Crosslinking of telokin to chicken gizzard smooth muscle myosin.
Telokin, which is a candidate for one of the factors stabilizing dephosphorylated myosin filaments in smooth muscle cells, was subjected to a chemical crosslinking study to determine its binding location on the myosin molecule. Telokin labeled with 5-iodoacetamidofluorescein (Fl-telokin) retained the nature of unlabeled telokin: it bound to dephosphorylated gizzard myosin with a stoichiometry of 1 mol/mol myosin and induced filament assembly. The carboxyl groups of Fl-telokin were activated with 1-ethyl-3-(3-dimethyl-amino-propyl) carbodiimide and N-hydroxysuccinimide and then crosslinked to myosin. The production of three fluorescent peptides was observed on an SDS-gel, accompanying a decrease in the amount of regulatory light chain (LC20). The molecular weights of these products estimated to be > 200, 62, and 41 kDa. When unlabeled telokin was crosslinked to myosin of which LC20 was exchanged with 5-[[2-[(iodoacetyl)amino]ethyl]amino]-naphthalene-1-sulfonic acid-labeled LC20, the 62- and 41-kDa bands were also fluorescent. These results suggest that the > 200, 62, and 41-kDa species are telokin crosslinked with a heavy chain, with 2 LC20, and with 1 LC20, respectively. Myosin crosslinked with unlabeled telokin showed an extra structure with a small projection at the head-rod junction on electron microscopy and this structure was proved to be telokin by decorating it with anti-telokin antibodies. In addition, dephosphorylated myosin crosslinked with Fl-telokin was incapable of folding into the 10S conformation at 0.2 NaCl in the presence of MgATP, and assembled into filaments at 0.15 M NaCl in the presence of MgATP. Thus, telokin may bind to LC20 and a heavy chain region at the head-rod junction and suppress folding into the 10S conformation, leading to the assembly of myosin into filaments.
Masato
T
T
Division of Chemistry, Graduate School of Science, Hokkaido University, Sapporo.
Numata
T
T
Katoh
T
T
Morita
F
F
Yazawa
M
M
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002234:Carbodiimides / Q000378:metabolism; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000378:metabolism; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005895:Gizzard, Avian / Q000378:metabolism; D008854:Microscopy, Electron; D009124:Muscle Proteins / Q000378:metabolism* / Q000648:ultrastructure; D009130:Muscle, Smooth / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism*; D010446:Peptide Fragments; D010455:Peptides; D011487:Protein Conformation; D017510:Protein Folding; D013388:Succinimides / Q000378:metabolism
D002234:Carbodiimides; D003432:Cross-Linking Reagents; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009124:Muscle Proteins; D010446:Peptide Fragments; D010455:Peptides; D013388:Succinimides; C074139:1-ethyl-3-(3-dimethylaminoethyl)carbodiimide; C060640:telokin; C015296:5-iodoacetamidofluorescein; D000255:Adenosine Triphosphate; D009219:Myosin-Light-Chain Kinase; D009218:Myosins; C001426:N-hydroxysuccinimide