Laurent A G, Krust B, Galabru J, Svab J, Hovanessian A G
Proc Natl Acad Sci U S A. 1985 Jul;82(13):4341-5. doi: 10.1073/pnas.82.13.4341.
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 Mr 69,000-72,000 protein in its phosphate-saturated state. By using a highly purified protein kinase fraction from interferon-treated human Daudi cells, we can now describe the preparation of murine monoclonal antibodies directed against this phosphoprotein, the Mr of which in its native state is found to be 68,000. 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 have 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]methionine-labeled extracts show the specific immunoprecipitation of p68. In addition, several other [35S]methionine-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. These results indicate that p68 by itself has no protein kinase activity. The induction of [35S]methionine-labeled p68 in Daudi cells occurs with as little as 1 unit of human alpha interferon, with maximal synthesis between 6 to 9 hr after the addition of interferon. Actinomycin D blocks this induction.
从经干扰素处理的人细胞中提取的物质显示，一种双链RNA依赖性蛋白激酶活性水平增强，这表现为一种内源性分子量为69,000 - 72,000的蛋白在其磷酸饱和状态下发生磷酸化。通过使用从经干扰素处理的人Daudi细胞中高度纯化的蛋白激酶组分，我们现在可以描述针对这种磷蛋白制备鼠单克隆抗体的过程，发现其天然状态下的分子量为68,000。这些单克隆抗体（IgG1类）可通过电泳转移印迹技术在聚丙烯酰胺凝胶中识别电泳后的蛋白（p68）。用单克隆抗体孵育经干扰素处理的人细胞提取物后形成的免疫沉淀物，可通过蛋白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——即加入[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发生磷酸化。来自[35S]甲硫氨酸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。此外，在与蛋白激酶活性测定相似的实验条件下制备的这些免疫复合物中，还有几种其他[35S]甲硫氨酸标记的蛋白非特异性结合。这些非特异性结合的蛋白可以用含有去污剂或高浓度氯化钾和醋酸镁的缓冲液洗去。用这些缓冲液类似地洗涤后的免疫复合物制剂仍保留p68，但失去了磷酸化p68或外源底物的能力。这些结果表明p68本身没有蛋白激酶活性。在Daudi细胞中，只需1单位的人α干扰素就能诱导[35S]甲硫氨酸标记的p68产生，在加入干扰素后6至9小时之间合成量最大。放线菌素D可阻断这种诱导作用。
