Characterization of ligand-binding activity of isolated murine Fc gamma receptor.
Fc gamma-binding macromolecules were isolated from 2 murine macrophage-like cell lines by affinity chromatography using various immunoglobulins coupled to Sepharose. P388D1 and J744.2 cells were radiolabeled with 125I using a modified lactoperoxidase method, washed cells were solubilized using NP-40, and the solubilized cell lysates were incubated at 4 degrees C with immunoadsorbents. Fc gamma receptor-like material was obtained from the IgG-Sepharose columns by elution at 4 degrees C with 0.5 N acetic acid containing 1% NP-40 into buffer for neutralization. Purified material thus obtained retained its ligand-binding activity, since approximately 30 to 60% rebound to IgG-Sepharose. Upon analysis by SDS polyacrylamide gel electrophoresis, putative Fc gamma receptor had an apparent m.w. of 50 to 65,000. Neither a comparable SDS-PAGE band nor receptor activity was isolated from a 3rd murine cell line, which lacks the Fc gamma receptor. Putative receptor obtained by elution from IgG2a-Sepharose rebound equally well to IgG2a-, IgG1- and IgG2b-Sepharose, but did not rebind to IgG3-, F(ab')2-, or BSA-Sepharose. Similarly, Fc gamma-binding macromolecules eluted from IgG2b-Sepharose rebound only to IgG2b-, IgG2a- and IgG1-Sepharose rebound only to IgG2b-, IgG2a- and IgG1-Sepharose. The rebinding of either receptor preparation was inhibited in a dose-dependent manner by both monomeric IgG2a and monomeric IgG2b, and myeloma protein from each subclass inhibited to the same extent. Therefore, the mouse Fc gamma receptor in its isolated state does not appear to discriminate between monomeric IgG2a and IgG2b.
D000818:Animals; D001665:Binding Sites; D002846:Chromatography, Affinity; D007074:Immunoglobulin G / Q000145:classification; D007164:Immunosorbents; D008024:Ligands / Q000378:metabolism*; D046911:Macromolecular Substances; D008264:Macrophages / Q000276:immunology; D051379:Mice; D011961:Receptors, Fc / Q000276:immunology* / Q000302:isolation & purification
D007074:Immunoglobulin G; D007164:Immunosorbents; D008024:Ligands; D046911:Macromolecular Substances; D011961:Receptors, Fc