ATP-dependent movement of myosin in vitro

Characterization of a quantitative assay

Michael Sheetz, R. Chasan, J. A. Spudich

Research output: Contribution to journalArticle

75 Citations (Scopus)

Abstract

Sheetz and Spudich (1983, Nature (Lond.), 303:31-35) showed that ATP-dependent movement on myosin along actin filaments can be measured in vitro using myosin-coated beads and oriented actin cables from Nitella. To establish this in vitro movement as a quantitative assay and to understand better the basis for the movement, we have defined the factors that affect the myosin-bead velocity. Beads coated with skeletal muscle myosin move at a rate of 2-6 μm/s, depending on the myosin preparation. This velocity is independent of myosin concentration on the bead surface for concentrations above a critical value (~ 20 μg myosin/2.5 x 109 beads of 1 μm in diameter). Movement is optimal between pH 6.8 and 7.5, at KCl concentrations <70 mM, ATP concentrations >0.1 mM, and at Mg2+ concentrations between 2 and 6 mM. From the temperature dependence of bead velocity, we calculate activation energies of 91 kJ/mol below 22°C and 40 kJ/mol above 22° C. Different myosin species move at their own characteristic velocities, and these velocities are proportional to their actin-activated ATPase activities. Further, the velocities of beads coated with smooth or skeletal muscle myosin correlate well with the known in vivo rates of myosin movement along actin filaments in these muscles. This in vitro assay, therefore, provides a rapid, reproducible method for quantitating the ATP-dependent movement of myosin molecules on actin.

Original languageEnglish (US)
Pages (from-to)1867-1871
Number of pages5
JournalJournal of Cell Biology
Volume99
Issue number5
DOIs
StatePublished - Jan 1 1984
Externally publishedYes

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Myosins
Adenosine Triphosphate
Skeletal Muscle Myosins
Actin Cytoskeleton
Actins
Nitella
In Vitro Techniques
Smooth Muscle Myosins
Muscles
Temperature

ASJC Scopus subject areas

  • Cell Biology

Cite this

ATP-dependent movement of myosin in vitro : Characterization of a quantitative assay. / Sheetz, Michael; Chasan, R.; Spudich, J. A.

In: Journal of Cell Biology, Vol. 99, No. 5, 01.01.1984, p. 1867-1871.

Research output: Contribution to journalArticle

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