| Abstract: | The 20-kDa fragment of myosin subfragment-1 heavy chain was cleaved with cyanogen bromide. Gel electrophoresis of the fragmented peptides indicated the presence of 20-, 18-, 16-, 14-, 12-, and 10-kDa peptides in addition to two peptides smaller than 10 kDa. The renaturation procedure of Muhlrad and Morales (Muhlrad, A., and Morales, M. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 1003-1007) was applied to the mixture of these peptides. The peptides larger than 10 kDa, which contain both the reactive SH1 and SH2 groups, were precipitated with F-actin by ultracentrifugation. The 10-kDa peptide was purified and was identified as p10 of Elzinga and Collins (Elzinga, M., and Collins, J. H. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 4281-4284). The renaturation procedure was applied to the purified 10-kDa peptide. The 10-kDa peptide was also precipitated with F-actin by ultracentrifugation. Affinity of the 10-kDa peptide for F-actin was determined with an increase of turbidity, and the apparent dissociation constant was 0.94 microM. Results are consistent with our proposition that a binding site for F-actin exists around the SH1 and SH2 groups of subfragment-1 (Katoh, T., Imae, S., and Morita, F. (1984) J. Biochem. 95, 447-454; Katoh, T., and Morita, F. (1984) J. Biochem. 96, 1223-1230). |
