STRUCTURE OF THE 265-KILODALTON COMPLEX FORMED UPON EDC CROSS-LINKINGOF SUBFRAGMENT-1 TO F-ACTIN

The conventional model of force generation in muscle requires the pres ence of at least two different contact areas between the myosin head ( S1) and the actin filament. It has been found that S1 has two sites av ailable for carbodiimide cross-linking, but it is generally believed t hat the myosin head can be cross-linked to only one actin through eith er site. We provide here, for the first time, evidence that one S1 can be cross-linked to two separate actin molecules. The covalent complex of one S1 with two actins was found to have an apparent molecular mas s of 265 kDa. The formation of the 265-kDa acto-S1 complex was strongl y dependent on the ratio of S1 to actin. Limited tryptic digestion con verted the 265-kDa product into the 240-kDa complex by releasing a 27- kDa N-terminal S1 fragment. Limited subtilisin digestion of the 265-kD a covalent acto-S1 complex yielded 29-, 93-, and 66-kDa peptides which corresponded to the 29-kDa N-terminal domain of S1, actin-44-kDa (cen tral domain of S1) and actin-22-kDa (C-terminal domain of S1) complexe s, respectively. These peptides could be generated only if a single S1 has been cross-linked to two separate actins. The 265-kDa acto-S1 com plex (S1:actin ratio = 0.5) had 60% of the ATPase activity of the 175- 185-kDa acto-S1 complex (S1:actin ratio = 1). The ability of the myosi n head to bind to one or to two actins suggests that during an active stroke the myosin head may first bind to one and then to two monomers in F-actin, producing a approximately 10-nm shift between thick and th in filaments.