Journal
CELL MOTILITY AND THE CYTOSKELETON
Volume 59, Issue 4, Pages 264-272Publisher
WILEY-LISS
DOI: 10.1002/cm.20040
Keywords
heavy meromyosin; light meromyosin; motility assay; ATPase assay; ionic strength
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Funding
- NHLBI NIH HHS [HL64381] Funding Source: Medline
- NIAMS NIH HHS [AR45604] Funding Source: Medline
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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 30degreesC. 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. (C) 2004 Wiley-Liss, Inc.
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