Use of a Real-Time, Coupled Assay to Measure the ATPase Activity of DNA Topoisomerase II

  • Janet E. Lindsley
Part of the Methods in Molecular Biology™ book series (MIMB, volume 95)


This chapter describes the use of a common spectrophotometric assay for following the rate of ATP hydrolysis as applied to type II DNA topoisomerases. It is called a “coupled assay,” because each time an ATP molecule is hydro-lyzed, a molecule of NADH is rapidly oxidized; ATP hydrolysis and NADH oxidation are therefore coupled. Although NADH absorbs strongly at 340 nm, the product of its oxidation, NAD+, does not. Therefore, the rate of ATP hydrolysis can be determined by following the decrease in optical absorbance of the reaction at 340 nm.


ATPase Activity Lactate Dehydrogenase Pyruvate Kinase NADH Oxidation Coupling Enzyme 
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.


  1. 1.
    Kornberg A. and Pricer W. E., Jr. (1951) Enzymatic phosphorylation of adenosine and 2,6-diaminopurine riboside. J. Biol. Chem. 193, 481–495.PubMedGoogle Scholar
  2. 2.
    Adams H. (1965) Adenosine-5′-diphosphate and adenosine-5′-monophosphate in Methods of Enzymatic Analysis (Bergmeyer H. U., ed.), Academic, New York, pp. 573–577.Google Scholar
  3. 3.
    Lindsley J. E. and Wang J. C. (1993) On the coupling between ATP usage and DNA transport by yeast DNA topoisomerase II. J. Biol. Chem. 268, 8096–8104.PubMedGoogle Scholar
  4. 4.
    Lowry O. H. and Passonneau J. V. (1972) A Flexible System of Enzyme Analysis, Academic, New York, pp. 4–20.Google Scholar
  5. 5.
    Worland S. T. and Wang J. C. (1989) Inducible overexpression, purification, and active site mapping of DNA topoisomerase II from the yeast Saccharomyces cerevisiae. J. Biol. Chem. 264, 4412–4416.PubMedGoogle Scholar
  6. 6.
    Osheroff N., Shelton E. R., and Brutlag D. L. (1983) DNA Topoisomerase II from Drosophila melanogaster: Relaxation of Supercoiled DNA. J. Biol. Chem. 258, 9536–9543.PubMedGoogle Scholar
  7. 7.
    Sambrook J., Fritsch E. F., and Maniatis T. (1989) Purification of plasmid DNA, in: Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, p. 1.42.Google Scholar
  8. 8.
    Tamura J. K. and Gellert M. (1990) Characterization of the ATP binding site on Escherichia coli DNA gyrase. J. Biol. Chem. 265, 21,342–21,349.PubMedGoogle Scholar
  9. 9.
    Tamura J. K., Bates A. D., and Gellert M. (1992) Slow Interaction of 5′-Adenylyl-β,γ-imidodiphosphate with Esherichia coli DNA gyrase. J. Biol. Chem. 267, 9214–9222.PubMedGoogle Scholar

Copyright information

© Humana Press Inc. 2001

Authors and Affiliations

  • Janet E. Lindsley

There are no affiliations available

Personalised recommendations