Biophysical Journal 68: 2419-2428 (1995)
© 1995 the Biophysical Society

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Mode of caldesmon binding to smooth muscle thin filament: possible projection of the amino-terminal of caldesmon from native thin filament.

Department of Fine Morphology, University of Tokyo, Japan.

ABSTRACT

The structure of smooth muscle thin filament was examined by various electron microscopy techniques, with special attention to the mode of caldesmon binding. Chemical cross-linking was positively used to avoid the dissociation of accessory proteins upon dilution. Caldesmon in reconstituted thin filament was observed as fine filamentous projections from thin filament. Native thin filament isolated from smooth muscle showed similarly numerous fine whisker-like projections by all the techniques employed here. Antibody against the amino-terminus of caldesmon labeled the end of such projections indicating the possibility that the amino-terminal myosin binding moiety might stick out from the shaft of the thin filament. Such whiskers are often projected out as a cluster to the same side of native thin filament. Further, we could visualize the assembly of dephosphorylated heavy meromyosin (HMM) with native or reconstituted thin filament forming "nonproductive" complex in the presence of ATP. The association of HMM to the shaft of thin filament was through subfragment-2 moiety, in accordance with biochemical studies. Some HMM particles bound closer to the thin filament shaft, possibly suggesting the presence of the second myosin-binding site on caldesmon. Occasionally two kinds of HMM association as such coexisted at a single site on this filament in tandem. Thus, we constructed a structural model of thin filament. The proposed molecular arrangement is not only compatible with all the biochemical results but also provides additional support for our recent findings (E. Katayoma, G. C. Scott-Woo, and M. Ikebe (1995) J. Biol. Chem. 270, 3919-3925) regarding the capability of caldesmon to induce dephosphorylated myosin filament, which explains the existence of thick filaments in relaxed smooth muscle cells.

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