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J Biochem. 1997 Feb;121(2):225-30.

Crosslinking of telokin to chicken gizzard smooth muscle myosin.

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  • 1Division of Chemistry, Graduate School of Science, Hokkaido University, Sapporo.

Abstract

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.

PMID:
9089394
[PubMed - indexed for MEDLINE]
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