Chemical and functional analysis of components of adenylyl cyclase from human platelets treated with phorbolesters.
Human platelets, prelabeled with [32P]phosphate were treated with tetradecanoylphorbol acetate (TPA) for 5 min at 37 degrees 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 alpha-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 of TPA. Lack of modification of the alpha-subunit of Gi was also indicated by the results of reconstitution experiments with purified Gi alpha from bovine brain: adenylyl cyclase in membranes from untreated platelets was significantly more inhibited by added G1 alpha, than that from TPA treated cells. While beta, gamma-subunits were like-wise inhibitory no difference dependent on platelet-pretreatment could be observed.
Simmoteit
R
R
Department of Physiological Chemistry, University of Würzburg, Medical School, Germany.
Schulzki
H D
HD
Palm
D
D
Mollner
S
S
Pfeuffer
T
T
D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000097:blood* / Q000737:chemistry / Q000187:drug effects; D001792:Blood Platelets / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D000067956:Adenylyl Cyclase Inhibitors; D046911:Macromolecular Substances; D008565:Membrane Proteins; D010710:Phosphates; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D013755:Tetradecanoylphorbol Acetate