COVALENT MODIFICATION OF PROTEINS BY ARACHIDONATE AND EICOSAPENTAENOATE IN PLATELETS

The posttranslational modification of proteins by fatty acids has been shown to involve long chain-saturated fatty acids, predominantly palm itate. In the present study, we demonstrated by metabolic labeling of human platelets with [H-3]arachidonate and [H-3]eicosapentaenoate that these polyunsaturated fatty acids can also become covalently linked t o proteins. The extent of binding of arachidonate to proteins was some what less than that of palmitate. Arachidonate binding to platelet pro teins was not significantly influenced by the inhibition of cyclooxyge nase and lipoxygenase. This finding and the high performance liquid ch romatography analysis of radiolabeled products removed from proteins b y selective cleavage techniques established that arachidonate, and not its metabolic products, was the protein-linked radiolabeled moiety in [H-3] arachidonate-labeled platelets. A 7.5-fold higher concentration of unlabeled palmitate competed to a small extent with [H-3]arachidon ate for protein labeling. Both arachidonate and eicosapentaenoate were bound to proteins almost exclusively through ester linkages. It was f urther demonstrated that 61 and 66% of total protein-linked arachidona te and eicosapentaenoate, respectively, were bound via thioester bonds . In contrast, 91% of the binding of palmitate to proteins occurred vi a thioester linkages. As demonstrated by SDS-polyacrylamide gel electr ophoresis and fluorography, the patterns of palmitoylated and arachido noylated proteins were similar but not identical, with selected protei ns only palmitoylated or only arachidonoylated. [H-3]Eicosapentaenate labeled the same set of proteins as [H-3]arachidonate. The fluorograph ic pattern of H-3-arachidonoylated proteins was not changed by cycloox ygenase and lipoxygenase inhibitors. The binding of a polyunsaturated fatty acid to a protein in place of a saturated fatty acid could signi ficantly influence the hydrophobic interactions of the protein and, th ereby, have important functional implications.