Chemical and functional analysis of components of adenylyl cyclase from human platelets treated with phorbolesters

Abstract

Human platelets, prelabeled with [³²P]phospate were treated with tetradecanoylphorbol acetate (TPA) for 5 min at 37°C. Phosphorylation of the components of adenylyl cyclase was determined in membranes using specific antibodies against G‐proteins and the catalytic moiety. Less than 0.01 mol of [³²P]phosphate/mol could be detected in immunoprecipitates using antibodies against sequences within the α‐subunit of the GTP binding protein G_i. TPA, however, caused the incorporation of 0.67–1.1 mol of [³²P]phosphate per mol of catalyst while 0.13‐0.2 mol were found in the absence or TPA. Lack of modification of the α‐subunit of G_i was also indicated by the results of reconstitution experiments with purified G_iα from bovine brain: adenylyl cyclase in membranes from untreated platelets was significantly more inhibited by added G_iα, than that from TPA treated cells. While β,γ‐subunits were like‐wise inhibitory no difference dependent on platelet‐pretreatment could be observed.