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To investigate the role of the myosin hinge region in muscle contraction, we examined the contraction characteristics and Mg-ATPase activity of glycerinated muscle fibers prepared from rabbit psoas in the presence and absence of polyclonal antibody directed against the subfragment 2 (S-2) region of myosin. The antibody-induced reduction of Ca2+-activated isometric force was always accompanied by a parallel decrease of muscle fiber stfess, so that the stiffness versus force relation remained unchanged by the antibody treatment.Force-velocity relations of the fibers, obtained by applying ramp decreases in force at steady isometric forces, indicated that the antibody had no effect on maximum shortening vdocity or on the shape of force-velocity curves. Simultaneous measurements of Mg-ATPase activity and Ca2+-activated force showed that Mg-ATPase activity of the fibers remained unchanged despite the antibody-induced reduction of isometric force even to zero. These results indicate that when anti-S-2 antibody attaches to the S-2 region of myosin molecules, their heads still hydrolyze ATP but no longer contribute to both force generation and muscle fiber stiffness.Muscle contraction results from alternate formation and breaking of cross-links between the myosin head (subfragment 1; S-1), extending from the thick filament and a neighboring thin filament (1, 2). The energy for contraction is supplied by ATP hydrolysis. Since the ATPase activity and actin binding site are localized in the S-1 region ofmyosin, S-1 is commonly believed to play a major role in muscle contraction. Recent in vitro motility assays have shown that S-1 alone is sufficient to produce force and move actin filaments (3, 4), but it is not clear whether the ATP-dependent actinmyosin sliding observed in the assay systems is the same as that actually taking place in living muscle.On the other hand, it has been proposed that melting and shortening in the proteolytically sensitive hinge region lying between the short subfragment 2 (S-2) and light meromyosin segments of the myosin tail contribute to force generation in muscle (5-8). In support of this hypothesis, polyclonal anti-S-2 antibody has been shown to reduce Ca2+-activated isometric force in glycerinated skeletal muscle fibers, while ATPase activity of the fibers and the initial unloaded shortening velocity of isolated myofibrils undergo little change (9, 10). More recently, it has been shown that, in the presence of antibody directed against a 20-amino acid peptide segment within the hinge region of cardiac myosin, movement of actin filaments in an in vitro motility assay is suppressed, while ATPase activity of myofibrils and purified S-1 remained unchanged (11).The present experiments were undertaken to further investigate the effect of anti-S-2 antibody on the contraction characteristics and ATPase activity of glycerinated muscle fibers prepared from rabbit psoas. It will be shown that anti-S-2 antibody produces a parallel decrease of muscle fiber stiffness and Ca2+-activated isometric f...

Temperature-jump studies on the long S-2 fragment (100,000 daltons) isolated from myosin show that this structure can undergo a-helix-random coil transitions in a time range approximating the cycle time of a crossbridge. Two relaxation times are observed after temperature jumps of 50C over the range 35-55°C, one in the submillisecond (Tf) and the other in the millisecond (r,) time ranges. Both processes exhibit maxima near the midpoint of the helix-coil transition (ttm = 45 + 2°C) as determined by optical rotation melt experiments. Similar results were observed for the low temperature transition (t. = 45°C) of the myosin rod. Viscosity studies reveal that the S-2 particle has significant flexibility at physiological temperature. Results are considered in terms of the Huxley-Simmons and helix-coil transition models for force generation in muscle. The subfragment 2 (S-2) region of the myosin molecule plays a major role in most current theories of muscle contraction. In the model of H. E. Huxley (1) for contraction, this segment, which forms the coiled-coil a-helical tail of heavy meromyosin, swings away from the thick filament surface during each contractile cycle of a crossbridge, thus allowing the myosin heads (S-1 subunits) to attach to neighboring thin filaments with the same stereospecific orientation over a wide range of interfilament spacing. S-2 is considered to be a rigid, inextensible rod which is fastened to the thick filament backbone by a hinge at the junction of light meromyosin (LMM) and heavy meromyosin (HMM) and is joined to the myosin head by a second hinge-the S-1/S-2 linkage. This arrangement provides the freedom required to accommodate the variable interfilament spacing as the sarcomere shortens. After attachment of the S-1 subunit to the thin filament, it rotates relative to this structure, generating a relative sliding force between the thick and thin filaments. During each cycle of a crossbridge, ATP is hydrolyzed. In the A. F. Huxley-Simmons model (2-4), S-2 serves as an elastic element which, by spring-like extension, acts to exert a part of the tensile force developed when a crossbridge interacts with the thin filament. The force-generating event is again considered to be rotation of the myosin head on the thin filament with the S-2 elastic element stretching instantaneously as the S-1 subunit alters its angle of attachment through a series of stable positions with progressively lower potential energy.In the Harrington model (5) for contraction, the force-generating event is considered to be an a helix-random coil transition within the S-2 element during each cycle of a crossbridge. In this model the myosin head remains fixed in orientation during its transient attachment to the thin filament and the melting of part of the double a helix near the LMM-HMM junction is effected by the ATP cleavage reaction occurring on a neighboring myosin head.Our crosslinking studies of synthetic myosin thick filaments and glycerinated rabbit muscle fibers in rigor indicate that the The publicatio...

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...