Abstract
Muscle contraction results from attachment-detachment cycles between myosin heads extending from myosin filaments and actin filaments. It is generally believed that a myosin head first attaches to actin, undergoes conformational changes to produce force and motion in muscle, and then detaches from actin. Despite extensive studies, the molecular mechanism of myosin head conformational changes still remains to be a matter for debate and speculation. The myosin head consists of catalytic (CAD), converter (CVD) and lever arm (LD) domains. To give information about the role of these domains in the myosin head performance, we have examined the effect of three site-directed antibodies to the myosin head on in vitro ATP-dependent actin-myosin sliding and Ca2+-activated contraction of muscle fibers. Antibody 1, attaching to junctional peptide between 50K and 20K heavy chain segments in the CAD, exhibited appreciable effects neither on in vitro actin-myosin sliding nor muscle fiber contraction. Since antibody 1 covers actin-binding sites of the CAD, one interpretation of this result is that rigor actin-myosin linkage is absent or at most a transient intermediate in physiological actin-myosin cycling. Antibody 2, attaching to reactive lysine residue in the CVD, showed a marked inhibitory effect on in vitro actin-myosin sliding without changing actin-activated myosin head (S1) ATPase activity, while it showed no appreciable effect on muscle contraction. Antibody 3, attaching to two peptides of regulatory light chains in the LD, had no significant effect on in vitro actin-myosin sliding, while it reduced force development in muscle fibers without changing MgATPase activity. The above definite differences in the effect of antibodies 2 and 3 between in vitro actin-myosin sliding and muscle contraction can be explained by difference in experimental conditions; in the former, myosin heads are randomly oriented on a glass surface, while in the latter myosin heads are regularly arranged within filament-lattice structures.
Highlights
More than 50 years have passed since the monumental discovery that muscle contraction results from relative sliding between actin and myosin filaments coupled with ATP hydrolysis [1,2], molecular mechanisms of the myofilament sliding are not yet fully understood
(2) Anti-lever arm domain (LD) antibody, which is believed to differ from anti-catalytic domain (CAD) antibody only at the epitope-binding site, shows reversible inhibitory effect on Ca2+-activated force development (Figs. 4–7), indicating that IgG molecules can diffuse into the fiber to exhibit their inhibitory effect by attaching to their epitope, and can diffuse out of the fiber after its removal from external solution
The same explanation may apply to both anti-CAD antibody and anti-RLR antibody, which has no effect on Ca2+-activated contraction of muscle fibers
Summary
More than 50 years have passed since the monumental discovery that muscle contraction results from relative sliding between actin and myosin filaments coupled with ATP hydrolysis [1,2], molecular mechanisms of the myofilament sliding are not yet fully understood. It is generally believed that a myosin head extending from myosin filaments first attaches to actin filaments, undergoes conformational changes to produce unitary myofilament sliding, and detaches from actin filaments [3,4] In accordance with this view, biochemical studies on reaction steps of actomyosin ATPase in solution [5] indicate that the myofilament sliding is caused by cyclic interaction between myosin heads and actin filaments; the myosin head (M) first attaches to actin (A) in the form of M?ADP?Pi to undergo a conformational change, i.e. power stroke, associated with release of Pi and ADP, and forms rigor linkage (AM) with A. We have further succeeded in recording myosin head power stroke in hydrated mixture of actin and myosin filaments [12,13]. These results constitute the first electron microscopic visualization of myosin head power and recovery strokes producing muscle contraction
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