Characterization of an interferon receptor on human lymphoblastoid cells.
Characterization of an interferon receptor on human lymphoblastoid cells.
复制标题
人淋巴母细胞上干扰素受体的表征。
DOI：
10.1073/pnas.80.11.3269
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发表时间：
1983
影响因子：
11.1
通讯作者：
Baglioni,C
中科院分区：
文献类型：
--
作者：
Faltynek,CR;Branca,AA;McCandless,S;Baglioni,C
建立了一种无细胞检测方法来检测碘标记的人干扰素-α2与淋巴母细胞Daudi细胞膜的结合。在0摄氏度和30摄氏度时，结合动力学相似，较高温度结合的干扰素多1.3倍。经Triton X-100处理的膜制剂是一种方便的增溶受体来源。建立了一种测定~(125)I标记的干扰素(~(125)I-干扰素)与增溶受体结合的方法，该方法基于干扰素-受体复合体与聚乙二醇的沉淀。通过区带沉淀、离心、凝胶过滤等方法对增溶受体进行了分析。在H2O和2H2O梯度下的沉降分析提供了受体-Triton X-100络合物的沉降系数和部分比体积。凝胶过滤层析提供了该络合物的斯托克斯半径。根据这些数据，我们计算了几个物理参数，包括复合体的蛋白质部分的MR=95,000。该受体是一种高度不对称和疏水性的膜蛋白。~(125)I-干扰素可与完整Daudi细胞的受体或离体膜上的受体通过琥珀酸二亚胺进行交联。用凝胶电泳法对共价连接的~(125)I-干扰素-受体复合物进行分析。在凝胶放射自显影中检测到一条MR=140,000的单一条带。如果在这个复合体中存在一个干扰素分子，那么受体的MR接近120,000。讨论了两种分析方法得到不同MR值的可能原因。
A cell-free assay was developed to measure the binding of iodinated human interferon-alpha 2 to membranes prepared from lymphoblastoid Daudi cells. The kinetics of binding were similar at 0 degrees C and 30 degrees C, with 1.3-fold more interferon bound at the higher temperature. Membrane preparations treated with Triton X-100 proved to be a convenient source of solubilized receptor. An assay was developed to measure the binding of 125I-labeled interferon (125I-interferon) to solubilized receptors, based on the precipitation of interferon-receptor complexes with polyethylene glycol. Optimal binding with this assay was obtained at 0 degrees C. The solubilized receptor was analyzed by zonal sedimentation centrifugation and gel filtration. Sedimentation analysis in H2O and 2H2O gradients provided the sedimentation coefficient and the partial specific volume of the receptor-Triton X-100 complex. Gel filtration chromatography provided the Stokes radius of this complex. From these data we calculated several physical parameters, including Mr = 95,000 for the protein portion of the complex. The receptor is a highly asymmetric and hydrophobic membrane protein. 125I-Interferon could be crosslinked to receptors of intact Daudi cells or of isolated membranes by use of disuccinimidyl suberate. The covalently linked 125I-interferon-receptor complexes were analyzed by gel electrophoresis. A single band with Mr = 140,000 was detected in gel autoradiographs. If one molecule of interferon is present in this complex, the Mr of the receptor is close to 120,000. Possible reasons for the different Mr values obtained with the two analytical procedures used are discussed.