Immune interferon receptor: chemical and enzymatic sensitivity.
Human immune interferon-gamma (HuIFN-gamma) labeled with 32P was used to study the structure of IFN-gamma receptor. When [32P]HuIFN-gamma was bound and crosslinked to IFN-gamma the receptor of human cells with a bifunctional crosslinker disuccinimidyl suberate (DSS), a single diffused 32P-labeled band corresponding to the IFN-gamma.receptor complex was visualized by SDS-polyacrylamide gel electrophoresis and autoradiography. The size of the [32P]-HuIFN-gamma.receptor complex was about 100-120 kD. Separation of crosslinked complex in reducing and nonreducing gels showed no size differences, suggesting the absence of interchain disulfide linkage. However, binding and formation of the crosslinked IFN-gamma. receptor complex on cells was diminished in the presence of the disulfide reducing agent dithiothreitol (DTT). The reduction was DTT-dose-dependent, suggesting that intramolecular disulfides of the receptor are important for binding. Also, [32P]HuIFN-gamma did not bind if cells were pretreated with and then washed free of DTT, suggesting an irreversible reduction of intrachain disulfide bonds, presumably of the receptor. [32P]HuIFN-gamma also specifically binds to human placental membranes. Each placenta has about 170 ng of IFN-gamma receptors. Covalent attachment of [32P]HuIFN-gamma to placental plasma membranes via DSS produced 2 crosslinked complexes with the molecular sizes of 100-120 kD and 60-70 kD. The IFN-gamma.receptor complex of placental membranes was solubilized with NP-40 after DSS treatment and partially purified with immobilized antibody to the carboxyl terminus of IFN-gamma. Treatment of the receptor complex with trypsin and papain was used to demonstrate its differential proteolytic sensitivity.
Rashidbaigi
A
A
University of Medicine and Dentistry of New Jersey, Department of Molecular Genetics and Microbiology, Robert Wood Johnson Medical School, Piscataway 08854.
Stefanos
S
S
Jung
V
V
Langer
J A
JA
D002460:Cell Line; D004229:Dithiothreitol; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D007371:Interferon-gamma; D007372:Interferons; D008566:Membranes; D010447:Peptide Hydrolases / Q000378:metabolism; D010920:Placenta / Q000032:analysis; D011247:Pregnancy; D011971:Receptors, Immunologic / Q000302:isolation & purification*; D017471:Receptors, Interferon; D011994:Recombinant Proteins
D011971:Receptors, Immunologic; D017471:Receptors, Interferon; D011994:Recombinant Proteins; D007371:Interferon-gamma; D007372:Interferons; D010447:Peptide Hydrolases; D004229:Dithiothreitol