Immunoprecipitation of the solubilized membrane receptor for IgE of human cultured lymphoblastoid cells.
The putative Fc receptor for IgE (Fc epsilon) of cultured human lymphoblastoid cells was characterized by using a goat anti-receptor antiserum. The antiserum was prepared against NP-40-solubilized cell components of Wil-2WT cells which bound to an IgE-Sepharose-4B immunoadsorbent column. The antiserum specifically inhibited binding of 125I-labelled IgE to Fc epsilon-positive RPMI-8866 lymphoblastoid cells. Absorption of the antiserum with Fc epsilon-negative Raji and Molt-4 cells and with IgE and IgG did not change this inhibitory activity. Antiserum extensively absorbed with Raji and Molt-4 cells precipitated 7% of the radioactivity of lysates of 125I-lactoperoxidase-labelled RPMI-8866 cells but only 0.5-1.8% of that of Raji and Molt-4 cells. Autoradiography of SDS-PAGE analyses demonstrated two major labelled peptides of 86,000 and 47,000 mol. wt. in reduced immunoprecipitates from RPMI-8866 but not from Raji or Molt-4 cell lysates. As determined by Sepharose-6B gel filtration, the approximate molecular weight of the solubilized radiolabelled membrane component that reacted with the antiserum was 250,000 solubilized in NP-40 and 125,000 in NP-40-4 M urea. Both the 250,000 and 125,000 mol. wt. material consisted of the 86,000 and 47,000 peptides. The data demonstrate that the anti-receptor antiserum inhibited binding of 125I-labelled IgE to Fc epsilon-receptor-positive lymphoblastoid cells and suggest that the receptor may consist of two non-covalently linked polypeptides that remain associated in NP-40 and NP-40-4 M urea.
D002478:Cells, Cultured; D011232:Chemical Precipitation; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology*; D008214:Lymphocytes / Q000276:immunology*; D008565:Membrane Proteins / Q000032:analysis; D008970:Molecular Weight; D010455:Peptides / Q000032:analysis; D011485:Protein Binding; D011961:Receptors, Fc / Q000032:analysis*
D008565:Membrane Proteins; D010455:Peptides; D011961:Receptors, Fc; D007073:Immunoglobulin E