Characterization of the receptors for IgE on membranes isolated from rat basophilic leukemia cells

Abstract

Mast cells, basophils and a tumor analog, rat basophilic leukemia cells, have a surface receptor for immunoglobulin E (IgE). Membranes from such cells should be highly useful for the study of how the α- and β-subunits of the receptor interact with the plasma membrane and function therein. Membranes were prepared from rat basophilic leukemia cells. The cells were reacted with radioiodinated IgE to saturate a fraction of their receptors and the radiolabel was used as a surface marker. When isolated from sucrose gradients, 40 ± 10% yields of plasma membranes were obtained and these were typically purified 13-fold with regard to cellular proteins. Unoccupied receptors for IgE were almost all recovered (as assessed on membranes solubilized with detergent) as were surface-labeled proteins in general. The membrane-bound IgE appeared fully exposed as assessed with anti-IgE antibodies but surprisingly 70–80% of the unoccupied receptors present were unable to bind IgE when unsolubilized vesicles were tested. By electron microscopy the preparations appeared free of recognizable intracellular organelles. Though some of the membranes appeared multivesicular many showed large breaks in continuity. Assessment of permeability, carried out on dextran gradients, indicated that a significant number (⩾50%) of the IgE-carrying membranes are not sealed. It is therefore most likely that both external and cytoplasmic aspects of the plasma membrane were exposed in many of the vesicles. Treatment of such membrane vesicles with trypsin or papain did not alter the apparent mol. wt of the α-chain of the receptor or its binding activity for IgE. Additionally, when the vesicles were examined by enzyme-catalysed iodination, no labeling of the receptor was observed under conditions where it is not labeled on intact cells (i.e. in the presence of IgE). These results suggest that either the α-chain of the receptor does not penetrate the membrane or that it cannot be modified by the particular probes we used.