[Studies of phosphorylation in rat mast cells (sixth report). Cyclic AMP-dependent protein phosphorylation in rat mast cell granule membranes].

Granules with broken membranes were isolated from sonicated, purified rat serosal mast cells on a Percoll gradient. When the granules were incubated with [gamma 32P]ATP and Mg2+ in the absence of exogenous protein kinases and substrates, one major radioactive band was recovered in the 44 K area after SDS/PAG electrophoresis. The phosphorylation reaction with ATP requires Mg2+ and 1 mM Mg2+ produces the maximal response. The initial reaction is rapid and the maximal response is seen at 30 degrees C. The reaction is enhanced by 0.05-0.5 microM cyclic AMP and is inhibited by Ca2+ (0.45 mM and higher). There is no obvious effect of phosphatidylserine and phorbol ester TPA on the reaction. The 44 K phosphorylated protein is made up of several components ranging in isoelectric point from approximately 7.6 to 6.6. This cyclic AMP-dependent phosphorylating activity in the granules may provide a mechanism by which the granules respond rapidly to cyclic AMP during mediator release.