Search

2',3'-Cyclic nucleotide-3'-phosphodiesterase (CNP1 and CNP2 with Mr of 46,000 and 48,000, respectively) is the major enzyme of central nervous system myelin. It is associated with oligodendroglial plasma membrane and uncompacted myelin (myelin-like fraction), which are in contact with glial cytoplasm. Proteins of the myelin-like fraction were labeled with [3H]palmitic acid in brain slices from 17-day-old rats and immunoprecipitated with anti-CNP antiserum. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of immunoprecipitated material revealed intense acylation of CNP1 and CNP2, and radioactivity was released by hydroxylamine. Palmitic acid was covalently bound to CNP because radioactivity was not removed by extraction of immunoprecipitated CNP with organic solvent or by boiling in sodium dodecyl sulfate and dithiothreitol. However, treatment of immunoprecipitated CNP with (a) hydroxylamine-released palmitohydroxamate and palmitic acid, (b) sodium borohydride-released hexadecanol, and (c) methanolic-KOH-released methyl palmitate. Synthesis, acylation, or transport of CNP was not affected by monensin or colchicine. However, acylation of CNP was inhibited 24-32% by cycloheximide. These results provide conclusive evidence that CNP1 and CNP2 are fatty acid acylated with palmitate through a thioester linkage and is posttranslationally modified sometime after synthesis.

Examination of brain myelin proteins by sodium dodecyl sulfate-gel electrophoresis followed by fluorography clearly showed that both proteolipid protein (PLP) and DM-20 were acylated 24 h after the intracerebral injection of 30-day-old rats with [3H]palmitic acid. The radioactivity associated with PLP remained after purification, re-electrophoresis, and fluorography. Most of the radioactivity associated with PLP was removed when the gels were treated with hydroxylamine and then fluorographed, indicating that fatty acids were bound to PLP by ester linkage. Cleavage of purified PLP with methanolic sodium hydroxide readily released almost all protein-bound radioactivity. Thin layer chromatography of this material on both silver nitrate and reverse-phase plates provided evidence that most of the radioactivity co-migrated with methyl palmitate (77%) and methyl stearate (19%); however, some radioactivity was associated with methyl oleate (4%). Gas-liquid chromatography of the fatty acids associated with PLP distinctly revealed the presence of methyl palmitate and a detectable peak of methyl stearate.

Examination of 15-day-old rabbit brain myelin proteins by sodium dodecyl sulfate-slab gel electrophoresis revealed the presence of a basic protein with a molecular weight of 21,000 (21K protein) which was not previously reported in this species. This protein exhibited characteristic bluish green color with amido black and gave an amino acid composition strikingly similar to large basic protein (LBP). It formed a line of identity with LBP when diffused against antiserum to chicken brain basic protein. The concentration of LBP (18.9 micrograms/mg of dry myelin) is 6-fold greater than that of the 21K protein(3.31 micrograms/mg of dry myelin) in rabbit brain myelin. After the intracerebral injection of [32P]orthophosphoric acid, both LBP and 21K protein were found to be phosphorylated. [32P]Phosphate in the purified preparations of these proteins was covalently linked by phosphoester bonds to serine and threonine residues. The specific radioactivity of the 21K protein (84,693 cpm/mg of protein) was not significantly higher than LBP (69,797 cpm/mg of protein).

The incorporation of fatty acid into myelin proteolipid protein (PLP) was studied by incubating brain slices from 17-day-old rats with [9,10-3H]palmitic acid. Myelin proteins were analyzed by gel electrophoresis. As in vivo (Agrawal, H. C., Randle, C. L., and Agrawal, D. (1982) J. Biol. Chem. 257, 4588-4592) most of the radioactivity co-migrated with PLP, with radioactivity also appearing in the regions of the DM-20 and Wolfgram proteins. Treatment of myelin proteins or purified PLP with 1 M hydroxylamine released most of the radioactivity, indicating that [3H]palmitic acid was covalently bound by ester linkage to the proteins. Cleavage of PLP with methanolic NaOH and examination of the released fatty acids by thin layer chromatography showed radioactivity associated with the methyl esters of palmitate, stearate, and oleate (76, 23, and 1%, respectively). A linear increase in radioactivity from [3H]palmitic acid was observed in PLP in both myelin and myelin-like fractions, while appearance of [14C]glycine-labeled PLP showed a lag of 45-60 minutes. These results indicate that lipid is added to PLP in the myelin and myelin-like membranes, or immediately prior to the appearance of PLP in these fractions. For both precursors, the specific activity of PLP was greater in the myelin-like fraction than in myelin, indicating that the myelin-like fraction may contain a precursor pool of PLP for myelin.

Separation of rat sciatic nerve myelin proteins by sodium dodecyl sulfate-slab gel electrophoresis 18 h after injection of [3H]palmitic acid into the nerve demonstrated acylation of the PO protein. When sciatic nerve myelin labeled with [3H]palmitic acid was extracted with acidified chloroform/methanol or chloroform/methanol, the radioactivity associated with PO was retained. These results provided evidence that the radioactivity derived from [3H]palmitic acid in PO protein was not due to labeled phospholipid bound to PO protein by strong physical interaction. Furthermore, the radioactivity associated with purified preparations of PO remained after dialysis and re-electrophoresis, providing additional evidence that [3H]palmitic acid was firmly bound to PO. Treatment of myelin proteins with hydroxylamine at pH 6.6 released most of the radioactivity, indicating that [3H]palmitic acid was covalently bound by ester linkage to PO. Cleavage of purified acylated PO with methanolic NaOH released 85% of the protein-bound radioactivity. Gas-liquid chromatography of the fatty acids released from PO showed labeling of methyl esters of palmitate, stearate, and oleate (56, 16, and 5%, respectively). In addition, a small unlabeled peak with retention time identical with methyl linoleate was also observed. Intraneural injection of a mixture of [3H]palmitic acid and [14C]fucose or [3H]palmitic acid and [35S]sulfate in vivo followed by disc gel electrophoresis and determination of incorporated radioactivity clearly showed that PO was glycosylated, sulfated, and acylated. The potential significance of fatty acids linked to PO is discussed.