55 Scopus citations

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.

Original languageEnglish
Pages (from-to)4837-4841
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume85
Issue number13
DOIs
StatePublished - Jul 1988

Fingerprint

Dive into the research topics of 'Purification and characterization of the human interferon-gamma receptor from placenta.'. Together they form a unique fingerprint.

Cite this