Abstract
Granules were isolated from sonicated rat mast cells on a Percoll gradient and incubated with [γ 32P] ATP in the presence of Mg2+. After stopping the reaction, lipid extraction of the intact membrane granules was performed with acidic medium and phospholipids were separated by thin layer chromatography on 1% (w/v) oxalic acid and potassium oxalate impregnated silica gel plates. Considerable amounts of radioactivity were found to be incorporated into DPI. The radioactivity in DPI was associated with the granule themselves and not detected in the granule supernates. Extensive washing of the granules did not significantly affect DPI formation and areas of the Percoll gradient containing cytosol fraction had no demonstrable DPI forming activity and did not increase the response when added back to the granules. When suspensions of intact and broken membrane granules which contained approximately equivalent amounts of granule membrane proteins were compared with respect to DPI formation, broken membrane granule preparations were found not to exceed in DPI production than intact membrane granules, suggesting that DPI formation is occuring on the cytoplasmic side of granule membranes.
