Abstract

We have measured kinetic parameters of smooth muscle myosin (SMM) filament interactions with actin, ADP, and ATP using stopped-flow fluorimetry. Our objective was to determine which parameters, if any, are different from those of monomeric myosins, S1 and HMM, using buffers at physiologic ionic strength. Previously we measured the ATP-dependence of SMM filament velocities (using EDC-cross-linked filaments that are stable to ATP-induced depolymerization) in an inverted in vitro motility assay with myosin filaments moving over surface-attached actin. We compared this to the ATP-dependence of solution actin-activated ATPase measurements. The two relationships are very similar, suggesting that they are both attachment-limited and regulated by the same kinetic step, presumably phosphate release (Haldeman et al JBC 2014). This is in contrast to the kinetics of actin moving over a surface of monomeric SMM (standard vitro motility assay), known to be predominately detachment-limited, limited by ADP release. We hypothesized that heads within moving filaments are attached to unhindered and thus flexible S2 regions putting minimal strain on heads put in the drag position. In contrast, the S2 region of monomeric myosin is bound to the surface and not as flexible allowing the relative stiff head domains to become strained, which affects the underlying kinetics of acto-SMM interactions. All kinetic parameters measured so far in solution for myosin or acto-myosin, ADP association, ADP dissociation, ATP binding, ATP-induced dissociation, and acto-myosin affinity, are very similar to those for S1 and HMM. Measurements of Pi release kinetics by limited turnover are planned. If Pi release kinetics is also similar, this will suggest that the attachment-limited kinetics that is unique to filaments is not due to alterations in any fundamental kinetic parameters, but instead is due to differences in the effective head or head/S2 stiffness during the assays.

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