MONOCLONAL-ANTIBODIES TO AN INTERFERON-INDUCED MR 68,000 PROTEIN AND THEIR USE FOR THE DETECTION OF DOUBLE-STRANDED RNA-DEPENDENT PROTEIN-KINASE IN HUMAN-CELLS
MONOCLONAL-ANTIBODIES TO AN INTERFERON-INDUCED MR 68,000 PROTEIN AND THEIR USE FOR THE DETECTION OF DOUBLE-STRANDED RNA-DEPENDENT PROTEIN-KINASE IN HUMAN-CELLS
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DOI：
10.1073/pnas.82.13.4341
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发表时间：
1985-01-01
影响因子：
11.1
通讯作者：
HOVANESSIAN, AG
中科院分区：
文献类型：
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作者：
LAURENT, AG;KRUST, B;HOVANESSIAN, AG
从干扰素处理的人类细胞提取物中显示出双链RNA依赖性蛋白激酶活性水平升高，这表现为内源性分子量为69,000 - 72,000的蛋白质在其磷酸饱和状态下的磷酸化。通过使用从干扰素处理的人类Daudi细胞中高度纯化的蛋白激酶组分，描述了针对这种磷蛋白的鼠单克隆抗体的制备，该磷蛋白在其天然状态下分子量为68,000。这些单克隆抗体（IgG1类）能够通过电泳转移印迹技术识别聚丙烯酰胺凝胶中的电泳蛋白质（p68）。将干扰素处理的人类细胞提取物与单克隆抗体孵育后形成的免疫沉淀物可以通过蛋白A - 琼脂糖方便地回收。这种免疫复合物制剂具有相关的蛋白激酶活性，即添加[γ - 32P]ATP会导致p68以及添加的底物（小牛胸腺组蛋白和真核起始因子2）的磷酸化。来自[35S]Met标记提取物的免疫复合物制剂显示出p68的特异性免疫沉淀。在与用于蛋白激酶活性测定的相似实验条件下制备的这些免疫复合物中，其他几种[35S]Met标记的蛋白质非特异性结合。这些非特异性结合的蛋白质可以通过使用含有去污剂或高浓度KCl和醋酸镁的缓冲液洗脱。用这些缓冲液类似洗涤的免疫复合物制剂仍然保留p68，但失去了磷酸化p68或外源性底物的能力。p68本身没有蛋白激酶活性。在Daudi细胞中，仅用1单位的人α干扰素就会诱导[35S]Met标记的p68产生，在添加干扰素后6 - 9小时之间合成达到最大值。放线菌素D会阻断这种诱导。
Extracts from interferon-treated human cells show an enhanced level of a double-stranded RNA-dependent protein kinase activity that is manifested by the phosphorylation of an endogenous MW 69,000-72,000 protein in its phosphate-saturated state. By using a highly purified protein kinase fraction from interferon-treated human Daudi cells, the preparation of murine monoclonal antibodies directed against this phosphoprotein, the MW of which in its native state is found to be 68,000, is described. These monoclonal antibodies (class IgG1) can identify the electrophoresed protein (p68) in polyacrylamide gels by the electrophoretic transfer blotting technique. Immunoprecipitates formed after incubation of extracts from interferon-treated human cells with the monoclonal antibodies can be conveniently recovered by protein A-Sepharose. Such immune complex preparations associated protein kinase activity, i.e., addition of [.gamma.-32P]ATP results in the phosphorylation of p68 and added substrates, calf thymus histone and eukaryotic initiation factor 2. Immune complex preparations from [35S]Met-labeled extracts show the specific immunoprecipitation of p68. Several other [35S]Met-labeled proteins are bound unspecifically in these immune complexes prepared under similar experimental conditions as for the assay of protein kinase activity. These unspecifically bound proteins can be washed out by using a buffer containing detergents or high concentrations of KCl and magnesium acetate. Immune complex preparations washed similarly with these buffers still retain p68 but lose their capacity to phosphorylate p68 or exogenous substrates. By itself p68 has no protein kinase activity. The induction of [35S]Met-labeled p68 in Daudi cells occurs with as little as 1 U of human .alpha. interferon, with maximal synthesis between 6-9 h after the addition of interferon. Actinomycin D blocks this induction.