Shape and substructure of skeletal muscle myosin light chain kinase.

To evaluate the shape and substructure of calmodulin-dependent myosin light chain kinase from skeletal muscle, the apo- and holoenzyme and three well-characterized proteolytic fragments were studied by enzymatic measurements, by hydrodynamic techniques, and by CD spectroscopy. For the native apoenzyme, a molecular weight of 70 300 was established by sedimentation equilibrium in contrast to greater than 80 000 estimated by electrophoresis. A highly asymmetric structure was evidenced from sedimentation and viscosity data. Examination of two slightly different calmodulin binding fragments of Mr approximately 36 000 showed that both are fairly globular, high in alpha-helix content, enzymatically active, and calmodulin regulated. They have been termed head fragments. The third fragment of Mr approximately 33 000 could be demonstrated to represent the remaining part of the native enzyme by its amino acid composition and CD spectrum. This enzymatically inactive fragment, although low in alpha-helix content and rich in proline, was shown to be highly asymmetric (a/b greater than 10). From the latter, termed tail fragment and one of the head fragments, a more active enzyme could be partially reconstituted. Modeling by spherical beads [Bloomfield, V., Dalton, W. O., & Van Holde, K. E. (1967) Biopolymers 5, 135-148] led to a close agreement in observed and calculated frictional ratios for all fragments as well as the apoenzyme built up by end to end arrangement of head and tail fragment, suggesting this headed structure for the enzyme. Holoenzyme formation by calmodulin binding to the head was accompanied by an increase in asymmetry and alpha-helix content and a decrease in apparent partial specific volume.