Abstract
Smooth muscle myosin acts as a molecular motor only if the regulatory light chain (RLC) is phosphorylated. This subunit can be removed from myosin by a novel method involving the use of trifluoperazine. The motility of RLC-deficient myosin is very slow, but native properties are restored when RLC is rebound. Truncating 6 residues from the COOH terminus of the RLC had no effect on phosphorylated myosin's motor properties, while removal of the last 12 residues reduced velocity by approximately 30%. Very slow movement was observed once 26 residues were deleted, or with myosin containing only the COOH-terminal RLC domain. These two mutants thus mimicked the behavior of RLC-deficient myosin, with the important difference that the mutant myosins were monodisperse when assayed by sedimentation velocity and electron microscopy. The decreased motility therefore cannot be caused by aggregation. A common feature of RLC-deficient myosin and the mutant myosins that moved actin slowly was an increased myosin ATPase compared with dephosphorylated myosin, and a lower actin-activated ATPase than obtained with phosphorylated myosin. These results suggest that the COOH-terminal portion of an intact RLC is involved in interactions that regulate myosin's "on-off" switch, both in terms of completely inhibiting and completely activating the molecule.