Substructure of the myosin molecule: II. The light chains of myosin

Abstract

The low molecular weight components (light chains) dissociated from rabbit skeletal muscle myosin under a variety of denaturing conditions have been characterized by chemical and physico-chemical methods. Three electrophoretic components have been identified on acrylamide gels run both in the presence and absence of sodium dodecyl sulphate, indicating differences in net charge and molecular weight. These three light chains have been separated by chromatography, and their thiol sequences determined. The results show that two of the three are chemically related in that they contain an identical thiol sequence, while the third component is chemically different. This third component can be removed from myosin by treatment with 5,5′-dithio-(bis-2-nitrobenzoic acid), without loss of ATPase activity, showing that this light chain (called the DTNB light chain) is not essential for activity. The other two light chains cannot be removed without total loss of ATPase activity, and these are termed the alkali light chains since they are dissociated from myosin at pH 11. The stoichiometry of these two classes of light chain has been determined by isotope dilution, using as markers the [ 14C]iodoacetate-labelled peptides of the different light chains, and isolating these peptides from purified light chains and from mixtures of [ 14C]iodoacetate-labelled myosin and 14C-labelled light chains. The results confirm that both myosin and heavy meromyosin contain two moles of the essential alkali light chains, while heavy meromyosin subfragment 1 has a single alkali light chain non-covalently bound to it. There also appear to be two moles of the DTNB light chain attached to myosin and to heavy meromyosin but heavy meromyosin subfragment-1 contains less than half the expected number, indicating that this light chain may be susceptible to papain digestion. The two different alkali light chains have been compared by molecular weight estimation, by peptide mapping and by amino-acid compositions. These data indicate close chemical similarity of the two light chains, but a difference in molecular weight of up to 9000 daltons. The chemical studies indicate that these two proteins may contain a large segment of similar sequence, and the possibility that the A2 component is a fragment of A1 is considered. The significance of the two different alkali light chains is discussed in terms of the existence of possible isoenzymes of myosin.