Identification of high-affinity phorbol ester receptor in cytosol of EL4 thymoma cells: requirement for calcium, magnesium, and phospholipids.
Identification of high-affinity phorbol ester receptor in cytosol of EL4 thymoma cells: requirement for calcium, magnesium, and phospholipids.
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EL4 胸腺瘤细胞胞浆中高亲和力佛波酯受体的鉴定：钙、镁和磷脂的需求。
DOI：
10.1073/pnas.80.9.2642
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发表时间：
1983
影响因子：
11.1
通讯作者：
Young,MC
中科院分区：
文献类型：
--
作者：
Sando,JJ;Young,MC
在EL4小鼠胸腺瘤细胞系的细胞质中发现了一种特异性的高亲和力磷酯结合成分，使用的条件类似于证明钙/磷脂依赖性蛋白激酶活性的条件。特异性结合完全依赖于酸性磷脂（最大结合为每毫升96微克磷脂酰丝氨酸或每毫升200微克磷脂酰肌醇），并通过添加钙（0.5 mM）和镁（75 mM）大大增强。细胞质结合成分的Scatchard分析表明，Kd为4.2 +/- 2.5 nM，每个细胞1.8 +/- 0.6 × 10(4)个位点，而膜受体的Kd为11.9 +/- 4.4 nM，每个细胞4.1 +/- 1.0 × 10(4)个位点。与EL4细胞中至少存在两个磷酯结合位点相一致的是观察到与整个匀浆结合的曲线Scatchard图。细胞质结合显示出与完整细胞中所观察到的结合磷酯类似物相同的效力顺序。这些结果进一步支持了磷酯受体与钙/磷脂依赖性蛋白激酶之间的相似性，并表明细胞质受体可能参与了EL4细胞中随后的磷酯效应，包括细胞质中激酶活性的丧失和t细胞生长因子的产生。
A specific high-affinity phorbol ester binding component has been identified in the cytosol of an EL4 mouse thymoma line by using conditions similar to those for demonstrating activity of a calcium/phospholipid-dependent protein kinase. Specific binding is absolutely dependent on acidic phospholipids (maximal binding at 96 micrograms of phosphatidylserine per ml or 200 micrograms of phosphatidylinositol per ml) and is greatly enhanced by addition of calcium (0.5 mM) and magnesium (75 mM). Scatchard analysis of the cytosolic binding component indicated a Kd of 4.2 +/- 2.5 nM with 1.8 +/- 0.6 x 10(4) sites per cell compared to a Kd of 11.9 +/- 4.4 nM and 4.1 +/- 1.0 x 10(4) sites per cell for the membrane receptor. Consistent with the existence of at least two phorbol ester binding sites in EL4 cells was the observation of curvilinear Scatchard plots for binding to whole homogenates. The cytosolic binding showed the same order of potency for binding phorbol ester analogs as has been observed for intact cells. These results further support the similarity between phorbol ester receptors and the calcium/phospholipid-dependent protein kinase and suggest that the cytosolic receptor may be involved in subsequent phorbol ester effects in EL4 cells including loss of the kinase activity from the cytosol and production of T-cell growth factor.