| Popis: |
Previously we have shown that the ATP-dependence of EDC-stabilized fluorescent smooth muscle myosin (SMM) filament motility is very similar to solution ATPase values (KATP = 8.5 and 9.2 µM, respectively). This suggests both processes are influenced by the same kinetic step, acto-myosin attachment not detachment, presumably linked to Pi release. This mechanism predicts that velocities will be linearly related to the number of working heads. Therefore, we prepared SMM- rod co-filaments that contain fewer molecules per filament length as well as longer SMM filaments that contain more molecules per filament to determine the relationship between the number of heads and velocity. SMM:rod co-filaments (at 50:50, 25:75, and 15:25 ratios) were approximately the same length as normal SMM filaments (∼0.6 µm), and the number of molecules incorporated was directly related to the ratio of SMM to rods. As the number of heads decreased, the maximal velocity at saturating ATP also decreased, and the KATP was lower for all of the co-filaments compared to the normal filaments. These findings provide evidence that the velocity of SMM filaments is influenced by attachment limited kinetics. The longer SMM filaments (average length of 2.3 ± 0.81 µm) will allow us to visualize the geometry of acto-myosin interactions during motion. Also, the relationship between velocity and filament length will be determined at various [ATP]. The long filaments also better demonstrate our previously described “parking” behavior where filaments move to the end of actin filaments but remain attached for long periods even at saturating [ATP] (Haldeman et al, JBC, 2014). Interestingly, most of the SMM filament hangs off the end of the actin suggesting the parking is mediated by a small fraction of the heads. |
