Tropomyosin inhibits the glutaraldehyde-induced cross-link between the central 48-kDa fragment of myosin head and segment 48-67 in actin subdomain 2.

The glutaraldehyde-induced cross-linking of the F-actin-myosin head (S1) complex, previously described [Bertrand et al. (1988) Biochemistry 27, 5728-5736], was investigated in the presence of tropomyosin (Tm) alone or associated with troponin (Tn), at a Tm-Tn/actin/S1 molar ratio of 1:7:3. Among the two acto-S1 cross-linked products with apparent masses of 165 and 200 kDa generated in the absence of the regulatory proteins, only the 165-kDa adduct was formed in the presence of Tm. An identical result was obtained with and without Tn regardless of the presence of Ca2+ and/or Mg(2+)-ADP. The abolition of the 200-kDa cross-linked acto-S1 species was independent of the S1/actin ratio since even a 3-fold excess of S1 over actin, sufficient for fully turning on the thin filament, could not restore the 200-kDa covalent complex. In addition, the acto-S1 contacts cross-linked in either the 165- or 200-kDa product were not involved in the Ca(2+)-linked regulation of the acto-S1 ATPase activity, as the enzymatic activities of both types of complexes were regulated to the same extent by Ca2+/EGTA, in the presence of the regulatory proteins. Cross-linking experiments performed with [14C]glutaraldehyde showed that both covalent complexes were composed of 1 mol of actin bound to 1 mol of S1 heavy chain. The use of proteolytic actin or S1 derivatives together with the direct proteolysis of the acto-S1 covalent adducts revealed that Tm abolished the cross-link between the central 48-kDa fragment of the S1 heavy chain and Lys50 of actin subdomain 2 that is responsible for the formation of the 200-kDa entity, while it did not affect the cross-link between the S1 heavy chain segment of residues 636-642 and Arg28 of actin that generates the 165-kDa derivative. These results provide experimental clues for the interaction of S1 with actin subdomain 2 and show that this contact is implicated in the weak acto-S1 binding state. Furthermore they demonstrate the ability of Tm to affect the structure of actin subdomain 2 even in the presence of S1 bound in the rigor state.