Suppression of contractile force in muscle fibers by antibody to myosin subfragment 2.

Abstract

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...

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.…”

Contraction of myofibrils in the presence of antibodies to myosin subfragment 2.

“…Polyclonal antibodies against the S-2 region of chicken pectoralis myosin produced in rabbit (rabbit anti-chicken S-2) and polyclonal antibodies against the S-2 region of rabbit psoas myosin produced in goat (goat anti-rabbit S-2) were prepared and purified as described (5). Rabbit myosin S-2 was isolated (13) and further purified by calcium precipitation (5).…”

“…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.…”

Evidence for the Essential Role of Myosin Subfragment-2 in Muscle Contraction: Functional Communication between Myosin Head and Subfragment-2

“…Helical and random coil structures of the S-2 hinge region are expressed as straight and zig-zag shapes, respectively [7]. -activated myofibrillar shortening [11,12]. In 1992, he asked me to work together using his anti-S-2 antibody, and we made the experiments to be described later with unexpected results.…”