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 (S 1) complex, previously described [Bertrand et al. (1988) Biochemistry 27, 5728-5736],was investigated in the presence of tropomyosin (Tm) al one 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 ma sses of 165 and 200 kDa generated in the absence of the regulatory pro teins, only the 165-kDa adduct was formed in the presence of Tm. An id entical result was obtained with and without Tn regardless of the pres ence of Ca2+ and/or Mg2+-ADP. The abolition of the 200-kDa cross-linke d 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 thi n filament, could not restore the 200-kDa covalent complex. In additio n, the acto-S1 contacts cross-linked in either the 165- or 200-kDa pro duct were not involved in the Ca2+-linked regulation of the acto-S1 AT Pase 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 [C-14]g lutaraldehyde showed that both covalent complexes were composed of 1 m ol of actin bound to 1 mol of S1 heavy chain. The use of proteolytic a ctin or S1 derivatives together with the direct proteolysis of the act o-S1 covalent adducts revealed that Tm abolished the cross-link betwee n the central 48-kDa fragment of the S1 heavy chain and Lys(50) of act in subdomain 2 that is responsible for the formation of the 200-kDa en tity, while it did not affect the cross-link between the S1 heavy chai n segment of residues 636-642 and Arg(28) of actin that generates the 165-kDa derivative. These results provide experimental clues for the i nteraction of S1 with actin subdomain 2 and show that this contact is implicated in the weak acto-S1 binding state. Furthermore they demonst rate the ability of Tm to affect the structure of actin subdomain 2 ev en in the presence of S1 bound in the rigor state.