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

Journal Article · Fri Jul 01 00:00:00 EDT 1988 · Proceedings of the National Academy of Sciences of the United States of America; (USA)

DOI:https://doi.org/10.1073/pnas.85.13.4837· OSTI ID:5565205

Calderon, J; Sheehan, K C.F.; Chance, C; Thomas, M L; Schreiber, R D ^[1]

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1. Washington Univ. School of medicine, St. Louis, MO (USA)

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} 10{sup 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 INF-{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 NaDodSO{sub 4}/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 {sup 125}I-labeled IFN-{gamma} following NaDodSO{sub 4}/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.

• OSTI ID: 5565205
• Journal Information: Proceedings of the National Academy of Sciences of the United States of America; (USA), Journal Name: Proceedings of the National Academy of Sciences of the United States of America; (USA) Vol. 85:13; ISSN PNASA; ISSN 0027-8424
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANIMALS
ANTIBODIES
BETA DECAY RADIOISOTOPES
CHEMICAL REACTIONS
CROSS-LINKING
DAYS LIVING RADIOISOTOPES
ELECTRON CAPTURE RADIOISOTOPES
ELECTROPHORESIS
FETAL MEMBRANES
GROWTH FACTORS
INTERFERON
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPES
LYMPHOKINES
MAMMALS
MAN
MEMBRANE PROTEINS
MEMBRANES
MITOGENS
MOLECULAR STRUCTURE
MONOCLONAL ANTIBODIES
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
PLACENTA
POLYMERIZATION
PRIMATES
PROTEINS
PURIFICATION
RADIOISOTOPES
RECEPTORS
VERTEBRATES