Preparation and characterization of mast cell cytoplasts

A new model system for studying biochemical reactions in mast cell plasma membranes was developed. Particles termed cytoplasts consisting of organelle-depleted cytoplasm surrounded by an intact plasma membrane were formed from cytochalasin B-treated mast cells ultracentrifuged through a discontinuous Ficoll gradient. Two cytoplasts were formed per mast cell and 95% were recovered. Mast cell cytoplasts had a mean diameter of 3.2 microns with a median volume of 38 microns 3. Enzyme marker studies indicated that subcellular recoveries in the mast cell cytoplast were: plasma membrane = 16%, cytoplasm = 39%, nucleus = 1.1%, granule = 0.5%. Analysis of IgE receptors indicated that mast cell cytoplasts retained the normal asymmetric orientation of the plasma membrane. Mast cell cytoplasts synthesized ATP, incorporated labeled fatty acids into complex lipids and retained fluorescein after deacylation of diacetylfluorescein. The quantity of cAMP (adenosine 3':5'-cyclic monophosphate) maintained in mast cell cytoplasts was 0.0304 pmol/10(6) original mast cells. Cytoplasts offer the opportunity to study plasma membrane and cytoplasmic biochemical events that occur during stimulation in a relatively physiologic environment.