Contraction characteristics and ATPase activity of skeletal muscle fibers in the presence of antibody to myosin subfragment 2.

Abstract

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...

Dynamic electron microscopy of ATP-induced myosin head movement in living muscle thick filaments

“…The large myosin head movement may not readily fit into the contraction model based on the myosin head rotation, and possibly would result either from cooperative action of myosin two heads (20,21) or from shortening of myosin subfragment-2 region (22,23). Much more experimental work is needed to clarify the myosin head movement responsible for muscle contraction.…”

Mutation of the myosin converter domain alters cross-bridge elasticity

“…Some authors considered elastic bending of the long, light-chain-binding ␣-helix an obvious candidate (4,9,10). The actin-myosin interface (11) or the junction between the lightchain domain and the catalytic domain of the myosin head (9) as well as subfragment 2 were also considered (12) as other possibilities for the location of elastic distortion.…”

Unfolding/refolding studies of the myosin rod

“…Such melting would be expected to give rise to a reduction in the overall length of the molecule, and this is consistent with electron-microscope observations of the effect of temperature on the length of the myosin tail (Walker and Trinick, 1986;Walzthony et al, 1986). This transition could therefore be an important force-generating event in muscle contraction and the subfragment-2 region as well as the myosin head would contribute to force production in actively contracting muscle (Harrington et al, 1990;Sugi et al, 1992).It has recently been proposed that the myosin rod contains six independent domains (Privalov, 1982 ;Lopez-Lacomba et al, 1989;Bertazzon and Tsong, 1990). The Cterminus of the heavy chain has been shown to be unfolded and mobile , and to play a crucial role in myosin assembly Kalbitzer et al (1991) have indicated that the two chains are not in exact register but are slightly staggered.…”

“…Such melting would be expected to give rise to a reduction in the overall length of the molecule, and this is consistent with electron-microscope observations of the effect of temperature on the length of the myosin tail (Walker and Trinick, 1986;Walzthony et al, 1986). This transition could therefore be an important force-generating event in muscle contraction and the subfragment-2 region as well as the myosin head would contribute to force production in actively contracting muscle (Harrington et al, 1990;Sugi et al, 1992).…”