Guinea-pig peritoneal macrophage receptor for IgG--II. Purification of the receptor and its partial characterization.
The Fc gamma receptor of guinea-pig peritoneal macrophages was purified by affinity chromatography by using rabbit IgG or guinea-pig IgG2 coupled to Sepharose. Lysates prepared by treatment of 125I-labeled macrophages with NP-40 were first applied to BSA-Sepharose and then to IgG-Sepharose and eluted with 0.5 M acetic acid containing 1% NP-40. The specific binding was determined by interaction of the 125I-labeled receptor with IgG-Sepharose in the presence and absence of soluble IgG. The specific binding of the purified receptor was 42-82%. Interactions of the purified receptor with IgG-Sepharose were equally well inhibited by soluble rabbit IgG or guinea-pig IgG2, but not by F(ab')2 fragments. Inclusion of NP-40 in the buffer used in the assay reduced nonspecific binding of the receptor to the affinity gels. The purified receptor can be stored for 20 days at 4 degrees C without a significant loss of the specific binding activity. Analysis of the receptor by SDS-polyacrylamide gel electrophoresis, under nonreducing and reducing conditions, revealed two major peaks of radioactivity corresponding to mol. wts of about 50,000 and 25,000, and one very minor peak corresponding to a mol. wt of about 30,000. The results obtained suggest that the protein of the second major peak is a product of the dissociation of the protein of the first major peak rather than a product of its reduction by 2-mercaptoethanol.
D000818:Animals; D001202:Ascitic Fluid; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006168:Guinea Pigs; D007074:Immunoglobulin G / Q000276:immunology*; D007163:Immunosorbent Techniques; D008264:Macrophages / Q000276:immunology*; D008970:Molecular Weight; D011817:Rabbits; D011961:Receptors, Fc / Q000302:isolation & purification*; D017452:Receptors, IgG
D007074:Immunoglobulin G; D011961:Receptors, Fc; D017452:Receptors, IgG