Isolation of nebulin from rabbit skeletal muscle and its interaction with actin.
Nebulin is about 800 kDa filamentous protein that binds the entire thin filament of vertebrate skeletal muscle sarcomeres. Nebulin cannot be isolated from muscle except in a completely denatured form by direct solubilization of myofibrils with SDS because nebulin is hardly soluble under salt conditions. In the present study, nebulin was solubilized by a salt solution containing 1 M urea and purified by DEAE-Toyopearl column chromatography via 4 M urea elution. Rotary-shadowed images of nebulin showed entangled knit-like particles, about 20 nm in diameter. The purified nebulin bound to actin filaments to form loose bundles. Nebulin was confirmed to bind actin, alpha-actinin, beta-actinin, and tropomodulin, but not troponin or tropomyosin. The data shows that full-length nebulin can be also obtained in a functional and presumably native form, verified by data from experiments using recombinant subfragments.
Chitose
Ryo
R
Department of Biology, Graduate School of Science, Chiba University, Chiba 263-8522, Japan.
Watanabe
Atsushi
A
Asano
Masato
M
Hanashima
Akira
A
Sasano
Kouhei
K
Bao
Yulong
Y
Maruyama
Koscak
K
Kimura
Sumiko
S
D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D000837:Anion Exchange Resins; D015153:Blotting, Western; D002852:Chromatography, Ion Exchange / Q000379:methods*; D009124:Muscle Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D018482:Muscle, Skeletal / Q000737:chemistry*; D011817:Rabbits; D014335:Tropomyosin; D014336:Troponin; D014508:Urea / Q000737:chemistry
D000199:Actins; D000837:Anion Exchange Resins; D009124:Muscle Proteins; D014335:Tropomyosin; D014336:Troponin; C035620:nebulin; D014508:Urea
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