The purification and characterization of CTP:phosphorylcholine cytidylyltransferase from rat liver.
The purification and characterization of CTP:phosphorylcholine cytidylyltransferase from rat liver.
复制标题
大鼠肝脏 CTP：磷酸胆碱胞苷酰转移酶的纯化和表征。
DOI：
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发表时间：
1986
影响因子：
4.8
通讯作者：
D. Feldman
中科院分区：
文献类型：
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作者：
P. Weinhold;M. E. Rounsifer;D. Feldman
我们已经从大鼠肝胞质溶胶中纯化CTP：磷酸胆碱胞苷酰转移酶2180倍至12，250 nmol/min/mg蛋白质的比活性。纯化的酶在Triton X-100和0.2M磷酸盐存在下在-70 ℃下是稳定的。纯化的酶在非变性聚丙烯酰胺电泳上显示单一蛋白质和活性带。十二烷基硫酸钠-聚丙烯酰胺电泳分离表明，纯化的酶含有亚基与39，000和48，000的Mr。凝胶过滤分析表明，天然酶是一个四聚体，含有两个39，000和两个48，000亚基。纯化的酶似乎结合到Triton X-100胶束，一个分子的四聚体/胶束。用100 μ M磷脂酰胆碱-油酸囊泡获得最大活性（8-10倍刺激）。磷脂酰甘油在10 μ M时产生4-5倍的活性增加。CTP和磷酸胆碱的最适pH值和真实Km值与先前报道的胞苷酰转移酶粗品制剂相似。胞苷酰转移酶在纯化过程中的整体行为和随后的分析表明，它具有类似于膜蛋白所表现出的疏水特性。
We have purified CTP:phosphorylcholine cytidylyltransferase from rat liver cytosol 2180-fold to a specific activity of 12,250 nmol/min/mg of protein. The purified enzyme was stable at -70 degrees C in the presence of Triton X-100 and 0.2 M phosphate. The purified enzyme gave a single protein and activity band on nondenaturing polyacrylamide electrophoresis. Separation by sodium dodecyl sulfate-polyacrylamide electrophoresis indicated that the purified enzyme contained subunits with Mr of 39,000 and 48,000. Gel filtration analysis indicated that the native enzyme was a tetramer containing two 39,000 and two 48,000 subunits. The purified enzyme appeared to bind to Triton X-100 micelles, one molecule of tetramer/micelle. Maximal activity was obtained with 100 microM phosphatidylcholine-oleic acid vesicles (8-10-fold stimulation). Phosphatidylglycerol produced a 4-5-fold increase in activity at 10 microM. The pH optimum and true Km values for CTP and phosphorylcholine were similar to those reported previously for crude preparations of cytidylyltransferase. The overall behavior of cytidylyltransferase during purification and subsequent analysis suggested that it has hydrophobic properties similar to those exhibited by membrane proteins.