Phase separation of myelin sheath in Triton X-114 solution: predominant localization of the 21.5-kDa isoform of myelin basic protein in the lipid raft-associated domain.
Myelin basic protein (MBP) isoforms in the myelin sheath are known to have distinct intracellular expression patterns, which are profoundly related to functional specificity. Determining the differential localization of MBP isoforms is therefore important for understanding their pathophysiological roles. In this study, we have developed a new method for phase separation of myelin. The non-ionic detergent Triton X-114 is used to solubilize myelin sheath which then undergoes phase separation to yield four fractions. The lipid raft-associated proteins and lipids in each fraction were analysed by immunoblotting and lipid analysis, respectively. The present method gives two lipid raft-enriched fractions, one of them was found to contain only lipid raft-associated galactocerebroside and cholesterol as the major lipids. The 21.5-kDa MBP isoforms (21.5 MBP), both unphosphorylated and phosphorylated, were exclusively contained in this fraction. Phosphorylated 21.5 MBP (21.5 pMBP) has been shown to specifically disappear from demyelinated loci. The present analytical method clearly indicated that disappearance of 21.5 pMBP corresponded to demyelination and its reappearance corresponded to prevention of demyelination. Demyelination was also associated with aging and was prevented by the myelin-protecting herbal medicine, Chinpi, a type of dried citrus peel.
Center for Kampo Medicine, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan; Tsumura Research Laboratories, Tsumura & Co., Yoshiwara 3586, Ami-machi, Inashiki-gun, Ibaraki 300-1192, Japan; and Department of Anatomy, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan.
Michihiro
M
Uruse
Masahiro
M
Yamamoto
Makoto
M
Sugawa
Keiko
K
Matsuura
Yurie
Y
Sato
Chika
C
Seiwa
Kenji
K
Watanabe
Sadakazu
S
Aiso
Hiroaki
H
Asou
D000375:Aging / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D002855:Chromatography, Thin Layer; D015151:Immunoblotting; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D004676:Myelin Basic Protein / Q000378:metabolism*; D009186:Myelin Sheath / Q000187:drug effects / Q000378:metabolism*; D017830:Octoxynol; D010766:Phosphorylation / Q000187:drug effects; D011092:Polyethylene Glycols / Q000494:pharmacology*; D020033:Protein Isoforms / Q000378:metabolism; D012996:Solutions
D004676:Myelin Basic Protein; D020033:Protein Isoforms; D012996:Solutions; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40
21.5-kDa isoform of myelin basic protein; Triton X-114; demyelination; detergent extraction; lipid raft; remyelination