The tail of myosin reduces actin filament velocity in the in vitro motility assay.
It has been observed that heavy meromyosin (HMM) propels actin filaments to higher velocities than native myosin in the in vitro motility assay, yet the reason for this difference has remained unexplained. Since the major difference between these two proteins is the presence of the tail in native myosin, we tested the hypothesis that unknown interactions between actin and the tail (LMM) slow motility in native myosin. Chymotryptic HMM and LMM were mixed in a range of molar ratios (0-5 LMM/HMM) and compared to native rat skeletal myosin in the in vitro motility assay at 30 degrees C. Increasing proportions of LMM to HMM slowed actin filament velocities, becoming equivalent to native myosin at a ratio of 3 LMM/HMM. NH4+ -ATPase assays demonstrated that HMM concentrations on the surface were constant and independent of LMM concentration, arguing against a simple displacement mechanism. Relationships between velocity and the number of available heads suggested that the duty cycle of HMM was not altered by the presence of LMM. HMM prepared with a lower chymotrypsin concentration and with very short digestion times moved actin at the same high velocity. The difference between velocities of actin filament propelled by HMM and HMM/LMM decreased with increasing ionic strength, suggesting that ionic bonds between myosin tail and actin filaments may play a role in slowing filament velocity. These data suggest the high velocities of actin filaments over HMM result from the absence of drag generated by the myosin tail, and not from proteolytic nicking of the motor domain.
Guo
Bin
B
Department of Biomedical Engineering, University of Virginia, Charlottesville, VA 22908, USA.
Guilford
William H
WH
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000465:Algorithms; D000818:Animals; D005456:Fluorescent Dyes / Q000737:chemistry; D018482:Muscle, Skeletal / Q000378:metabolism*; D015879:Myosin Subfragments / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats
D005456:Fluorescent Dyes; D015879:Myosin Subfragments; D009218:Myosins