Kinetic Characterization of Interactions between Stabilized Smooth Muscle Myosin Filaments and Actin

Abstract

Using our previously described EDC-cross-linked smooth muscle myosin (SMM) filaments that are stable to ATP-induced depolymerization, we measured kinetic parameters of the SMM filament interactions with actin comparing the filamentous physiological state with monomeric forms of SMM. We use stopped-flow spectrometry, actin-activated steady state Mg-ATPase, and in vitro motility assays to measure the fundamental kinetic differences between SMM monomers and filaments. Using stopped-flow, we show that the apparent second order rate constant for ATP binding to the complex of pyrene-actin with SMM filaments is 0.21 µM-1s-1, similar to HMM monomers with two heads, at 0.46 µM-1s-1, and single-headed S1 at 0.47 µM-1s-1. Using TIRF microscopy of filaments bound to surface-attached F-actin, we show ATP decreases the number of unphosphorylated SMM filaments bound per µm actin, giving an apparent KATP of 4.8 µM. We show that the ATP concentration that gives half-maximal velocities of actin moving over surface-attached phosphorylated SMM monomers is 21.2 µM (KATP). Using inverted-geometry to measure the velocities of phosphorylated SMM filaments moving over surface-attached actin gave a much lower KATP = 8.5 µM. Actin-activated steady-state Mg-ATPase of phosphorylated filaments gave a KATP = 9.2 µM. The similarities in the two KATPs for filaments suggest that filament velocities and ATPase are limited by the same kinetic step, which best fits an attachment-limited over a detachment-limited model. Using TIRF, we show that phosphorylated SMM filaments move over surface-attached actin filaments until they reach the ends from which they do not detach, even upon dephosphorylation of the SMM. This behavior is specific to SMM filaments since skeletal filaments readily detach from actin filament ends. These observations suggest that phosphorylated SMM filaments that have been dephosphorylated are in a different state than unphosphorylated filaments in the presence of ATP.