|
pubmed:abstractText |
A method has been developed by which rat basophilic leukemia (RBL) cells can be permeabilized to small molecules while maintaining their ability to degranulate in response to aggregation of IgE receptors. alpha-Toxin was isolated from culture supernatants of Staphylococcus aureus by precipitation with (NH4)2SO4 and chromatography on phenyl-Sepharose. The isolated toxin binds to the plasma membrane of RBL cells and polymerizes to form a transmembrane pore that allows small molecules (Mr less than 1000), but not macromolecules, to diffuse freely across the membrane. There was no spontaneous release of the contents of RBL cell secretory granules during permeabilization or subsequent incubations. Substantial IgE receptor-mediated exocytosis occurred in the absence of Ca2+, and degranulation was maximal at 0.1-1.0 microM Ca2+, the physiologically important range of Ca2+ concentrations. Using these permeabilized cells, small molecules (i.e., substrates and inhibitors of various enzymes) normally excluded by the plasma membrane can be introduced into the cell. Moreover, the intracellular concentration of ions (such as Ca2+) can be precisely controlled. This method will allow a detailed examination of the individual biochemical events involved in degranulation of mast cells.
|
