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• W2010287574 abstract "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% H218O. The uptake of 18O 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 18O, ending with 2.9% 18O enrichment after 6 h of incubation.18O incorporation into myelin free palmitic acid also increased over the course of the incubation (67.2% 18O enrichment). After correcting for the specific activity of the18O-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. 18O 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. 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% H218O. The uptake of 18O 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 18O, ending with 2.9% 18O enrichment after 6 h of incubation.18O incorporation into myelin free palmitic acid also increased over the course of the incubation (67.2% 18O enrichment). After correcting for the specific activity of the18O-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. 18O 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. 1Towler D.A. Gordon J.I. Glaser L. Annu. Rev. Biochem. 1988; 57: 69-99Crossref PubMed Google Scholar, 2Schultz A.M. Henderson L.E. Oroszlan S. Annu. Rev. Cell Biol. 1988; 4: 611-647Crossref PubMed Scopus (159) Google Scholar, 3Schmidt M.F.G. Biochim. Biophys. Acta. 1989; 988: 411-426Crossref PubMed Scopus (193) Google Scholar, 4Grand R.J.A. Biochem. J. 1989; 258: 625-638Crossref PubMed Scopus (167) Google Scholar, 5Bizzozero O.A. Tetzloff S.U. Bharadwaj M. Neurochem. Res. 1994; 19: 923-933Crossref PubMed Scopus (37) Google Scholar, 6Casey P.J. Science. 1995; 268: 221-225Crossref PubMed Scopus (727) Google Scholar). 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 (7James G. Olson E.N. J. Biol. Chem. 1989; 264: 20998-21006Abstract Full Text PDF PubMed Google Scholar, 8Huang E.M. Biochim. Biophys. Acta. 1989; 1011: 134-139Crossref PubMed Scopus (27) Google Scholar, 9Jochen A. Hays J. Lianos E. Hager S. Biochem. Biophys. Res. Commun. 1991; 177: 797-801Crossref PubMed Scopus (15) Google Scholar) or pharmacological stimuli (10Mouillac B. Caron M. Bonin H. Dennis M. Bouvier M. J. Biol. Chem. 1992; 267: 21733-21737Abstract Full Text PDF PubMed Google Scholar, 11Kennedy M.E. Limbird L.E. J. Biol. Chem. 1994; 269: 31915-31922Abstract Full Text PDF PubMed Google Scholar, 12Robinson L.J. Busconi L. Michel T. J. Biol. Chem. 1995; 270: 995-998Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 13Degtyarev M.Y. Spiegel A.M. Jones T.L.Z. J. Biol. Chem. 1993; 268: 23769-23772Abstract Full Text PDF PubMed Google Scholar, 14Wedegaertner P.B. Bourne H.R. Cell. 1994; 77: 1063-1070Abstract Full Text PDF PubMed Scopus (305) Google Scholar). To date, the metabolic features of palmitoylation have only been studied by labeling cultured cells with [3H]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 [3H]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 experiment could either represent the periodic repair of thioester linkages on a few protein molecules or the physiologically relevant exchange of the fatty acids on many molecules.In the central nervous system, proteolipid protein (PLP) 1The abbreviations used are: PLP, myelin proteolipid protein; GLC, gas-liquid chromatography; FAME, fatty acid methyl ester; MS, mass spectrometry; FFA, free fatty acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine. 1The abbreviations used are: PLP, myelin proteolipid protein; GLC, gas-liquid chromatography; FAME, fatty acid methyl ester; MS, mass spectrometry; FFA, free fatty acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine. accounts for more than 50% of the total myelin protein (15Lees M.B. Macklin W.B. Marangos P.J. Campbell I.C. Neuronal and Glial Proteins: Structure, Function and Clinical Applications. Academic Press, New York1988: 267-294Google Scholar). Although the specific function of this tetraspan membrane protein is unclear, its physiological importance is demonstrated by the requirement of normal PLP synthesis during myelination (16Hudson L.D. Nadon N.L. Martenson R. Myelin: Biology and Chemistry. CRC Press, Boca Raton, FL1992: 677-702Google Scholar). PLP contains between 2 and 3 mol of fatty acids, mainly palmitic, oleic, and stearic acid (17Stoffyn P. Folch J. Biochem. Biophys. Res. Commun. 1971; 44: 157-161Crossref PubMed Scopus (90) Google Scholar), which are bound to several intracellular cysteine residues via labile thioester linkages (18Bizzozero O.A. Good L.K. J. Neurochem. 1990; 55: 1986-1992Crossref PubMed Scopus (18) Google Scholar, 19Bizzozero O.A. Good L.K. Evans J.E. Biochem. Biophys. Res. Commun. 1990; 170: 375-382Crossref PubMed Scopus (23) Google Scholar, 20Weimbs T. Stoffel W. Biochemistry. 1992; 31: 12289-12296Crossref PubMed Scopus (189) Google Scholar). Studies employing [3H]palmitate have shown that the attachment of fatty acid to the polypeptide backbone takes place close to or within the myelin membrane (21Townsend L.E. Agrawal D. Benjamins J.A. Agrawal H.C. J. Biol. Chem. 1982; 257: 9745-9750Abstract Full Text PDF PubMed Google Scholar, 22Bizzozero O.A. Soto E.F. Pasquini J.M. Neurochem. Int. 1983; 5: 729-736Crossref PubMed Scopus (28) Google Scholar). The half-life of the palmitate bound to PLP measured in vivo was found to be approximately 3 days, a value significantly smaller than that of the protein backbone (t½ > 30 days) (23Bizzozero O.A. Good L.K. J. Biol. Chem. 1991; 266: 17092-17098Abstract Full Text PDF PubMed Google Scholar). Moreover, pulse-chase experiments in cell-free systems have shown that PLP-bound palmitate turns over within a few minutes (23Bizzozero O.A. Good L.K. J. Biol. Chem. 1991; 266: 17092-17098Abstract Full Text PDF PubMed Google Scholar). The occurrence of dynamic palmitoylation of PLP in myelin is also supported by the presence of substantial levels of PLP acylesterase activity in this subcellular fraction (24Bizzozero O.A. Leyba J. Nuñez D.J. J. Biol. Chem. 1992; 267: 7886-7894Abstract Full Text PDF PubMed Google Scholar). However, the occurrence of PLP in a metabolically stable membrane such as myelin makes it difficult to envision the function that such a dynamic modification may have, and therefore it raises questions as to how many molecules do indeed participate in deacylation-reacylation cycles.To specifically address the question regarding the number of fatty acids being incorporated into PLP during a period of time, we used the elegant technique of H218O exchange initially developed by Schmid and co-workers (25Kuwae T. Schmid P.C. Schmid H.H.O. Biochem. Biophys. Res. Commun. 1987; 142: 86-91Crossref PubMed Scopus (24) Google Scholar, 26Schmid P.C. Johnson S.B. Schmid H.H.O. Chem. Phys. Lipids. 1988; 46: 165-170Crossref PubMed Scopus (10) Google Scholar, 27Kuwae T. Schmid P.C. Johnson S.B. Schmid H.H.O. J. Biol. Chem. 1990; 265: 5002-5007Abstract Full Text PDF PubMed Google Scholar) to determine phospholipid acyl chain turnover in macrophages. In this study, we incubated rat brain white matter slices in medium containing H218O, a molecule that readily equilibrates into the cells and participates in all normal hydrolytic reactions. As fatty acyl-esters in phospholipids are hydrolyzed in the presence of H218O, the isotopic oxygen becomes either the hydroxyl or the carbonyl oxygen of the resultant free fatty acid (FFA) (Fig. 1). The carbonyl 18O-labeled fatty acids are then activated to fatty acyl-CoA, and reesterified creating phospholipids and acylproteins with the stable isotope incorporated at the carbonyl oxygen of the newly formed oxyester and thioester linkage, respectively. The incorporation of these 18O-labeled fatty acids into PLP and lipids is quantitatively determined via GLC/MS of the methyl esters released by alkaline methanolysis. Using this technique, we found that a significant proportion of the palmitate (7.6%) and stearate (2.5%) in PLP from 20-day-old animals are incorporated during the course of 6 h. These values cannot be attributed to changes in the stoichiometry of acylation or to the acylation of newly synthesized protein, but to the replacement of unlabeled fatty acids for 18O-labeled fatty acids. The value of this approach is also evident when considering that, whereas significant amounts of radioactivity are incorporated into PLP in the adult (60-day-old) animal when [3H]palmitic acid is used as tracer (23Bizzozero O.A. Good L.K. J. Biol. Chem. 1991; 266: 17092-17098Abstract Full Text PDF PubMed Google Scholar), only a minute proportion of PLP molecules are palmitoylated when using endogenously generated 18O-labeled fatty acids. To the best of our knowledge, this is the first time that the metabolism of acyl chains in palmitoylated proteins has been studied with a stable isotope technique.DISCUSSIONIn this study, we have determined the minimal amount of fatty acids that are incorporated into myelin PLP in a particular period of time. These experiments are novel in that they assess the palmitoylation of a protein utilizing the endogenous fatty acids, thus eliminating the problems associated with the entry of the [3H]palmitic acid into cells, its equilibration with specific metabolic pools, and its interconversion into other radiolabeled metabolites. We have found that an amount close to 3% of palmitate and 1% of the stearate bound to PLP is incorporated in 6 h and 4 h, respectively. These are minimal rates of incorporation since they do not include adjustments for the specific activity of the donor fatty acid pool. When these values are corrected by changes in the specific activity of the myelin FFAs, the incorporation of palmitic and stearic acid into PLP increased to approximately 7.6% and 2.6%, respectively. Since PLP has six potential acylation sites (20Weimbs T. Stoffel W. Biochemistry. 1992; 31: 12289-12296Crossref PubMed Scopus (189) Google Scholar), the amount of fatty acids incorporated into the protein cannot be equated to the number of protein molecules that are being acylated. Consequently, it was not possible to distinguish whether only 7.6% of PLP molecules exchange all of their palmitate or whether a larger proportion of the molecules becomes palmitoylated to a lesser extent.Both this study and those using [3H]palmitic acid (21Townsend L.E. Agrawal D. Benjamins J.A. Agrawal H.C. J. Biol. Chem. 1982; 257: 9745-9750Abstract Full Text PDF PubMed Google Scholar,22Bizzozero O.A. Soto E.F. Pasquini J.M. Neurochem. Int. 1983; 5: 729-736Crossref PubMed Scopus (28) Google Scholar) have shown that acylation of myelin PLP is not affected by cycloheximide. The persistence of normal levels of palmitate incorporation long after the arrest of the protein synthesis can be attributed to: (a) palmitoylation occurring at a site temporally distant from the rough endoplasmic reticulum of the oligodendroglial cell, where PLP synthesis takes place; (b) an increase in the stoichiometry of acylation; or (c) fatty acid turnover. The first of these possibilities is rather unlikely since the rate of PLP acylation with 18O-labeled fatty acids exceeds by at least 5-fold the rate at which the newly synthesized protein molecules are accumulated into myelin. The second alternative can also be ruled out because the amount of fatty acids covalently bound to PLP remains unchanged during the incubation and throughout development. Thus, the data can solely be explained by exchange of unlabeled fatty acids for 18O-fatty acids. Interestingly, the amount of [18O]palmitic acid incorporated into PLP did not decrease upon replacement of H218O by H216O, indicating that the half-life of the protein-bound palmitate is greater than that of free palmitic acid (t½ ∼ 2 h). Because of this relatively low turnover rate, incubation for 6 h was insufficient to achieve isotopic equilibrium between the PLP-bound palmitic acid and the myelin free [18O]palmitic acid. However, the rate of uptake of 18O into PLP-derived palmitic acid showed a gradual decrease during the course of the incubation. Assuming that this decline was caused by the approach to the equilibrium, it is possible to calculate both the maximal number of fatty acids that can be incorporated and the half-life of the protein-bound palmitic acid. When the corrected 18O incorporation values shown in Fig. 2 C were fitted to a first-rate equation, the curve reached a limit at 12–14%18O enrichment with a half-life of 4–5 h.Profound differences in the incorporation of 18O-labeled fatty acids into PLP were observed between young and adult animals. The % 18O enrichment in palmitic and stearic acid derived from PLP was greatly diminished in the older, slowly myelinating animals. This reduction is too large to be only explained by the 3-fold increase in the concentration of PLP, and therefore protein-derived16O-fatty acids, that occurs between 20 and 60 days of age (40Agrawal H.C. Fujimoto K. Burton R.M. Biochem. J. 1976; 154: 265-269Crossref PubMed Scopus (12) Google Scholar). Consequently, the age-associated changes are, to a large extent, due to a reduction in the number of PLP molecules undergoing palmitoylation with age. The limited incorporation of [18O]palmitic acid into PLP in the older animal was an unexpected finding since there are no noticeable developmental differences in acylation of PLP with [3H]palmitic acid (23Bizzozero O.A. Good L.K. J. Biol. Chem. 1991; 266: 17092-17098Abstract Full Text PDF PubMed Google Scholar). One possibility is that the presence of exogenously added palmitic acid may have influenced the normal fatty acid metabolism. However, we found that the addition of [18O]palmitic acid to an incubation medium containing 50% H218O does not change the % 18O enrichment of PLP-derived palmitic acid (data not shown). This result also suggests that the radioactivity normally incorporated into PLP in the adult, slowly myelinating animal represents almost negligible amounts of palmitate. In light of this new finding and contrary to our original view (23Bizzozero O.A. Good L.K. J. Biol. Chem. 1991; 266: 17092-17098Abstract Full Text PDF PubMed Google Scholar), it is fair to hypothesize that PLP palmitoylation plays some role in myelin formation and/or compaction rather than in the maintenance of this membrane.Little is known regarding the subcellular site of protein palmitoylation, and it appears to depend upon the protein in question. Initial studies have shown that acylation of viral and cellular membrane glycoproteins occurs in membranes from the endoplasmic reticulum/Golgi complex (41Schmidt M.F.G. Schlesinger M.J. J. Biol. Chem. 1980; 255: 3334-3339Abstract Full Text PDF PubMed Google Scholar, 42Dolci E.D. Palade G.E. J. Biol. Chem. 1985; 260: 10728-10735Abstract Full Text PDF PubMed Google Scholar, 43Schmidt J.W. Catterall W.A. J. Biol. Chem. 1987; 262: 13713-13723Abstract Full Text PDF PubMed Google Scholar, 44Berger M. Schmidt M.F.G. FEBS Lett. 1985; 187: 289-294Crossref PubMed Scopus (39) Google Scholar). However, for proteins that participate in rapid deacylation-reacylation cycles, the attachment of the fatty acid is likely to take place at the plasma membrane, where the presence of protein acyltransferase activity has been recently demonstrated (45Kinet J.P. Quatro R. Perez-Montfort R. Metzger H. Biochemistry. 1985; 24: 7342-7348Crossref PubMed Scopus (24) Google Scholar, 46Staufenbiel M. Mol. Cell Biol. 1987; 7: 2981-2984Crossref PubMed Scopus (39) Google Scholar, 47Dunphy J.T. Greentree W.K. Manahan C.L. Linder M.E. J. Biol. Chem. 1996; 271: 7154-7159Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 48Berthiaume L. Resh M.D. J. Biol. Chem. 1995; 270: 22399-22405Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 49Schroeder H. Leventis R. Shahinian S. Walton P. Silvius J.R. J. Cell Biol. 1996; 134: 647-660Crossref PubMed Scopus (66) Google Scholar, 50Liu L. Dudler T. Gelb M.H. J. Biol. Chem. 1996; 271: 23269-23276Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). In the case of PLP, studies involving biosynthetic labeling with [3H]palmitic acid and cellular fractionation have suggested that acylation occurs at a locus close to or within the myelin membrane (22Bizzozero O.A. Soto E.F. Pasquini J.M. Neurochem. Int. 1983; 5: 729-736Crossref PubMed Scopus (28) Google Scholar, 23Bizzozero O.A. Good L.K. J. Biol. Chem. 1991; 266: 17092-17098Abstract Full Text PDF PubMed Google Scholar). Both the dynamic nature of PLP acylation and the finding that palmitoylation is reduced in adult animals suggest that the reaction is likely to occur in specialized regions of the myelin sheath, such Schmidt-Lantermann incisures; inner, outer, and paranodal loops; and the network of cytoplasmic channels, where PLP may be accessible to the acylating/deacylating machinery as well as fatty acyl-CoA. Experiments combining 18O labeling and fractionation of the myelin membranes are being undertaken to localize more precisely the subcompartment(s) where acylation takes place.In this study, we also showed that a significant proportion of the acyl chains in myelin phospholipids acquire 18O during the course of the incubation. No attempts were made to determine whether the uptake of 18O into phospholipid-derived fatty acids takes place by the de novo synthesis through the acylation of glycerophosphate and/or via deacylation-reacylation reactions. However, the high levels of isotope incorporation relative to the proportion of membrane lipids synthesized during incubation suggests that the exchange of fatty acids on preexisting phospholipid molecules constitutes a major mechanism. In general, 18O enrichment profiles of PLP-fatty acids resemble those of the major myelin phospholipids. In both cases, the incorporation of 18O was time-dependent, unaffected by cycloheximide, and greatly reduced in the adult animal. At present, the reason(s) for the similarities in the metabolic behavior of PLP and phospholipid acyl chains are unknown. The possibility, however, that phospholipids could be contaminating the PLP preparations can be safely excluded based on the following observations. (a) Addition of tritiated phospholipids, glycolipids, or palmitic acid to unlabeled PLP prior to chromatography on Sephadex LH-60 does not result in the appearance of radioactivity in the protein peak (30Bizzozero O.A. Besio-Moreno M. Pasquini J.M. Soto E.F. Gomez C.J. J. Chromatogr. 1982; 227: 33-44Crossref PubMed Scopus (63) Google Scholar); (b) chemical analysis of isolated PLP yields less than 0.035% (w/w) lipid phosphorous (30Bizzozero O.A. Besio-Moreno M. Pasquini J.M. Soto E.F. Gomez C.J. J. Chromatogr. 1982; 227: 33-44Crossref PubMed Scopus (63) Google Scholar); (c) incubation of isolated PLP with phospholipase A2 does not remove the protein-bound fatty acid (24Bizzozero O.A. Leyba J. Nuñez D.J. J. Biol. Chem. 1992; 267: 7886-7894Abstract Full Text PDF PubMed Google Scholar); and (d) the fatty acid composition of PLP does not resemble that of any myelin lipids (34Lees M.B. Bizzozero O.A. Odykirk T. McGarry J.F. J. Neurochem. 1989; 52: 195Google Scholar).As mentioned above, a benefit of the H218O method developed by Schmid and co-workers (25Kuwae T. Schmid P.C. Schmid H.H.O. Biochem. Biophys. Res. Commun. 1987; 142: 86-91Crossref PubMed Scopus (24) Google Scholar, 26Schmid P.C. Johnson S.B. Schmid H.H.O. Chem. Phys. Lipids. 1988; 46: 165-170Crossref PubMed Scopus (10) Google Scholar, 27Kuwae T. Schmid P.C. Johnson S.B. Schmid H.H.O. J. Biol. Chem. 1990; 265: 5002-5007Abstract Full Text PDF PubMed Google Scholar) is that it eliminates the problems associated with the slow entry of [3H]palmitic acid into cells and its equilibration with specific metabolic pools. This becomes evident when considering recent findings revealing that some fatty acid stores in the cell are resistant to labeling upon incubation with radiolabeled fatty acids (51Chilton F.H. Connell T.R. J. Biol. Chem. 1988; 263: 5260-5265Abstract Full Text PDF PubMed Google Scholar). However, in our opinion, the major advantage of this stable isotope technique is that it allows one to estimate the minimal number of molecules being modified during the course of an experiment. Knowledge of acylation rates and the proportion of protein molecules that undergo palmitoylation is critical when making biological assumptions regarding the function of the modification. Consequently, it would be of considerable interest to extend this approach to study the palmitoylation of other proteins. Although the use of H218O to conduct a systematic study of protein palmitoylation is somewhat limited by the necessity for substantial amounts of purified protein (nanomoles), with the advent of effective expression systems in eukaryotic cells, heavy isotope labeling should become increasingly feasible. 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. 1Towler D.A. Gordon J.I. Glaser L. Annu. Rev. Biochem. 1988; 57: 69-99Crossref PubMed Google Scholar, 2Schultz A.M. Henderson L.E. Oroszlan S. Annu. Rev. Cell Biol. 1988; 4: 611-647Crossref PubMed Scopus (159) Google Scholar, 3Schmidt M.F.G. Biochim. Biophys. Acta. 1989; 988: 411-426Crossref PubMed Scopus (193) Google Scholar, 4Grand R.J.A. Biochem. J. 1989; 258: 625-638Crossref PubMed Scopus (167) Google Scholar, 5Bizzozero O.A. Tetzloff S.U. Bharadwaj M. Neurochem. Res. 1994; 19: 923-933Crossref PubMed Scopus (37) Google Scholar, 6Casey P.J. Science. 1995; 268: 221-225Crossref PubMed Scopus (727) Google Scholar). 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 (7James G. Olson E.N. J. Biol. Chem. 1989; 264: 20998-21006Abstract Full Text PDF PubMed Google Scholar, 8Huang E.M. Biochim. Biophys. Acta. 1989; 1011: 134-139Crossref PubMed Scopus (27) Google Scholar, 9Jochen A. Hays J. Lianos E. Hager S. Biochem. Biophys. Res. Commun. 1991; 177: 797-801Crossref PubMed Scopus (15) Google Scholar) or pharmacological stimuli (10Mouillac B. Caron M. Bonin H. Dennis M. Bouvier M. J. Biol. Chem. 1992; 267: 21733-21737Abstract Full Text PDF PubMed Google Scholar, 11Kennedy M.E. Limbird L.E. J. Biol. Chem. 1994; 269: 31915-31922Abstract Full Text PDF PubMed Google Scholar, 12Robinson L.J. Busconi L. Michel T. J. Biol. Chem. 1995; 270: 995-998Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 13Degtyarev M.Y. Spiegel A.M. Jones T.L.Z. J. Biol. Chem. 1993; 268: 23769-23772Abstract Full Text PDF PubMed Google Scholar, 14Wedegaertner P.B. Bourne H.R. Cell. 1994; 77: 1063-1070Abstract Full Text PDF PubMed Scopus (305) Google Scholar). To date, the metabolic features of palmitoylation have only been studied by labeling cultured cells with [3H]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 [3H]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 experiment could either represent the periodic repair of thioester linkages on a few protein molecules or the physiologically relevant exchange of the fatty acids on many molecules. In the central nervous system, proteolipid protein (PLP) 1The abbreviations used are: PLP, myelin proteolipid protein; GLC, gas-liquid chromatography; FAME, fatty acid methyl ester; MS, mass spectrometry; FFA, free fatty acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine. 1The abbreviations used are: PLP, myelin proteolipid protein; GLC, gas-liquid chromatography; FAME, fatty acid methyl ester; MS, mass spectrometry; FFA, free fatty acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine. accounts for more than 50% of the total myelin protein (15Lees M.B. Macklin W.B. Marangos P.J. Campbell I.C. Neuronal and Glial Proteins: Structure, Function and Clinical Applications. Academic Press, New York1988: 267-294Google Scholar). Although the specific function of this tetraspan membrane protein is unclear, its physiological importance is demonstrated by the requirement of normal PLP synthesis during myelination (16Hudson L.D. Nadon N.L. Martenson R. Myelin: Biology and Chemistry. CRC Press, Boca Raton, FL1992: 677-702Google Scholar). PLP contains between 2 and 3 mol of fatty acids, mainly palmitic, oleic, and stearic acid (17Stoffyn P. Folch J. Biochem. Biophys. Res. Commun. 1971; 44: 157-161Crossref PubMed Scopus (90) Google Scholar), which are bound to several intracellular cysteine residues via labile thioester linkages (18Bizzozero O.A. Good L.K. J. Neurochem. 1990; 55: 1986-1992Crossref PubMed Scopus (18) Google Scholar, 19Bizzozero O.A. Good L.K. Evans J.E. Biochem. Biophys. Res. Commun. 1990; 170: 375-382Crossref PubMed Scopus (23) Google Scholar, 20Weimbs T. Stoffel W. Biochemistry. 1992; 31: 12289-12296Crossref PubMed Scopus (189) Google Scholar). Studies employing [3H]palmitate have shown that the attachment of fatty acid to the polypeptide backbone takes place close to or within the myelin membrane (21Townsend L.E. Agrawal D. Benjamins J.A. Agrawal H.C. J. Biol. Chem. 1982; 257: 9745-9750Abstract Full Text PDF PubMed Google Scholar, 22Bizzozero O.A. Soto E.F. Pasquini J.M. Neurochem. Int. 1983; 5: 729-736Crossref PubMed Scopus (28) Google Scholar). The half-life of the palmitate bound to PLP measured in vivo was found to be approximately 3 days, a value significantly smaller than that of the protein backbone (t½ > 30 days) (23Bizzozero O.A. Good L.K. J. Biol. Chem. 1991; 266: 17092-17098Abstract Full Text PDF PubMed Google Scholar). Moreover, pulse-chase experiments in cell-free systems have shown that PLP-bound palmitate turns over within a few minutes (23Bizzozero O.A. Good L.K. J. Biol. Chem. 1991; 26" @default.
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• W2010287574 creator A5030241928 @default.
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• W2010287574 date "1998-01-01" @default.
• W2010287574 modified "2023-10-11" @default.
• W2010287574 title "Palmitoylation of Proteolipid Protein from Rat Brain Myelin Using Endogenously Generated 18O-Fatty Acids" @default.
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