Velocity-dependent actomyosin ATPase cycle revealed by in vitro motility assay with kinetic analysis

Masaaki K. Sato, Takashi Ishihara, Hiroto Tanaka, Akihiko Ishijima, Yuichi Inoue

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

The actomyosin interaction plays a key role in a number of cellular functions. Single-molecule measurement techniques have been developed to study the mechanism of the actomyosin contractile system. However, the behavior of isolated single molecules does not always reflect that of molecules in a complex system such as a muscle fiber. Here, we developed a simple method for studying the kinetic parameters of the actomyosin interaction using small numbers of molecules. This approach does not require the specialized equipment needed for single-molecule measurements, and permits us to observe behavior that is more similar to that of a complex system. Using an in vitro motility assay, we examined the duration of continuous sliding of actin filaments on a sparsely distributed heavy meromyosin-coated surface. To estimate the association rate constant of the actomyosin motile system, we compared the distribution of experimentally obtained duration times with a computationally simulated distribution. We found that the association rate constant depends on the sliding velocity of the actin filaments. This technique may be used to reveal new aspects of the kinetics of various motor proteins in complex systems.

Original languageEnglish
Pages (from-to)711-718
Number of pages8
JournalBiophysical Journal
Volume103
Issue number4
DOIs
Publication statusPublished - Aug 22 2012
Externally publishedYes

Fingerprint

Actomyosin
Myosins
Actin Cytoskeleton
Myosin Subfragments
Equipment and Supplies
Muscles
In Vitro Techniques
Proteins

ASJC Scopus subject areas

  • Biophysics

Cite this

Velocity-dependent actomyosin ATPase cycle revealed by in vitro motility assay with kinetic analysis. / Sato, Masaaki K.; Ishihara, Takashi; Tanaka, Hiroto; Ishijima, Akihiko; Inoue, Yuichi.

In: Biophysical Journal, Vol. 103, No. 4, 22.08.2012, p. 711-718.

Research output: Contribution to journalArticle

Sato, Masaaki K. ; Ishihara, Takashi ; Tanaka, Hiroto ; Ishijima, Akihiko ; Inoue, Yuichi. / Velocity-dependent actomyosin ATPase cycle revealed by in vitro motility assay with kinetic analysis. In: Biophysical Journal. 2012 ; Vol. 103, No. 4. pp. 711-718.
@article{328f92674a0b42238213ecef843bfecb,
title = "Velocity-dependent actomyosin ATPase cycle revealed by in vitro motility assay with kinetic analysis",
abstract = "The actomyosin interaction plays a key role in a number of cellular functions. Single-molecule measurement techniques have been developed to study the mechanism of the actomyosin contractile system. However, the behavior of isolated single molecules does not always reflect that of molecules in a complex system such as a muscle fiber. Here, we developed a simple method for studying the kinetic parameters of the actomyosin interaction using small numbers of molecules. This approach does not require the specialized equipment needed for single-molecule measurements, and permits us to observe behavior that is more similar to that of a complex system. Using an in vitro motility assay, we examined the duration of continuous sliding of actin filaments on a sparsely distributed heavy meromyosin-coated surface. To estimate the association rate constant of the actomyosin motile system, we compared the distribution of experimentally obtained duration times with a computationally simulated distribution. We found that the association rate constant depends on the sliding velocity of the actin filaments. This technique may be used to reveal new aspects of the kinetics of various motor proteins in complex systems.",
author = "Sato, {Masaaki K.} and Takashi Ishihara and Hiroto Tanaka and Akihiko Ishijima and Yuichi Inoue",
year = "2012",
month = "8",
day = "22",
doi = "10.1016/j.bpj.2012.07.014",
language = "English",
volume = "103",
pages = "711--718",
journal = "Biophysical Journal",
issn = "0006-3495",
publisher = "Biophysical Society",
number = "4",

}

TY - JOUR

T1 - Velocity-dependent actomyosin ATPase cycle revealed by in vitro motility assay with kinetic analysis

AU - Sato, Masaaki K.

AU - Ishihara, Takashi

AU - Tanaka, Hiroto

AU - Ishijima, Akihiko

AU - Inoue, Yuichi

PY - 2012/8/22

Y1 - 2012/8/22

N2 - The actomyosin interaction plays a key role in a number of cellular functions. Single-molecule measurement techniques have been developed to study the mechanism of the actomyosin contractile system. However, the behavior of isolated single molecules does not always reflect that of molecules in a complex system such as a muscle fiber. Here, we developed a simple method for studying the kinetic parameters of the actomyosin interaction using small numbers of molecules. This approach does not require the specialized equipment needed for single-molecule measurements, and permits us to observe behavior that is more similar to that of a complex system. Using an in vitro motility assay, we examined the duration of continuous sliding of actin filaments on a sparsely distributed heavy meromyosin-coated surface. To estimate the association rate constant of the actomyosin motile system, we compared the distribution of experimentally obtained duration times with a computationally simulated distribution. We found that the association rate constant depends on the sliding velocity of the actin filaments. This technique may be used to reveal new aspects of the kinetics of various motor proteins in complex systems.

AB - The actomyosin interaction plays a key role in a number of cellular functions. Single-molecule measurement techniques have been developed to study the mechanism of the actomyosin contractile system. However, the behavior of isolated single molecules does not always reflect that of molecules in a complex system such as a muscle fiber. Here, we developed a simple method for studying the kinetic parameters of the actomyosin interaction using small numbers of molecules. This approach does not require the specialized equipment needed for single-molecule measurements, and permits us to observe behavior that is more similar to that of a complex system. Using an in vitro motility assay, we examined the duration of continuous sliding of actin filaments on a sparsely distributed heavy meromyosin-coated surface. To estimate the association rate constant of the actomyosin motile system, we compared the distribution of experimentally obtained duration times with a computationally simulated distribution. We found that the association rate constant depends on the sliding velocity of the actin filaments. This technique may be used to reveal new aspects of the kinetics of various motor proteins in complex systems.

UR - http://www.scopus.com/inward/record.url?scp=84865369390&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84865369390&partnerID=8YFLogxK

U2 - 10.1016/j.bpj.2012.07.014

DO - 10.1016/j.bpj.2012.07.014

M3 - Article

VL - 103

SP - 711

EP - 718

JO - Biophysical Journal

JF - Biophysical Journal

SN - 0006-3495

IS - 4

ER -