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

Proc Natl Acad Sci U S A. 1988 Jul;85(13):4837-41. doi: 10.1073/pnas.85.13.4837.

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 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Epitopes / immunology
  • Female
  • Glycoproteins / immunology
  • Glycoproteins / isolation & purification
  • Humans
  • Interferon-gamma / metabolism*
  • Placenta / analysis*
  • Receptors, Immunologic / immunology
  • Receptors, Immunologic / isolation & purification*
  • Receptors, Immunologic / metabolism
  • Receptors, Interferon

Substances

  • Epitopes
  • Glycoproteins
  • Receptors, Immunologic
  • Receptors, Interferon
  • Interferon-gamma