| Abstract: | 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. |
