Covalent modification of proteins by arachidonate and eicosapentaenoate in platelets

Laszlo Muszbek, Michael Laposata

Research output: Contribution to journalArticle

33 Citations (Scopus)

Abstract

The posttranslational modification of proteins by fatty acids has been shown to involve long chain-saturated fatty acids, predominantly palmitate. In the present study, we demonstrated by metabolic labeling of human platelets with [3H]arachidonate and [3H]eicosapentaenoate that these polyunsaturated fatty acids can also become covalently linked to proteins. The extent of binding of arachidonate to proteins was somewhat less than that of palmitate. Arachidonate binding to platelet proteins was not significantly influenced by the inhibition of cyclooxygenase and lipoxygenase. This finding and the high performance liquid chromatography analysis of radiolabeled products removed from proteins by selective cleavage techniques established that arachidonate, and not its metabolic products, was the protein-linked radiolabeled moiety in [3H] arachidonate-labeled platelets. A 7.5-fold higher concentration of unlabeled palmitate competed to a small extent with [3H]arachidonate for protein labeling. Both arachidonate and eicosapentaenoate were bound to proteins almost exclusively through ester linkages. It was further demonstrated that 61 and 66% of total protein-linked arachidonate and eicosapentaenoate, respectively, were bound via thioester bonds. In contrast, 91% of the binding of palmitate to proteins occurred via thioester linkages. As demonstrated by SDS-polyacrylamide gel electrophoresis and fluorography, the patterns of palmitoylated and arachidonoylated proteins were similar but not identical, with selected proteins only palmitoylated or only arachidonoylated. [3H]Eicosapentaenate labeled the same set of proteins as [3H]arachidonate. The fluorographic pattern of 3H-arachidonoylated proteins was not changed by cyclooxygenase and lipoxygenase inhibitors. The binding of a polyunsaturated fatty acid to a protein in place of a saturated fatty acid could significantly influence the hydrophobic interactions of the protein and, thereby, have important functional implications.

Original languageEnglish (US)
Pages (from-to)18243-18248
Number of pages6
JournalJournal of Biological Chemistry
Volume268
Issue number24
StatePublished - Aug 25 1993
Externally publishedYes

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Platelets
Blood Platelets
Proteins
Palmitates
Fatty Acids
Unsaturated Fatty Acids
Labeling
Photofluorography
Lipoxygenase Inhibitors
Lipoxygenase
Cyclooxygenase Inhibitors
High performance liquid chromatography
Post Translational Protein Processing
Prostaglandin-Endoperoxide Synthases
Electrophoresis
Hydrophobic and Hydrophilic Interactions
Polyacrylamide Gel Electrophoresis

ASJC Scopus subject areas

  • Biochemistry

Cite this

Covalent modification of proteins by arachidonate and eicosapentaenoate in platelets. / Muszbek, Laszlo; Laposata, Michael.

In: Journal of Biological Chemistry, Vol. 268, No. 24, 25.08.1993, p. 18243-18248.

Research output: Contribution to journalArticle

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abstract = "The posttranslational modification of proteins by fatty acids has been shown to involve long chain-saturated fatty acids, predominantly palmitate. In the present study, we demonstrated by metabolic labeling of human platelets with [3H]arachidonate and [3H]eicosapentaenoate that these polyunsaturated fatty acids can also become covalently linked to proteins. The extent of binding of arachidonate to proteins was somewhat less than that of palmitate. Arachidonate binding to platelet proteins was not significantly influenced by the inhibition of cyclooxygenase and lipoxygenase. This finding and the high performance liquid chromatography analysis of radiolabeled products removed from proteins by selective cleavage techniques established that arachidonate, and not its metabolic products, was the protein-linked radiolabeled moiety in [3H] arachidonate-labeled platelets. A 7.5-fold higher concentration of unlabeled palmitate competed to a small extent with [3H]arachidonate for protein labeling. Both arachidonate and eicosapentaenoate were bound to proteins almost exclusively through ester linkages. It was further demonstrated that 61 and 66{\%} of total protein-linked arachidonate and eicosapentaenoate, respectively, were bound via thioester bonds. In contrast, 91{\%} of the binding of palmitate to proteins occurred via thioester linkages. As demonstrated by SDS-polyacrylamide gel electrophoresis and fluorography, the patterns of palmitoylated and arachidonoylated proteins were similar but not identical, with selected proteins only palmitoylated or only arachidonoylated. [3H]Eicosapentaenate labeled the same set of proteins as [3H]arachidonate. The fluorographic pattern of 3H-arachidonoylated proteins was not changed by cyclooxygenase and lipoxygenase inhibitors. The binding of a polyunsaturated fatty acid to a protein in place of a saturated fatty acid could significantly influence the hydrophobic interactions of the protein and, thereby, have important functional implications.",
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