Palmitoylation of proteolipid protein from rat brain myelin using endogenously generated 18O-fatty acids.
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 the 18O-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.
Tetzloff
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Department of Cell Biology and Physiology, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131-5218, USA.
Bizzozero
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D000818:Animals; D001921:Brain / Q000378:metabolism*; D005227:Fatty Acids / Q000096:biosynthesis / Q000378:metabolism*; D066298:In Vitro Techniques; D018991:Myelin Proteolipid Protein / Q000378:metabolism*; D010103:Oxygen Isotopes; D019308:Palmitic Acid / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
D005227:Fatty Acids; D018991:Myelin Proteolipid Protein; D010103:Oxygen Isotopes; D010743:Phospholipids; D019308:Palmitic Acid