Covalent binding of arachidonate to G protein α subunits of human platelets

Hazem Hallak, Laszlo Muszbek, Michael Laposata, Elizabeth Belmonte, Lawrence F. Brass, David R. Manning

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Abstract

The α subunits of GTP-binding regulatory proteins (G proteins) are subject to lipid modifications required for anchorage to membrane and/or interactions with other proteins. With the knowledge that α subunits are palmitoylated, which we demonstrate here for human platelets, we sought to determine whether these subunits also bind arachidonate and myristate in a covalent, post-translational manner. All α subunits examined were found to incorporate radioactivity upon incubation of human platelets with [3H]palmitate, [3H]arachidonate, and [3H]myristate. The identity of [3H]palmitate and [3H]arachidonate as covalently bound fatty acids was confirmed by high pressure liquid chromatography following alkaline methanolysis. With [3H]myristate, however, the bound fatty acid proved to be [3H]palmitate, presumably generated by a 2-carbon chain elongation. Protein- bound [3H]palmitate and [3H]arachidonate were released by hydroxylamine at neutral pH, implying a thioester linkage between protein and fatty acid. Thus, post-translational modifications of G protein α subunits include palmitoylation and arachidonoylation, but not myristoylation. Given the different physical properties of saturated and unsaturated fatty acids and the large-scale release of arachidonate during platelet activation, changes in arachidonate incorporation may serve as an important regulator of α subunit function.

Original languageEnglish (US)
Pages (from-to)4713-4716
Number of pages4
JournalJournal of Biological Chemistry
Volume269
Issue number7
StatePublished - Feb 18 1994
Externally publishedYes

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ASJC Scopus subject areas

  • Biochemistry

Cite this

Hallak, H., Muszbek, L., Laposata, M., Belmonte, E., Brass, L. F., & Manning, D. R. (1994). Covalent binding of arachidonate to G protein α subunits of human platelets. Journal of Biological Chemistry, 269(7), 4713-4716.