Agonist-enhanced palmitoylation of platelet proteins.
When washed human platelets were incubated with [3H]palmitic acid, radioactivity was incorporated into a major 38 kDa doublet and several minor proteins that were resolved on polyacrylamide gels. The radioactivity associated with the proteins remained after extractions with organic solvents, but it was lost after hydroxylamine treatment or mild alkali methanolysis. The products of these reactions were analyzed by thin-layer chromatography and HPLC. They were identified as palmitohydroxamate and methyl palmitate, respectively, indicating that the palmitic acid was covalently linked to the proteins via oxygenester or thioester bonds. In resting platelets, radioactivity was detected in the 38 kDa proteins 2 min after the addition of [3H]palmitic acid. A plateau was reached between 5 and 11 min, at which time radioactivity was also detected in a 23 kDa protein. Thrombin elicited faster and greater incorporation of label into both proteins. Phorbol 12-myristate 13-acetate (PMA) led to a similar, but slower increase of radioactivity in the 38 kDa proteins, while collagen and A23187 were less effective. Enhanced palmitoylation may be closely linked to platelet activation, as suggested by the following observations: (1) in thrombin- or PMA-activated platelets, the time-course of aggregation correlated with the time-course of enhanced palmitoylation of the 38 kDa proteins; (2) in platelets activated by various concentrations of thrombin with or without prostacyclin, aggregation was correlated with the enhanced incorporation of radioactivity into the 38 kDa proteins.
Huang
E M
EM
Department of Biochemistry, SUNY Health Science Center, Brooklyn 11203.
D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D001798:Blood Proteins / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D011464:Epoprostenol / Q000494:pharmacology; D006801:Humans; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D066298:In Vitro Techniques; D008970:Molecular Weight; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000097:blood*; D010974:Platelet Aggregation / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000502:physiology
D001798:Blood Proteins; D006898:Hydroxylamines; D010169:Palmitic Acids; D019811:Hydroxylamine; D019308:Palmitic Acid; D011464:Epoprostenol; D013917:Thrombin; D013755:Tetradecanoylphorbol Acetate