Purification and characterization of the human interferon-gamma receptor from placenta.
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 X 10(12) 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 chromatography 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 degrees 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.
Calderon
J
J
Department of Pathology, Washington University School of Medicine, Saint Louis, MO 63110.
Sheehan
K C
KC
Chance
C
C
Thomas
M L
ML
Schreiber
R D
RD
D000939:Epitopes / Q000276:immunology; D005260:Female; D006023:Glycoproteins / Q000276:immunology / Q000302:isolation & purification; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism*; D010920:Placenta / Q000032:analysis*; D011971:Receptors, Immunologic / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D017471:Receptors, Interferon
D000939:Epitopes; D006023:Glycoproteins; D011971:Receptors, Immunologic; D017471:Receptors, Interferon; D007371:Interferon-gamma