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Polyclonal antibody directed against the subfragment-2 region of myosin was purified by affinity chromatography. Skinned muscle fibers that had been preincubated with antibody were able to sustain only 7% of the active isometric force generated by control fibers. The effect of antibody on force production could not be accounted for by inhibition of ATP turnover.The classical rotating-head sliding filament model for force generation in activated muscle fibers (1-3) proposes that the force-generating event results from a structural change in the subfragment 1 (S-1) region (the myosin head) while it is attached to actin. The helix-coil model (4, 5) for force generation proposes that melting and shortening in a section [the heavy meromyosin (HMM)/light meromyosin (LMM) hinge domain] of subfragment 2 (S-2) occurs as the actin-attached cross-bridge swings away from the thick filament surface in an active bridge cycle. It has proved difficult to decide conclusively between these two models; indeed, it seems possible that aspects of both processes are fundamental to the tension-generating mechanism in muscle. Evidence has been provided that beads coated with HMM (6) as well as soluble HMM fragments (7) can move along actin filaments energized by ATP at speeds approaching those obtained with muscle fibers under no-load conditions. Fluorescent actin filaments have also been shown to slide along single-headed myosin filaments bound to a glass support (8) and isolated S-1 subunits bound to a nitrocellulose film (9) (11,12). Although other interpretations are possible, this finding suggests that release of S-2 from the thick filament surface in a normal bridge cycle may be an essential element of the force-generating mechanism in muscle.In the work to be described below, we examine the effect of anti-S-2 polyclonal antibodies on the contractile force of activated, skinned psoas fibers. As in the cross-linking studies, our primary goal was to determine if modulation of the interaction between S-2 and the thick-filament backbone could suppress the isometric force in the absence of any direct effect on the ability of the S-1 subunit to cycle through actin. MATERIALS AND METHODSPreparation of Muscle Proteins and Immunogens. Chicken myosin was prepared by extraction of chicken breast muscle with Guba Straub solution (0.3 M KC1/0.09 M KH2PO4/ 0.06 M K2HPO4) followed by repeated washes in low-ionicstrength buffer (13). HMM and LMM were prepared by digestion of chicken myosin (10 mg/ml) with chymotrypsin (0.1 mg/ml) in 20 mM NaP,/3 mM MgC12/0.6 M NaCl, pH 6.9 for 6 hr at 40C. Digestion was quenched by the addition of phenylmethylsulfonyl fluoride to 1 mM. LMM and undigested myosin were precipitated by dialysis of the digest against 20 mM imidazole hydrochloride/i mM CaCl2, pH 6.2. LMM was further purified by ethanol fractionation. S-2 was prepared from HMM by the method of Sutoh et al. (14) and further purified by chromatography on Sepharose 4B in 20 mM imidazole hydrochloride (pH 6.2). Addition of CaCl2 (to 6 mM) to the c...

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In a muscle-based version of in vitro motility assays, the unloaded shortening velocity of rabbit skeletal myofibrils has been determined in the presence and absence of affinity-column-purified polyclonal antibodies directed against the subfragment-2 region of myosin. Contraction was initiated by photohydrolysis of caged ATP and the time dependence of shortening was monitored by an inverted microscope equipped with a video camera. Antibody-treated myofibrils undergo unloaded shortening in a fast phase with initial rates and half-times comparable to control (untreated) myofibrils, despite a marked reduction in the isometric force of skinned muscle fibers in the presence of the antibodies. In antibodytreated myofibrils, this process is followed by a much slower phase of contraction, terminating in elongated structures with well-defined sarcomere spacings (-1 ,um) in contrast to the supercontracted globular state of control myofibrils. These results suggest that although the unloaded shortening of myofibrils (and in vitro motility of actin filaments over immobilized myosin heads) can be powered by myosin heads, the subfragment-2 region as well as the myosin head contributes to force production in actively contracting muscle.The classical rotating cross-bridge model proposes that the contractile force in activated muscle originates from a structural transition within the myosin head [subfragment-1 (S-1) subunit] while it is attached to actin (1, 2). The helix-coil model proposes that melting within subfragment-2 (S-2; the a-helical tail segment of heavy meromyosin) is triggered and generates force when the actin-attached cross-bridge swings away from the thick filament surface (3, 4). In earlier work (5), it was shown that purified anti-S-2 antibody can markedly depress the isometric tension generated by skinned psoas fibers. The reduction in tension (to about 7% ofcontrol fibers) occurs in the absence of any direct effect on the ability of S-1 to undergo cyclic interaction with actin as measured by the ATPase of antibody-treated fibers. One interpretation of this experiment is that S-2 contributes to force production in vivo, possibly through a helix-coil transition in the S-2 hinge domain of the myosin molecule (for review, see ref. 6). However, it seems clear from recent in vitro model studies that S-1 alone, energized by ATP, can produce force (7) and slide actin filaments at speeds approaching those obtained with muscle fibers under no-load conditions (8,9). One possibility to reconcile these observations is that the force generated by S-1 in the model systems is sufficient to produce sliding motion but not sufficient for the expression of the full isometric force developed in working muscle. To test this explanation, we have measured the relative unloaded sliding velocity of overlapping thin and thick filaments in myofibrils, the intact basic structural units of skeletal muscle, in the presence and absence of polyclonal antibodies directed against the S-2 region of myosin. In these experiments, we have...

Ox muscle troponin was shown by equilibrium- and velocity-sedimentation studies to undergo concentration-dependent dissociation into its constituent subunits as well as self-association in imidazole buffers, pH 6.9. The extent of troponin association was found to be strongly dependent on ionic strength and also to exhibit a dependence on pH and temperature; under conditions physiological in regard to pH, temperature and ionic strength the extent of polymerization of troponin is considerable in 2 mg/ml solutions. The ability of polymeric troponin to bind to tropomyosin has been inferred from studies of mixtures containing actin-tropomyosin and an excess of troponin over the amount required for the normal 7:1:1 actin-tropomyosin-troponin complex. These findings should be relevant to studies of reconstituted actin-tropomyosin-troponin preparations, since they signify possible chemical as well as physical differences between the gel, paracrystalline and filamentous states of the complex that result from adoption of different preparative procedures for analogues of the native thin filament.