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

• 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
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)67585-4

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.