(rabbit psoas muscle/antibody to subfragment 2/muscle contraction)

Polyclonal antibody directed against the sub- fragment-2 region of myosin was purified by affinity chroma- tography. 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 gen- eration 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) in the presence of ATP. It is not clear at the moment if this movement is a reflection of only a part of the force-generating mechanism or if it represents the complete force-producing event. This question should be viewed in the light of enzyme-probe studies that provide evidence for local melting at several sites spanning the LMM/HMM hinge region when rabbit psoas muscle is switched on. These studies reveal (10) that the rate constant for (chymotryptic) cleavage within the hinge region of activated, glycerinated fibers, in an ATP- regenerating backup system, is 100 times larger at each temperature examined over the range 5-37?C than for re- laxed or rigor fibers. A close coupling between the force generated by actively cycling bridges and the fraction of bridges undergoing local melting in the S-2 hinge domain was observed. Evidence has been provided (11) that the isomet- ric contractile force is abolished in parallel with chemical (dimethyl suberimidate) cross-linking within the thick fila- ment backbone of glycerinated rabbit psoas fibers. Yet the myosin heads in such cross-linked activated fibers appear to cycle through neighboring actin filaments, cleaving ATP at an undiminished rate (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