Purification and characterization of nebulin subfragments produced by 0.1 mM CaCl2.
Nebulin, which forms a long inextensible filament in sarcomeres, was fragmented into 200-, 180-, 40-, 33-, and 23-kDa subfragments on treatment with 0.1 mM CaCl2. The subfragments released from myofibrils were successfully purified by immunoaffinity column chromatography. The 200-, 40-, 33-, and 23-kDa subfragments were released from myofibrils and occupied 80% of the nebulin filaments. The remainder comprised the 180-kDa subfragment bound to the myofibrils. There is a possibility that an entire nebulin filament is constructed from the 200-, 180-, 40-, 33-, and 23-kDa subfragments. We have developed a new "fluorescence-method" to detect the binding of calcium ions to a protein using quin2, and clarified that nebulin is a calcium-binding protein, and that calcium ions bind to the 200-, 40-, and 23-kDa subfragments. Nebulin filaments are probably fragmented on the binding of large amounts of calcium ions to the 200-, 40-, and 23-kDa subfragments.
Tatsumi
R
R
Department of Animal Science, Faculty of Agriculture, Hokkaido University.
Hattori
A
A
Takahashi
K
K
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002122:Calcium Chloride / Q000494:pharmacology; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D007700:Kinetics; D008970:Molecular Weight; D009124:Muscle Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D009132:Muscles / Q000737:chemistry / Q000648:ultrastructure*; D009210:Myofibrils / Q000737:chemistry / Q000648:ultrastructure*; D011485:Protein Binding; D012518:Sarcomeres / Q000737:chemistry / Q000648:ultrastructure*
D000596:Amino Acids; D009124:Muscle Proteins; C035620:nebulin; D002122:Calcium Chloride; D002118:Calcium