Assessment of the mechanical activity of cardiac isomyosins V1 and V3 by the in vitro motility assay with regulated thin filament

BIOPHYSICS ◽  
2008 ◽  
Vol 53 (6) ◽  
pp. 510-514 ◽  
Author(s):  
L. V. Nikitina ◽  
G. V. Kopylova ◽  
D. V. Shchepkin ◽  
L. B. Katsnel’son
Keyword(s):  
Thin Filament ◽  
Motility Assay ◽  
Mechanical Activity ◽  
In Vitro Motility Assay ◽  
In Vitro Motility ◽  
Regulated Thin Filament

The effects of phosphate and acidosis on regulated thin-filament velocity in an in vitro motility assay

2012 ◽  
Vol 113 (9) ◽  
pp. 1413-1422 ◽  
Author(s):  
Edward P. Debold ◽  
Thomas J. Longyear ◽  
Matthew A. Turner
Keyword(s):  
Molecular Basis ◽  
Bound States ◽  
Contractile Activity ◽  
Motility Assay ◽  
Shortening Velocity ◽  
In Vitro Motility Assay ◽  
In Vitro Motility ◽  
Specific Step ◽  
Regulated Thin Filament

Muscle fatigue from intense contractile activity is thought to result, in large part, from the accumulation of inorganic phosphate (Pi) and hydrogen ions (H+) acting to directly inhibit the function of the contractile proteins; however, the molecular basis of this process remain unclear. We used an in vitro motility assay and determined the effects of elevated H+ and Pi on the ability of myosin to bind to and translocate regulated actin filaments (RTF) to gain novel insights into the molecular basis of fatigue. At saturating Ca++, acidosis depressed regulated filament velocity ( VRTF) by ∼90% (6.2 ± 0.3 vs. 0.5 ± 0.2 μm/s at pH 7.4 and 6.5, respectively). However, the addition of 30 mM Pi caused VRTF to increase fivefold, from 0.5 ± 0.2 to 2.6 ± 0.3 μm/s at pH 6.5. Similarly, at all subsaturating Ca++ levels, acidosis slowed VRTF, but the addition of Pi significantly attenuated this effect. We also manipulated the [ADP] in addition to the [Pi] to probe which specific step(s) of cross-bridge cycle of myosin is affected by elevated H+. The findings are consistent with acidosis slowing the isomerization step between two actomyosin ADP-bound states. Because the state before this isomerization is most vulnerable to Pi rebinding, and the associated detachment from actin, this finding may also explain the Pi-induced enhancement of VRTF at low pH. These results therefore may provide a molecular basis for a significant portion of the loss of shortening velocity and possibly muscular power during fatigue.

scholarly journals Calcium regulation of thin filament movement in an in vitro motility assay

1996 ◽  
Vol 70 (4) ◽  
pp. 1881-1892 ◽  
Author(s):  
E. Homsher ◽  
B. Kim ◽  
A. Bobkova ◽  
L.S. Tobacman
Keyword(s):  
Thin Filament ◽  
Calcium Regulation ◽  
Motility Assay ◽  
In Vitro Motility Assay ◽  
In Vitro Motility

scholarly journals Ca++-sensitizing mutations in troponin, Pi, and 2-deoxyATP alter the depressive effect of acidosis on regulated thin-filament velocity

2014 ◽  
Vol 116 (9) ◽  
pp. 1165-1174 ◽  
Author(s):  
Thomas J. Longyear ◽  
Matthew A. Turner ◽  
Jonathan P. Davis ◽  
Joseph Lopez ◽  
Brandon Biesiadecki ◽  
...  
Keyword(s):  
Thin Filament ◽  
Contractile Activity ◽  
Dependent Manner ◽  
In Vitro Motility Assay ◽  
Depressive Effect ◽  
In Vitro Motility ◽  
Acidic Conditions ◽  
Underlying Mechanisms ◽  
Regulated Thin Filament

Repeated, intense contractile activity compromises the ability of skeletal muscle to generate force and velocity, resulting in fatigue. The decrease in velocity is thought to be due, in part, to the intracellular build-up of acidosis inhibiting the function of the contractile proteins myosin and troponin; however, the underlying molecular basis of this process remains poorly understood. We sought to gain novel insight into the decrease in velocity by determining whether the depressive effect of acidosis could be altered by 1) introducing Ca++-sensitizing mutations into troponin (Tn) or 2) by agents that directly affect myosin function, including inorganic phosphate (Pi) and 2-deoxy-ATP (dATP) in an in vitro motility assay. Acidosis reduced regulated thin-filament velocity ( VRTF) at both maximal and submaximal Ca++ levels in a pH-dependent manner. A truncated construct of the inhibitory subunit of Tn (TnI) and a Ca++-sensitizing mutation in the Ca++-binding subunit of Tn (TnC) increased VRTF at submaximal Ca++ under acidic conditions but had no effect on VRTF at maximal Ca++ levels. In contrast, both Pi and replacement of ATP with dATP reversed much of the acidosis-induced depression of VRTF at saturating Ca++. Interestingly, despite producing similar magnitude increases in VRTF, the combined effects of Pi and dATP were additive, suggesting different underlying mechanisms of action. These findings suggest that acidosis depresses velocity by slowing the detachment rate from actin but also by possibly slowing the attachment rate.

Caged FEDA-ATP: a new tool in the measurement of ATP turnover during the in vitro motility assay

1995 ◽  
Vol 23 (3) ◽  
pp. 401S-401S ◽  
Author(s):  
Daren S. Jeffreys ◽  
Robert J. Eaton ◽  
Clive R. Bagshaw
Keyword(s):  
Motility Assay ◽  
In Vitro Motility Assay ◽  
In Vitro Motility ◽  
Atp Turnover

scholarly journals An automated in vitro motility assay for high-throughput studies of molecular motors

Lab on a Chip ◽  
2018 ◽  
Vol 18 (20) ◽  
pp. 3196-3206 ◽  
Author(s):  
Till Korten ◽  
Elena Tavkin ◽  
Lara Scharrel ◽  
Vandana Singh Kushwaha ◽  
Stefan Diez
Keyword(s):  
High Throughput ◽  
Molecular Motors ◽  
Lab On A Chip ◽  
Force Generation ◽  
Motility Assay ◽  
In Vitro Motility Assay ◽  
In Vitro Motility ◽  
Cargo Transport

Molecular motors, essential to force-generation and cargo transport within cells, are invaluable tools for powering nanobiotechnological lab-on-a-chip devices.

scholarly journals Determination of the Maximum Velocity of Filaments in the in vitro Motility Assay

2019 ◽  
Vol 10 ◽  
Author(s):  
Nasrin Bopp ◽  
Lisa-Mareike Scheid ◽  
Rainer H. A. Fink ◽  
Karl Rohr
Keyword(s):  
Maximum Velocity ◽  
Motility Assay ◽  
In Vitro Motility Assay ◽  
In Vitro Motility
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