Homologous Metal-Binding Sites of Myosin Regulatory Light Chains Revealed by the Paramagnetic Manganous Ion

Abstract

After the discovery in molluscan muscles of the myosin-linked regulatory system of muscle contraction (Kendrick-Jones et al., 1970), there has been much interest in the Caz+-binding sites of both molluscan and vertebrate myosins. Scallop myosin binds two Caz+ ions specifically, and exhibits a Caz+-sensitive actin-activated ATPaseJ whereas rabbit skeletal myosin binds two Ca’+ ions non-specifically and has a Ca2+-insensitive ATPase. However, the rabbit Nbs2 light chain, which bears the non-specific Caz+ site (Bagshaw, 1977), appears to substitute for the scallop regulatory light chain in hybrid myofibrils (Kendrick-Jones, 1974). It would be of interest to know if these Ca2+-binding sites are related. Has the property of specificity been lost during the preparation or evolution of the rabbit myosin? Do the properties of the non-specific Ca2+-binding site of the rabbit Nbs, light chain change when it is hybridized with scallop myofibrils? The paramagnetic Mn2+ ion is a convenient probe to study the metal-ion-binding sites of proteins. Generally the e.p.r. spectrum of bound Mnz+ is broad; however, the sites of rabbit myosin are an exception (Bagshaw & Reed, 1976). Here the intensity of the derivative spectrum is only decreased to about 20% of that of Mn(HzO),2+. The characteristic spectrum of the bound Mn2+ is readily observed for the isolated Nbs, light chain (Bagshaw, 1977) and dominates the signals from all other sites within a glycerinated muscle fibre (Bagshaw &Reed, 1977). Mn” therefore seemed an ideal probe for a comparative investigation of myosins from other sources. Studies with scallop myofibrils showed that Mn2+ does not compete for the Ca2+specific sites, and the characteristic intense e.p.r. spectrum was not observed. Although this situation ruled out a direct investigation of the Ca2+-specific sites, MnZ+ could be used to probe scallop hybrid myofibrils containing a rabbit Nbs, light chain. Figs. l (a)l(d) show that the characteristic e.p.r. spectrum is only observed when the rabbit light chain is present. The lineshape is comparable with that of the isolated Nbs2 light chain (Fig. l h ) and rabbit myofibrils themselves (Fig. If) . Addition of Caz+ or Mg2+ to the hybrid myofibrils caused a decrease in the signal from the bound MnZ+. It is concluded that the bivalent-metal-ion site of the Nbs, light chain remains non-specific in the hybrid, and confirms that this subunit plays an indirect role in the Caz+-resensitization process (Kendrick-Jones et al., 1976). Myofibrils from other species of mollusc, notably the clams, possess Mnz+-binding sites homologous with those of the rabbit (Fig. l e ) . The location of these sites also proved to be the regulatory class of light chains (Fig. Ig) . To clarify the relationship between these sites, the total number of high-affinity, bivalent-metal-ion sites of rabbit, scallop and clam myosins were estimated by equilibration against various concentrations of Ca2+, Mg2+ and Mn2+. Rabbit skeletal myosin possesses two non-specific sites