Isolation of cellular membranes from rat mast cells

Large amounts of membranes enriched either in perigranular membranes or in plasma membranes have been successfully isolated from rat peritoneal mast cells. A cycle consisting of a single sonication pulse to disrupt the mast cells followed by centrifugation to separate the released granules was repeated until 90% of the mast cells were disrupted. This technique resulted in a high yield of intact granules since the released granules were only exposed to the single sonication pulse. The intact granules were separated from plasma membrane fragments by centrifugation through a Percoll gradient. The perigranular membranes were then obtained by osmotic lysis of the purified intact granules. The plasma membrane fraction was enriched 4.5-fold (range, 4.1-6.1) in 5'-nucleotidase activity, a plasma membrane marker enzyme. No suitable marker enzyme activity was found for the perigranular membrane fraction. An important aspect of this procedure is its potential for obtaining both a plasma and perigranular membrane preparation in high yield and purity from the same mast cell preparation.