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

Murine immune interferon (Mu-IFN-γ) can be radiolabeled with [γ-³²P]ATP by the catalytic subunit of cAMP-dependent protein kinase. The resulting ³²P-labeled Mu-IFN-γ (³²P-Mu-IFN-γ) with high radiological specific activity (60-260 μCi/μg) retains biological activity. Acid hydrolysis of ³²P-Mu-IFN-γ or ³²P-labeled human IFN-γ leads to the release of [³²P]phosphoserine but not phosphothreonine or phosphotyrosine. With ³²P-Mu-IFN-γ, we have demonstrated that there are 5 x 10³ to 1.5 x 10⁴ receptors per-cell on several murine cell lines of diverse origin and that the K(d) at 24°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-γ 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-γ and its receptor (or its binding subunit), the murine interferon γ receptor has a molecular weight of 75,000-85,000.