Cell-free acylation of rat brain myelin proteolipid protein and DM-20.
Incubation of rat brain myelin with [3H]palmitic acid in the presence of ATP, CoA and MgCl2 or [14C]-palmitoyl-CoA in a cell-free system resulted in the selective labelling of 'PLP' [proteolipid protein; Folch & Lees (1951) J. Biol. Chem. 191, 807-817] and 'DM-20' [Agrawal, Burton, Fishman, Mitchell & Prensky (1972) J. Neurochem. 19, 2083-2089] which, after polyacrylamide-gel electrophoresis in SDS, were revealed by fluorography. These results provide evidence of the association of fatty acid-CoA ligase and acyltransferase in isolated myelin. Palmitic acid is covalently bound to PLP and DM-20, because 70 and 92% of the radioactivity was removed from proteolipid proteins after treatment with hydroxylamine and methanolic NaOH respectively. Incubation of myelin with [3H]palmitic acid in the absence of ATP, CoA, MgCl2, or all three, decreased incorporation of fatty acid into PLP to 3, 55, 18 and 2% respectively. The cell-free system exhibits specificity with respect to the chain length of the fatty acids, since myristic acid is incorporated into PLP at a lower rate when compared with palmitic and oleic acids. The acylation of PLP is an enzymic reaction, since (1) maximum incorporation of [3H]palmitic acid into PLP occurred at physiological temperatures and decreased with an increase in the temperature; (2) acylation of PLP with [3H]palmitic acid and [14C]palmitoyl-CoA was severely inhibited by SDS (0.05%); and (3) the incorporation of fatty acid and palmitoyl-CoA into PLP was substantially decreased by the process of freezing-thawing and freeze-drying of myelin. We have provided evidence that all of the enzymes required for acylation of PLP and DM-20 are present in isolated rat brain myelin. Acylation of PLP in a cell-free system with fatty acids and palmitoyl-CoA suggests that a presynthesized pool of non-acylated PLP and DM-20 is available for acylation.
D000215:Acylation; D000217:Acyltransferases / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002474:Cell-Free System / Q000187:drug effects; D002850:Chromatography, Gel; D003066:Coenzyme A Ligases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005227:Fatty Acids / Q000378:metabolism; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D009185:Myelin Proteins / Q000378:metabolism*; D018991:Myelin Proteolipid Protein; D009419:Nerve Tissue Proteins; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism; D010171:Palmitoyl Coenzyme A / Q000378:metabolism; D011510:Proteolipids / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D000098859:Long-Chain-Fatty-Acid-CoA Ligase
D005227:Fatty Acids; D006898:Hydroxylamines; D009185:Myelin Proteins; D018991:Myelin Proteolipid Protein; D009419:Nerve Tissue Proteins; D010169:Palmitic Acids; C489071:Plp1 protein, rat; D011510:Proteolipids; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D010171:Palmitoyl Coenzyme A; D019811:Hydroxylamine; D019308:Palmitic Acid; D000217:Acyltransferases; D003066:Coenzyme A Ligases; C090799:FAA2 protein, S cerevisiae; D000098859:Long-Chain-Fatty-Acid-CoA Ligase