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Measurement of ATPase Activities of Myosin at the Level of Tracks and Single Molecules

  • Paul B. Conibear
  • Philip A. Kuhlman
  • Clive R. Bagshaw
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 453)

Abstract

In order to determine the degree of mechanochemical coupling in actomyosin in vitro motility assays, it is desirable to measure the sliding velocity and the associated ATP turnover simultaneously at the single filament level. Actin sliding over tracks of immobilised heavy meromyosin (HMM) has been initiated by flash photolysis of caged ATP. Flash photolysis has also been used to displace fluorescent Cy3-EDA-nucleotides from HMM tracks to monitor the ATPase activity. These assays are now being combined using total internal reflectance fluorescence (TIRF) microscopy with a dual-view detection system for Cy3/Cy5 labels on ATP and actin respectively.

In other experiments, we are exploring the use of the single molecule kinetic technique developed by Funatsu et al. (Nature 374, 555-559, 1995) to scale down ATPase assays of Dictyostelium myosin fragments and to elucidate the mechanism of regulation of the molluscan (scallop) myosin ATPase. Although fluctuations occur from the binding and release of Cy3-EDA-nucleotides during turnover and might provide a measure of the ATPase activity, other sources of fluctuations also need to be considered.

Keywords

ATPase Activity Actin Filament Myosin Head Flash Photolysis Motility Assay 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Plenum Press, New York 1998

Authors and Affiliations

  • Paul B. Conibear
    • 1
  • Philip A. Kuhlman
    • 1
  • Clive R. Bagshaw
    • 1
  1. 1.Department of BiochemistryUniversity of LeicesterLeicesterUK

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