂酰肌醇4 - 磷酸和磷脂酰肌醇4,5 - 二磷酸会发生显著水解。在生理浓度的钙条件下，磷脂酰肌醇4 - 磷酸和磷脂酰肌醇4,5 - 二磷酸是优先底物。针对PI - PLC I的特异性抗体与豚鼠子宫胞质和膜部分中的一种62 kDa蛋白质发生反应。免疫印迹定量分析表明，62 kDa蛋白质的25%与膜相关，而总酶活性中只有5%与膜相关。大约20%的膜结合磷脂酶C活性和免疫反应性物质是松散结合的，其余部分则需要用去污剂提取才能完全溶解。与膜部分相关的62 kDa蛋白质不结合凝集素亲和柱，这表明它没有糖基化。通过二维凝胶电泳然后进行免疫印迹，在[32P]正磷酸盐标记的大鼠嗜碱性白血病（RBL - 1）细胞中，PI - PLC I被鉴定为一种磷蛋白。在未处理的细胞中，32P标记的PI - PLC I存在于胞质部分。用已知能激活Ca2 + /磷脂依赖性酶蛋白激酶C的佛波酯处理RBL - 1细胞，会导致膜结合和胞质PI - PLC I的磷酸化随时间增加。因此，在RBL - 1细胞中，蛋白激酶C可能通过蛋白质磷酸化在磷脂酶C的调节中发挥重要作用。
