CROSS-LINKING OF TELOKIN TO CHICKEN GIZZARD SMOOTH-MUSCLE MYOSIN

Telokin, which is a candidate for one of the factors stabilizing depho sphorylated 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 dephosp horylated gizzard myosin with a stoichiometry of 1 mol/mol myosin and induced filament assembly. The carboxyl groups of Fl-telokin were acti vated with 1-ethyl-3-(3-dimethyl-amino-propyl)carbodiimide and N-hydro xysuccinimide and then crosslinked to myosin. The production of three fluorescent peptides was observed on an SDS-gel, accompanying a decrea se in the amount of regulatory light chain (LC20). The molecular weigh ts of these products were estimated to be >200, 62, and 41 kDa. When u nlabeled telokin was crosslinked to myosin of which LC20 was exchanged with doacetyl)amino]ethyl]amino]-naphthalene-1-sulfonic acid-labeled LC20, the 62- and 41-kDa bands were also fluorescent. These results su ggest that the >200, 62, and 41-kDa species are telokin crosslinked wi th a heavy chain, with 2 LC20, and with 1 LC20, respectively. Myosin c rosslinked with unlabeled telokin showed an extra structure with a sma ll 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 M Na Cl in the presence of MgATP, and assembled into filaments at 0.15 M Na Cl in the presence of MgATP. Thus, telokin may bind to LC20 and a heav y chain region at the head-rod junction and suppress folding into the 10S conformation, leading to the assembly of myosin into filaments.