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Identification of the Palmitoylation Site in Rat Myelin P 0 Glycoprotein
Author(s) -
Bizzozero Oscar A.,
Fridal Kirsa,
Pastuszyn Andrzej
Publication year - 1994
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1994.62031163.x
Subject(s) - biochemistry , chemistry , cysteine , iodoacetamide , hydroxylamine , residue (chemistry) , thioester , glycoprotein , peptide , enzyme
P 0 glycoprotein, the major protein of PNS myelin, contains approximately 1 mol of covalently bound long‐chain fatty acids. To determine the chemical nature of the fatty acid‐protein linkage, P 0 was labeled in rat sciatic nerve slices with [ 3 H]palmitic acid and subsequently treated with various reagents. The protein‐bound palmi‐tate was released by incubation with the reducing agents dithiothreitol and 2‐mercaptoethanol, and with 1 M hydrox‐ylamine at pH 7.5. In addition, P 0 was deacylated by treatment with 10 m M NaBH 4 with the concomitant production of [ 3 H]hexadecanol, indicating that the fatty acid is bound in a thioester linkage. This conclusion was supported further by the fact that deacylation with hydroxylamine generated free thiol groups, which were titrated with [ 14 C]‐iodoacetamide. To identify the cysteine residue involved in the thioester linkage, [ 14 C]carboxyamidomethylated P 0 was digested with trypsin and the resulting peptides analyzed by reversed‐phase HPLC. Identification of the radioactive protein fragments by amino acid analysis and amino‐terminal peptide sequencing revealed that Cys 153 in rat P 0 glycoprotein is the acylation site. The acylated cysteine is located at the junction of the putative transmem‐brane and cytoplasmic domains. This residue is also present in the P 0 glycoprotein of other species, including human, bovine, mice, and chicken.