Substructure of Myosin Subfragment-1 as Revealed by Digestion with Proteolytic Enzymes

Abstract

To elucidate the substructure of myosin subfragment-1, we digested it with trypsin and papain and determined the sites of its heavy chain and light chains which were readily attacked by these proteolytic enzymes. It was found that a region separated from the N-terminal by about 96 K was easily attacked by trypsin, chymotrypsin, and papain. Since the specificities of these enzymes are quite different, the region may have a special structure, and it seems to be located at the flexible hinge between subfragment-1 and the rod. A close relation between the susceptibility of the region and the presence of DTNB light chain was found. The result suggests that some portion of the DTNB light chain is associated with subfragment-1 at this region. Another region separated from the N-terminal by about 26 K was found to be attacked readily by trypsin and papain. Since papain has a broad specificity, the region also seems to have some special structure which allows easy access of the enzyme. Alkali light chain 1 was degraded to a roughly 23 K fragment by both papain and trypsin, whereas alkali light chain 2 was resistant to these enzymes. The results indicate that the sites attacked by papain and trypsin were located in the N-terminal extra peptide of alkali light chain 1. When DTNB light chain was degraded to a roughly 17 K fragment by trypsin, Cys 157 was not dissociated from the DTNB light chain. This means that trypsin did not attack Lys 154 of the light chain. It is suggested, therefore, that trypsin attacked both Arg 7 and Lys 166 when it produced the 17 K fragment.