Purification and characterization of the human interferon-gamma receptor from placenta.

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Abstract
Purification of the human interferon-.gamma. (IFN-.gamma.) 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 .times. 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-.gamma. receptor from octyl glucoside-solubilized membranes: (i) sequential affinity cohromatography over wheat germ agglutinin- and IFN-.gamma.-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-.gamma. 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-.gamma. following NaDodSO4/PAGE and transfer to nitrocellulose. Certain receptor preparations converted into a 55-kDa fragment either during purification or upon storage at 4.degree. C. On the basis of N-glycanase digestion studies, the IFN-.gamma. receptor appeared to contain 17 kDa of N-linked carbohydrate. 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.