Proteolipid protein (PLP), the major protein of central nervous system myelin, contains covalently bound fatty acids, predominantly palmitic acid. This study adapts a stable isotope technique (Kuwae, T., Schmid, P. C., Johnson, S. B., and Schmid, H. O. (1990) J. Biol. Chem. 265, 5002-5007) to quantitatively determine the minimal proportion of PLP molecules which undergo palmitoylation. In these experiments, brain white matter slices from 20-day-old rats were incubated for up to 6 h in a physiological buffer containing 50% H 2 18 O. The uptake of 18 O into the carbonyl groups of fatty acids derived from PLP, phospholipids, and the free fatty acid pool was measured by gas-liquid chromatography/mass spectrometry of the respective methyl esters. Palmitic acid derived from PLP acquired increasing amounts of 18 O, ending with 2.9%18 O enrichment after 6 h of incubation.18 O incorporation into myelin free palmitic acid also increased over the course of the incubation (67.2% 18 O enrichment). After correcting for the specific activity of the 18 O-enriched free palmitic acid pool, 7.6% of the PLP molecules were found to acquire palmitic acid in 6 h. This value is not only too large to be the result of the palmitoylation of newly synthesized PLP molecules, it was also unchanged upon the inhibition of protein synthesis with cycloheximide.18 O enrichment in less actively myelinating 60-day-old rats was significantly reduced. In conclusion, our experiments suggest that a substantial proportion of PLP molecules acquire palmitic acid via an acylation/deacylation cycle and that this profile changes during development.A number of integral membrane proteins are modified after their synthesis by the covalent attachment of long-chain fatty acids (mostly palmitic acid) to one or more cytoplasmically oriented cysteine residues (for review, see Refs. 1-6). In the majority of the cases, the chemically bound acyl chains turn over much faster than the protein backbone, implying that palmitoylation is a regulatory modification. In fact, fatty acylation of this and other types of proteins has been shown to be modulated by physiological (7-9) or pharmacological stimuli (10 -14). To date, the metabolic features of palmitoylation have only been studied by labeling cultured cells with [ 3 H]palmitic acid, and the half-life of the palmitate has been estimated from the disappearance of the protein-bound radioactivity after isotopic dilution with the unlabeled fatty acid. Unfortunately, labeling experiments using [ 3 H]palmitic acid are difficult to interpret, particularly when considering the possibility that exogenous and endogenous palmitate may not have equal access to the fatty acid donor pools. Furthermore, since the specific radioactivity of the donor pool of palmitate used for protein palmitoylation cannot be estimated, it is not possible to determine the number of protein molecules participating in such rapid deacylation-reacylation cycles. Consequently, the radioactivity that becomes associated with a polypeptide during the course of an...