Binding of rat IgE with the subcellular components of normal rat mast cells

Attempts were made to obtain subcellular components of rat mast cells which contain receptors for IgE. Rat peritoneal cells were treated with ¹²⁵I-labeled rat IgE to label mast cells, and disrupted by ultrasonication. More than 90% of cell-bound IgE was recovered in the 20,000 g supernatant fraction which had the ability to block passive cutaneous anaphylaxis with mouse and rat reaginic antibodies. Gel-filtration of the supernatant fraction through a Sepharose 6B column showed that both radioactivity and PCA blocking activity were associated with subcellular components which were eluted in the void volume. When the 20,000 g supernatant fraction of normal peritoneal cells was incubated with ¹²⁵I-rat IgE, the protein combined with the subcellular components. Incubation of the 20,000 g supernatant with an IgE-rich rat serum inhibited the binding of ¹²⁵I-IgE with the components, whereas normal rat serum failed to do so. When the sepharose void volume fraction was obtained from purified mast cells and labeled with ¹²⁵I, approximately 25% of radioactive material was coprecipitated with IgE-anti-IgE complexes. Evidence was obtained that subcellular components of macrophages were not coprecipitated with the antigen-antibody complexes. These results indicated that the components eluted in the void volume fraction contained receptors for IgE. In another experiment, the 20,000 g supernatant fraction was obtained from rat peritoneal cells or purified mast cells whose membrane was labeled with ¹²⁵I, and fractioned by gel filtration followed by DEAE cellulose chromatography. More than 2 3 of the radioactivity and PCA-blocking activity associated with the sepharose void volume fraction were recovered in the 3 M NaCl fraction which contained 12-15% of total protein. When the 3 M fraction obtained from purified mast cells was labeled with ¹²⁵I and incubated with an IgE-rich serum, a significant amount of radioactive material in the fraction was precipitated with IgE-anti-IgE complexes. The results indicate that blocking of the PCA reaction by this fraction is due to the presence of receptors for IgE.