Nature of the cysteinyl residues in lipophilin from human myelin.
Ellman's reagent was used to investigate the status and exposure of the cysteinyl residues in lipophilin, a proteolipid apoprotein from human myelin. The hydrolyzed protein contained 3.5 to 4.5 cysteines per molecule, which increased to 11 after complete reduction. The native protein was thought to contain three disulfide bonds and five free sulfhydryl groups, which undergo partial oxidation during purification. Exposure of -SH groups in the aqueous protein was minimal, even in the presence of 6 M guanidinium chloride, suggesting a location in hydrophobic domains not disrupted by this reagent. In the helicogenic solvent 2-chloroethanol, the full complement of -SH groups could not be revealed, even with the addition of sodium dodecyl sulfate; a difference of two sulfhydryls between intact and hydrolyzed protein was consistently observed. Similar sulfhydryl reactivity toward iodoacetamide was also established in this solvent. Sulfhydryl assays on whole myelin in 2-chloroethanol indicated that the occurrence of -SH groups in the proteolipid component was at least as high as in the purified apoprotein. Lipophilin was reduced and alkylated with 4-vinylpyridine at 10 of its cysteinyl residues. The modified protein adopted a beta structure under conditions where lipophilin is normally highly alpha-helical, and was also less helical than usual in 2-chloroethanol; however, it was still abnormally resistant to denaturation by guanidinium chloride. Modified lipophilin contained as many ester groups as the intact protein; thus, it appeared unlikely that the long chain fatty acids associated with the protein were attached to cysteine residues.
D000109:Acetylcholine; D000122:Acetylthiocholine; D001923:Brain Chemistry; D002942:Circular Dichroism; D003553:Cystine / Q000032:analysis*; D004228:Dithionitrobenzoic Acid; D006801:Humans; D006898:Hydroxylamines; D008970:Molecular Weight; D009103:Multiple Sclerosis / Q000378:metabolism; D009185:Myelin Proteins / Q000032:analysis*; D009186:Myelin Sheath / Q000032:analysis*; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011510:Proteolipids / Q000032:analysis*; D014598:Uteroglobin
D006898:Hydroxylamines; D009185:Myelin Proteins; D011510:Proteolipids; D000122:Acetylthiocholine; D003553:Cystine; D014598:Uteroglobin; D004228:Dithionitrobenzoic Acid; D000109:Acetylcholine