No abstract

A cell-free assay was developed to measure the binding of iodinated human interferon-a2 to membranes prepared from lymphoblastoid Daudi cells. The kinetics of binding were similar at 0C and 30WC, 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 solubiized receptor. An assay was developed to measure the binding of 'MIlabeled interferon ("'I-interferon) to solubilized receptors, based on the precipitation of interferon-receptor complexes with polyethylene glycol. Optimal binding with this assay was obtained at OC. The solubilized receptor was analyzed by zonal sedimentation centrifugation and gel filtration. Sedimentation analysis in H20 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 (1)(2)(3)(4)(6)(7)(8)(9) and correlated with biological responses to IFN (6, 9, 10). These studies were carried out with intact cells, and the receptors were defined in terms of their affinity for IFN (1)(2)(3)(4) (pH 8.5) was allowed to react for 2 hr at 4°C with 1 mCi of Bolton-Hunter 125I reagent (New England Nuclear; 2,000 Ci/mmol; 1 Ci = 3.7 x 101' Bq). After addition of 0.5 ml of 0.2 M glycine/0.1 M sodium borate, pH 8.5, the reaction mixture was chromatographed on a Sephadex G-75 column (0. 7 x 26 cm) equilibrated with 0.25% gelatin in phosphate-buffered saline (PiNaCl) at pH 7. Fractions containing 25I-IFN were pooled as described (2). The initial specific activity of the 125I-IFN was 740 Ci/mmol; recovery of antiviral activity was >30%. For use in binding assays the 125I-IFN was diluted 1:10 with 20 mM Hepes KOH, pH 7.4/0.1% bovine serum albumin (Hepesalb.) and filtered through a Millex-GV filter (Millipore).Binding of 125I-IFN to Plasma Membranes. Plasma membranes were prepared from Daudi cells by the hypoosmotic borate method of Thom et al. (14). The purified membranes were suspended at 10 mg/ml in 10 mM Hepes buffer (pH 7.4) and stored at -70°C. For a standard binding assay, 1-3 ,ul of membrane suspension was incubated for 30 min at 30°C with 0.5 ng of 125I-IFN and Hepes-alb. in 50-,ul reaction mixtures.The incubations were diluted with 0.2 ml of cold Hepes-alb. and filtered through GVWP Durapore filters (Millipore). The filters were washed with 15 ml of Hepes-alb. and counted in a y-counter. An incubation without membranes was used as a blank; this corresponded to <2% of the input radioactivity. Nonspecific binding was measured in the presence of a 50-fold excess of unlabeled HuIFN-a2 at different time points and 125I-IFN concentrations. This nonspecific binding was consistently <15% of the total binding and was subtracted from the data reported.Abbreviations: IFN, interferon; HuIFN, human IF...

No abstract

No abstract