Abstract
Skeletal muscle actin was lightly digested by proteinase K, which cleaved the peptide bond between Met-47 and Gly-48, producing a C-terminal 35 kDa fragment. Proteinase K-cleaved actin (proK-actin) did not polymerize into F-actin upon addition of salt. In the presence of phalloidin, however, it polymerized slowly into F-actin (proK-F-actin), indicating that the cleaved actin did not dissociate into the individual cleaved fragments but retained the global structure of actin. Electron microscopy showed that proK-F-actin had the typical double-stranded structure of a normal actin filament and formed the arrowhead structure when decorated with HMM. Heavy meromyosin ATPase was weakly activated by proK-F-actin: Vmax=0.24s-1, and Kapp=2.8μM, while Vmax=7.6s-1, and Kapp=13μM by F-actin. Correspondingly, in vitro this proK-F-actin slid very slowly on HMM attached to a glass surface at an average velocity of 0.47μm/s, or 1/12 of that of intact F-actin. The fraction of sliding filaments was less than 50%. Assuming that the nonmotile filaments attached to HMM were not involved in ATPase activation, the sliding velocity correlated with the ATPase activity activated by proK-F-actin.
