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

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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 Gi. 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 Gi was also indicated by the results of reconstitution experiments with purified Giα from bovine brain: adenylyl cyclase in membranes from untreated platelets was significantly more inhibited by added Giα, than that from TPA treated cells. While β,γ‐subunits were like‐wise inhibitory no difference dependent on platelet‐pretreatment could be observed.