Characterization of the target cell receptor for IgE. II. Polyacrylamide gel analysis of the surface IgE receptor from normal rat mast cells and from rat basophilic leukemia cells

Abstract

Purified rat peritoneal mast cells (RMC) and cultured rat basophilic leukemia (RBL) cells were surface labeled with 125I by using lactoperoxidase, incubated with unlabeled rat monoclonal IgE and subjected to solubilization by treatment with Nonidet P-40 (NP-40). With both cell types significant amounts of radioiodinated material could be specifically precipitated by a "sandwich" system consisting of rabbit anti-rat epsilon-chain and goat anti-rabbit Ig. The precipitates were dissociated with sodium dodecyl sulfate (SDS) and urea and subsequently analyzed by SDS-polyacrylamide gel electrophoresis. With RMC three radioactive bands were seen. One corresponded to IgE present on the RMC at the time of isolation. A small band migrating in the region of light chain was seen with both sepcific (anti-IgE) and control precipitates. It showed no demonstrable relationship to IgE. The major radioactive band corresponded to a m.w. of 62,000. This band was dependent upon the presence of IgE and was not found when non-IgE binding control cells were used. With RBL cells, only the IgE-dependent 62,000 dalton peak was present. Saturation of the IgE receptor sites of the RMC or RBL cells before lactoperoxidase labeling almost totally eliminated this radioactive band, indicating that cell-bound IgE rendered this membrane component inaccessible to the radiolabel. These results strongly suggest that this cellular component is identical, at least in part, with the target cell surface receptor for reaginic antibody. The data also further support the hypothesis that the neoplastic RBL cells have a normal surface receptor for IgE.