Purification and characterization of the human interferon-. gamma. receptor from placenta
- 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), Vol. 85:13; ISSN 0027-8424
- Country of Publication:
- United States
- Language:
- English
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PLACENTA
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ELECTROPHORESIS
IODINE 125
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PURIFICATION
ANIMALS
ANTIBODIES
BETA DECAY RADIOISOTOPES
CHEMICAL REACTIONS
DAYS LIVING RADIOISOTOPES
ELECTRON CAPTURE RADIOISOTOPES
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INTERMEDIATE MASS NUCLEI
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LYMPHOKINES
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MITOGENS
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
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550201* - Biochemistry- Tracer Techniques