Faltynek C R, Branca A A, McCandless S, Baglioni C
Proc Natl Acad Sci U S A. 1983 Jun;80(11):3269-73. doi: 10.1073/pnas.80.11.3269.
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.
开发了一种无细胞测定法来测量碘化人α-2干扰素与从淋巴母细胞样Daudi细胞制备的膜的结合。在0℃和30℃下结合动力学相似，在较高温度下结合的干扰素多1.3倍。用Triton X-100处理的膜制剂被证明是溶解受体的方便来源。基于用聚乙二醇沉淀干扰素-受体复合物，开发了一种测定法来测量125I标记的干扰素（125I-干扰素）与溶解受体的结合。该测定法在0℃下获得最佳结合。通过区带沉降离心和凝胶过滤分析溶解的受体。在H2O和2H2O梯度中进行沉降分析可提供受体-Triton X-100复合物的沉降系数和比容。凝胶过滤色谱法可提供该复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，包括该复合物蛋白质部分的Mr = 95,000。该受体是一种高度不对称的疏水膜蛋白。通过使用辛二酸二琥珀酰亚胺酯，125I-干扰素可与完整Daudi细胞或分离膜的受体交联。通过凝胶电泳分析共价连接的125I-干扰素-受体复合物。在凝胶放射自显影片中检测到一条Mr = 140,000的单带。如果该复合物中存在一分子干扰素，则受体的Mr接近120,000。讨论了使用两种分析方法获得不同Mr值的可能原因。
