Palmitylation of the glycoprotein IIb-IIIa complex in human blood platelets.
The presence of covalently bound palmitic acid in fibrinogen receptors, glycoproteins (GP) IIb and IIIa, has been explored in human blood platelets. Membrane fractions were isolated from fresh blood platelets labeled with [9,10-3H]palmitic acid and then analyzed for radioactive proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein bands were visualized by staining with Coomassie Brilliant Blue, excised, and counted in a liquid scintillation counter. The results indicate that membrane proteins with electrophoretic mobility corresponding to glycoproteins IIb and IIIa incorporate [9,10-3H]palmitic acid. The palmitylated glycoproteins IIb and IIIa were immunoprecipitated by specific anti-GP IIb and GP IIIa antisera. It is interesting to note that the palmitylation of these glycoproteins occurred rapidly in platelets activated with 0.5 unit of thrombin or 30 microM ADP. At the concentration used (100 micrograms/ml), cycloheximide did not inhibit incorporation of [3H]palmitate into the glycoproteins showing that this process is not dependent upon protein synthesis. The acyl moiety was resistant to denaturating detergents, delipidation with organic solvents, and hydrolyzable with hydroxylamine. In the case of membrane protein with the electrophoretic mobility of GP IIb, the radioactive label was significantly decreased after reduction with 2-mercaptoethanol. Final identification of GP IIIa as an acylated product in human platelets incubated with [9,10-3H]palmitic acid was provided by two-dimensional polyacrylamide gel electrophoresis. In contrast to GP IIb alpha, GP IIIa isolated by this method showed the presence of attached radioactive palmitic acid residues. Analysis by high performance liquid chromatography after methanolysis of the [3H]palmitate-labeled glycoproteins confirmed the fatty acid nature of the label. Palmitylation is a newly identified post-translational modification of the fibrinogen receptor which may play an important role in its interaction with the membrane and/or its biological function.
Cierniewski
C S
CS
Department of Biophysics, Medical School, Lodz, Poland.
Krzeslowska
J
J
Pawlowska
Z
Z
Witas
H
H
Meyer
M
M
D000328:Adult; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003513:Cycloheximide / Q000494:pharmacology; D006801:Humans; D019811:Hydroxylamine; D006898:Hydroxylamines; D066298:In Vitro Techniques; D008297:Male; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000097:blood*; D010980:Platelet Membrane Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D013917:Thrombin / Q000502:physiology; D014316:Tritium
D006898:Hydroxylamines; D010169:Palmitic Acids; D010980:Platelet Membrane Glycoproteins; D014316:Tritium; D019811:Hydroxylamine; D019308:Palmitic Acid; D003513:Cycloheximide; D013917:Thrombin