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We compared the mechanisms by which caldesmon inhibits actin and actin-tropomyosin activation of myosin subfragment 1 (S1) MgATPase activity. Caldesmon always inhibited actin activation by displacing S1.ADP.Pi from actin and inhibition required at least 0.7 caldesmon molecules bond per actin for 90% inhibition. Caldesmon inhibited actin-tropomyosin without any displacement of S1.ADP.Pi; thus it inhibits a rate-limiting step. Inhibition is highly cooperative, requiring no more than one caldesmon bound per 10 actins for 90% inhibition of activation by actin and smooth muscle tropomyosin. The degree of cooperativity is defined by the tropomyosin since inhibition by skeletal tropomyosin requires up to one caldesmon bound per 4 actins for 90% inhibition under identical conditions. Both noncooperative inhibition of actin and cooperative, tropomyosin-dependent, inhibition are manifested by a fragment of caldesmon containing only the C-terminal 99 amino acids (658C), although this fragment does not itself bind to tropomyosin. The functional properties of 658C are very similar to striated muscle troponin I, consequently we propose a similar mechanism for tropomyosin-dependent regulation by caldesmon. Caldesmon binding switches actin-tropomyosin to the "off" or "weak" state and Ca2+/calmodulin binding to caldesmon blocks this switch and thus reactivates the actin filament.

The effects of Ca2+ and myosin subfragment 1 on the conformation of tropomyosin and actin in regulated actin filaments in ghost fibers were investigated by means of the polarized fluorescence technique. Regulated thin filaments were reconstituted in skeletal muscle ghost fibers by incorporation into the fibers of either skeletal muscle troponin-tropomyosin or smooth-muscle caldesmon-calmodulin-tropomyosin complexes. Tropomyosin and actin were specifically labeled with fluorescent probes, 1,5-IAEDANS and phalloidin-rhodamine, respectively. Analysis of the fluorescence parameters indicated that the binding of Ca2+ to regulated actin filaments induces conformational changes in tropomyosin and actin that lead to the strengthening of the interaction between these two proteins and weakening of the binding of actin monomers in the filament. These changes become larger when regulated actin forms rigor links with myosin subfragment 1. No notable alterations in the position of tropomyosin relative to actin in the frontal plane of the fiber were detected either upon binding of Ca2+ or upon the additional binding of myosin subfragment 1 to regulated actin.