Tightly (covalently) bound fatty acids in cell membrane proteins

Abstract

We find tightly (covalently) bound fatty acids in membrane proteins from human red cells and polymorphonuclear cells and several tissues of the rat. These fatty acids are not removed by exhaustive extraction with organic solvents, by phospholipase A 2 treatment, or by sodium dodecyl sulfate (SDS). The fatty acids are released by refluxing with hot methanolic-HCl. They account for 0.2–6.8% of the total fatty acids. 50–60% of the tightly bound fatty acids are removed by mild alkaline hydrolysis. [ 3H]Palmitic acid is incorporated into lipids and proteins of the red cell membrane. In red cell ghosts incorporation is stimulated by ATP and CoA but maximal incorporation is achieved with [ 14C]palmityl-CoA. The labeling of lipids and proteins by [ 14C]palmityl-CoA is greater in ghosts than in cells, however basal incorporation of [ 3H]palmitic acid into lipids and proteins is greater in cells than in ghosts. [ 3H]Palmitic acid is also incorporated into proteins of polymorphonuclear cells. The incorporation in polymorphonuclear cell proteins is inhibited by cycloheximide but not by actinomycin D. Labeled stearic, linoleic, linoleic, and palmitic acid are incorporated into red cell membrane proteins. These incorporated fatty acids are not removed by exhaustive solvent extraction, by prolonged dialysis or by SDS. On SDS-polyacrylamide gel electrophoresis several protein bands are labeled by all the fatty acids although each fatty acid gives a different pattern of labeling. In all cases, the major protein band which is labeled stains very weakly with Coomassie blue and runs between band 4.2 and 5. Other proteins which have appreciable label are bands 1, 2 and 3. We postulate that fatty acids are covalently attached to certain proteins as a post-translational event and act to direct, insert, and anchor the proteins in cell membranes. A similar postulate has been enunciated by Schmidt and Schlessinger (Cell 17, 813–819 (1979)).