EFFECT OF UNPHOSPHORYLATED SMOOTH-MUSCLE MYOSIN ON CALDESMON-MEDIATED REGULATION OF ACTIN FILAMENT VELOCITY

The effect of smooth muscle myosin at different levels of light chain phosphorylation on caldesmon-mediated movement of actin filaments was investigated using an in vitro motility assay. Myosin at different levels of phosphorylation was obtained by mixing different proportions of fully phosphorylated and unphosphorylated myosin in monomeric form, while keeping the total myosin concentration constant. The average velocity of actin filaments containing tropomyosin was 1.20 +/- 0.046 mu ms(-1) at 30 degrees C with fully phosphorylated myosin. This velocity was not altered when the percentage of unphosphorylated myosin coated on the nitrocellulose surface was increased to 80%; further increases lowered the velocity. When the act-in filaments with caldesmon bound at stoichiometric levels were used, filament velocity was unaffected until 50% of the myosin was unphosphorylated, but further increases in the percentage of unphosphorylated myosin induced a decrease in the velocity, and at 95% unphosphorylated myosin, filament movement had ceased. The decreased filament velocity in the presence of caldesmon was also observed when phosphorylated myosin was mixed with myosin rod instead of unphosphorylated myosin, but was not observed when the 38 kDa caldesmon C-terminal fragment, which lacks the myosin-binding domain, was used instead of intact caldesmon. These data indicate that the decreased filament velocity in the presence of caldesmon reflects the mechanical load produced by the tethering of actin to myosin through the interaction of the caldesmon N-terminal domain and the myosin S-2 region. The tethering effect mediated by caldesmon may play a role in smooth muscle contraction when a large number of myosin heads are dephosphorylated, as in force maintenance.