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 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.
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism*; D008623:Mercaptoethanol / Q000494:pharmacology; D051379:Mice; D008970:Molecular Weight; D009373:Neoplasms, Germ Cell and Embryonal / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism; D011971:Receptors, Immunologic / Q000378:metabolism*; D017471:Receptors, Interferon
D010710:Phosphates; D011971:Receptors, Immunologic; D017471:Receptors, Interferon; D008623:Mercaptoethanol; D007371:Interferon-gamma; D000255:Adenosine Triphosphate; D011494:Protein Kinases