Structural studies on the membrane-bound immunoglobulin E-receptor complex. 1. Characterization of large plasma membrane vesicles from rat basophilic leukemia cells and insertion of amphipathic fluorescent probes

Abstract

In order to investigate the properties of the membrane-bound IgE-receptor complex, a simple procedure has been adapted for preparing large plasma membrane vesicles from rat basophilic leukemia cells. These vesicles pinch off from the adherent cells after treatment with 2 mM N-ethylmaleimide or 50 mM formaldehyde and 1 mM dithiothreitol, and they are isolated from the supernatant after two centrifugation steps with yields of 20-25% of the initial cell-bound 125I-IgE. With phase and fluorescence microscopy, micron-size vesicles are seen which are unilamellar and spherically shaped and devoid of intracellular organelles. On dextran gradients at least 70% of the 125I-IgE is bound to membranes which band at low density, indicating large, intact vesicles that are impermeable to macromolecules. Between 60 and 75% of the bound 125I-IgE is accessible to the external medium, showing the vesicles to be predominantly right side out. This preparation was found to be suitable for resonance energy-transfer measurements. We have determined that amphipathic, fluorescent donor and acceptor probes partition into the vesicle bilayer in a randomly distributed, noninteracting manner. The densities of the probes can be ascertained directly from the amount of energy transfer that is observed as a function of acceptor concentration. This experimental system will allow energy-transfer measurements to determine distances between sites on receptor-bound IgE and the membrane surface.