Phosphoinositide-specific phospholipase C-delta 1 binds with high affinity to phospholipid vesicles containing phosphatidylinositol 4,5-bisphosphate.
Phosphoinositide-specific phospholipase C-delta 1 binds with high affinity to phospholipid vesicles containing phosphatidylinositol 4,5-bisphosphate.
复制标题
磷酸肌醇特异性磷脂酶 C-delta 1 以高亲和力与含有磷脂酰肌醇 4,5-二磷酸的磷脂囊泡结合。
DOI：
10.1021/bi00166a005
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发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
McLaughlin,S
中科院分区：
文献类型：
--
作者：
Rebecchi,M;Peterson,A;McLaughlin,S
材料和方法酶制剂.使用具有以下修改的Reichi和罗森（1987）的方法，从牛脑细胞质中分离出均一的PLC。(1)在硫酸铵沉淀后，用DEAE-Sepharose代替步骤6之前的三个分级分离程序，步骤6为Fastchi和罗森（1987）的Fast-S Sepharose步骤。在约70 mM KCl下从DEAE-Sepharose柱洗脱PLC活性。(2)用肝素-琼脂糖代替羟基磷灰石和PI-琼脂糖亲和层析步骤：将蛋白质应用于100 mM NaCl、1 mM EGTA、0.5 mM二硫苏糖醇（DTT）、20 mM HEPES（pH 7.2）中，并用NaCl（100-700 mM）的线性梯度洗脱。在500和600 mM NaCl之间回收PLC。使用具有50-kDa膜的Millipore Minitan超滤歧管透析
MATERIALS AND METHODSEnzyme Preparation. PLC-was isolated to homogeneity from bovine brain cytoplasm using the method of Rebecchi and Rosen (1987) with the following modifications.(1) Following ammonium sulfate precipitation, DEAE-Sepharose was substituted for the three fractionation procedures that precede step 6, the Fast-S Sepharose step of Rebecchi and Rosen (1987). The PLC-activity eluted at about 70 mM KC1 from the DEAE-Sepharose column.(2) Heparin-agarose was substituted for the hydroxylapatite and PI-Sepharose affinity chromatography steps: the protein was applied in 100 mM NaCl, 1 mM EGTA, 0.5 mM dithiothreitol (DTT), 20 mM HEPES, pH 7.2, and eluted with a linear gradient of NaCl (100-700 mM). PLC-was recovered between 500 and 600 mM NaCl. A Millipore Minitan ultrafiltration manifold with 50-kDa membranes was used to dialyze the