Evidence that Actin-Myosy Cycling in Muscle may not Pass through Rigor Configuration

Abstract

In 1989, Sutoh, Tokunaga and Wakabayashi (J Mol Biol 206:357) prepared a monoclonal antibody (IgG) directed to junctional peptide between 50- and 20-kDa segments of myosin heavy chain, and showed that the antibody attaches around the tip of myosin heads facing actin filaments in muscle. using the same antibody, we successfuly position-mark the tip of myosin heads in synthetic myosin filaments kept in hydrated state in the gas environmental chamber, and succeeded in recording ATP-induced myosin head movement electron microscopically (Sugi et al. PNAS 105: 17396, 2008). We have examined the effect of this antibody on both ATP-dependent in vitro actin-myosin sliding and Ca-activated contraction of permealized rabbit psoas muscle fibers. Unexpectedly, the antibody (concentration, up to 2mg/ml) had no appreciable effect on the velocity profile of actin-myosin sliding and the contraction characteristics of muscle fibers, including magnitude of the maximum Ca-activated isometric force, the maximum unloaded shortening velocity, ans MgATPase activity during contraction. Since the junctional peptide is located between the two main actin- binding sites of myosin heads during formation of rigor linkages, attachment of massive antibodies to myosin heads completely covers their actin- binding sites. The possibility that the antibody does not diffuse into muscle fibers is excluded by the fact that other kinds of antibody diffuse into muscle fibers to inhibit Ca-activated force generation (e.g., Sugi et al. PNAS 89: 6134, 1992). Our work, therefore, suggests strongly that attachment-detachment cycle between actin and myosin in muscle may not pass through rigor configuration. It seems possible that so-called strong actin-myosin binding might be due to some electrostatic mechanism.