Purification of Fc(γ) receptor from rabbit alveolar macrophages that retains ligand-binding activity

The Fc(γ) receptor of rabbit alveolar macrophages was purified by affinity chromatography by using rabbit γ-globulin (Rab(γ)G) coupled to Sepharose. Macrophage preparations were efficiently labeled with ¹²⁵I by using a modified lactoperoxidase method. After incubation of NP-40 cell lysates with Rab(γ)G-Sepharose, elution at 4°C with 0.5 N acetic acid containing 1% NP-40 and rapid neutralization allowed recovery of active Fc(γ) receptor. Purified Fc(γ) receptor retained its ligand-binding activity, since ~41 to 72% of labeled material specifically rebound to Rab(γ)G-Sepharose. Active receptor also rebound to human IgG- and rat IgG-Sepharose. Active Fc(γ) receptor did not bind to Sepharose coupled to rabbit Fab, rabbit F(ab)'₂, or human F(ab)'₂ fragments, nor to Sepharose coupled to chicken IgG. analysis of Fc(γ) receptor by SDS-polyacrylamide gels demonstrated a broad peak of radioactivity in the apparent m.w. range of 50,000 to 70,000 in 5.6% acrylamide gels and 35,000 to 55,000 in 9% gels. Labeled receptor with similar structural characteristics and ligand-binding activity was also obtained from highly purified adherent cell populations and from macrophages biosynthetically-labeled with [¹⁴C]glucosamine in culture.