2',3'-cyclic nucleotide-3'-phosphodiesterase in the central nervous system is fatty-acylated by thioester linkage.
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.
Agrawal
H C
HC
Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110.
Sprinkle
T J
TJ
Agrawal
D
D
D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D000215:Acylation; D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal; D001921:Brain / Q000201:enzymology*; D003078:Colchicine / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004952:Esters; D006801:Humans; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D007700:Kinetics; D008985:Monensin / Q000494:pharmacology; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism*; D051381:Rats
D000906:Antibodies; D000911:Antibodies, Monoclonal; D004952:Esters; D006898:Hydroxylamines; D010169:Palmitic Acids; D019811:Hydroxylamine; D019308:Palmitic Acid; D008985:Monensin; D003513:Cycloheximide; D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases; D003078:Colchicine