Muszbek L, Laposata M
Department of Pathology, Massachusetts General Hospital, Boston 02114.
J Biol Chem. 1993 Aug 25;268(24):18243-8.
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]Eicosapentaenoate 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.
脂肪酸对蛋白质的翻译后修饰已被证明涉及长链饱和脂肪酸，主要是棕榈酸。在本研究中，我们通过用[3H]花生四烯酸和[3H]二十碳五烯酸对人血小板进行代谢标记证明，这些多不饱和脂肪酸也可以与蛋白质共价连接。花生四烯酸与蛋白质的结合程度略低于棕榈酸。花生四烯酸与血小板蛋白质的结合不受环氧化酶和脂氧合酶抑制的显著影响。这一发现以及通过选择性裂解技术从蛋白质中去除的放射性标记产物的高效液相色谱分析表明，花生四烯酸而非其代谢产物是[3H]花生四烯酸标记的血小板中与蛋白质相连的放射性标记部分。7.5倍高浓度的未标记棕榈酸在一定程度上与[3H]花生四烯酸竞争蛋白质标记。花生四烯酸和二十碳五烯酸几乎完全通过酯键与蛋白质结合。进一步证明，与蛋白质结合的总花生四烯酸和二十碳五烯酸中，分别有61%和66%是通过硫酯键结合的。相比之下，棕榈酸与蛋白质结合的91%是通过硫酯键连接的。如SDS-聚丙烯酰胺凝胶电泳和荧光自显影所示，棕榈酰化和花生四烯酰化蛋白质的模式相似但不完全相同，有些蛋白质仅被棕榈酰化或仅被花生四烯酰化。[3H]二十碳五烯酸标记的蛋白质与[3H]花生四烯酸标记的是同一组蛋白质。环氧化酶和脂氧合酶抑制剂不会改变3H-花生四烯酰化蛋白质的荧光自显影模式。多不饱和脂肪酸取代饱和脂肪酸与蛋白质的结合可能会显著影响蛋白质的疏水相互作用，从而具有重要的功能意义。
