Purification and characterization of the human interferon-γ receptor from placenta

Purification of the human interferon-γ (IFN-γ) receptor was facilitated by identification of human placenta as a large-scale receptor source. When analyzed in radioligand binding experiments, intact placental membranes and detergent-solubilized membrane proteins expressed 1.3 and 5.9 x 1012 receptors per mg of protein, respectively, values that were 13-163 times greater than that observed for U937 membranes. Two protocols were followed to purify the IFN-γ receptor from octyl glucoside-solubilized membranes: (i) sequential affinity chromatography over wheat germ agglutinin- and INF-γ-Sepharose and (ii) affinity chromatography over columns containing receptor-specific monoclonal antibody and wheat germ agglutinin. Both procedures resulted in fully active preparations that were 70-90% pure. Purified receptor migrated as a single molecular species of 90 kDa either when analyzed on silver-stained NaDodSO4/polyacrylamide gels or when subjected to electrophoretic transfer blot analysis using a labeled IFN-γ receptor-specific monoclonal antibody. The identity of the 90-kDa component as the receptor was confirmed by demonstrating its ability to specifically bind 125I-labeled IFN-γ following NaDodSO4/PAGE and transfer to nitrocellulose. The ligand binding site, the epitope for the receptor-specific monoclonal antibody, and all of the N-linked carbohydrate could be localized to the 55-kDa domain of the molecule