Preparation of 32P-labeled murine immune interferon and its binding to the mouse immune interferon receptor

Murine immune interferon (Mu-IFN-gamma) can be radiolabeled with [gamma-32P]ATP by the catalytic subunit of cAMP-dependent protein kinase. The resulting 32P-labeled Mu-IFN-gamma (32P-Mu-IFN-gamma) with high radiological specific activity (60-260 muCi/micrograms) retains biological activity. Acid hyd...

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Bibliographic Details
Published in:The Journal of biological chemistry 1986-07, Vol.261 (21), p.9801-9804
Main Authors: Langer, J A, Rashidbaigi, A, Pestka, S
Format: Article
Language:English
Subjects:
Adenosine Triphosphate - metabolism
Analysis of the immune response. Humoral and cellular immunity
Animals
Biological and medical sciences
Cell Line
Electrophoresis, Polyacrylamide Gel
Fundamental and applied biological sciences. Psychology
Fundamental immunology
Humans
Immunobiology
Interferon-gamma - metabolism
Lymphokines, interleukins ( function, expression)
Mercaptoethanol - pharmacology
Mice
Molecular Weight
Neoplasms, Germ Cell and Embryonal - metabolism
Phosphates - metabolism
Protein Kinases - metabolism
Receptors, Immunologic - metabolism
Receptors, Interferon
Regulatory factors and their cellular receptors
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Summary:Murine immune interferon (Mu-IFN-gamma) can be radiolabeled with [gamma-32P]ATP by the catalytic subunit of cAMP-dependent protein kinase. The resulting 32P-labeled Mu-IFN-gamma (32P-Mu-IFN-gamma) with high radiological specific activity (60-260 muCi/micrograms) retains biological activity. Acid hydrolysis of 32P-Mu-IFN-gamma or 32P-labeled human IFN-gamma leads to the release of [32P]phosphoserine but not phosphothreonine or phosphotyrosine. With 32P-Mu-IFN-gamma, we have demonstrated that there are 5 X 10(3) to 1.5 X 10(4) receptors per-cell on several murine cell lines of diverse origin and that the Kd at 24 degrees C for these cells is in the range of 1 X 10(-10) to 1 X 10(-9) M. Covalent binding of 32P-Mu-IFN-gamma to its receptor results in the formation of several specific high-molecular weight products, the major one of which has an apparent molecular weight of 90,000-100,000. If this represents a 1:1 complex of Mu-IFN-gamma and its receptor (or its binding subunit), the murine interferon gamma receptor has a molecular weight of 75,000-85,000.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(18)67585-4