The role of the overlap region at the ends of tropomyosin molecules in the properties of regulated thin filaments has been investigated by substituting nonpolymerizable tropomyosin for tropomyosin in a reconstituted troponin-tropomyosin-actomyosin subfragment 1 ATPase assay system. A previous study [Heeley, Golosinka & Smillie (1987) J. Biol. Chem. 262, 9971-9978] has shown that at an ionic strength of 70 mM, troponin will induce full binding of nonpolymerizable tropomyosin to F-actin both in the presence and absence of calcium. At a myosin subfragment 1-to-actin ratio of 2:1 ([actin] = 4 microM) and an ionic strength of 50 mM, comparable levels of ATPase inhibition were observed with increasing levels of tropomyosin or the truncated derivative in the presence of troponin (-Ca2+). Large differences were noted, however, in the activation by Ca2+. Significantly lower ATPase activities were observed with nonpolymerizable tropomyosin and troponin (+Ca2+) over a range of subfragment 1-to-actin ratios from 0.25 to 2.5. The concentration of subfragment 1 required to generate ATPase activities exceeding those seen with actomyosin subfragment 1 alone under these conditions was 3-4-fold greater when nonpolymerizable tropomyosin was used. Similar effects were seen at the much lower ionic strength of 13 mM and are consistent with the reduced ATPase activity with nonpolymerizable tropomyosin observed previously [Walsh, Trueblood, Evans & Weber (1985) J. Mol. Biol. 182, 265-269] at low ionic strength and a subfragment 1-to-actin ratio of 1:100. Little cooperativity in activity as a function of subfragment 1 concentration with either intact tropomyosin or its truncated derivative was observed under the present conditions. Further studies are directed towards an understanding of these effects in terms of the two-state binding model for the attachment of myosin heads to regulated thin filaments.
A mosaic multiple-binding model for the binding of caldesmon and myosin subfragment-1 to actin
