Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Videos

A continuous, nonradioactive assay for histone acetyltransferases.

Y Kim1, K G Tanner, J M Denu

  • 1Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, Oregon, 97201-3098, USA.

Analytical Biochemistry
|May 3, 2000
PubMed
Summary

New continuous assays for histone acetyltransferases (HATs) accurately measure enzyme activity. These nonradioactive methods using coupled enzymes offer a reliable alternative to traditional radioactive assays for studying gene regulation.

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Investigating Histone Acetylation Stoichiometry and Turnover Rate.

Methods in enzymology·2016
Same author

Molecular reactions of protein phosphatases--insights from structure and chemistry.

Chemical reviews·2001
Same author

Calcium-mediated inactivation of the MAP kinase pathway in sea urchin eggs at fertilization.

Developmental biology·2001
Same author

Mutational analysis of conserved residues in the GCN5 family of histone acetyltransferases.

The Journal of biological chemistry·2001
Same author

Histone acetyltransferases.

Annual review of biochemistry·2001
Same author

Transition state analysis and requirement of Asp-262 general acid/base catalyst for full activation of dual-specificity phosphatase MKP3 by extracellular regulated kinase.

Biochemistry·2001

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Histone acetyltransferases (HATs) are crucial enzymes involved in gene regulation by modifying histone proteins.
  • Chromatin remodeling enzymes, including HATs, play a significant role in controlling gene activation.
  • Mechanistic studies of HATs are essential for understanding their role in biological processes.

Purpose of the Study:

  • To develop continuous, nonradioactive assays for quantifying the activity of the GCN5 HAT.
  • To provide a reliable and accessible method for mechanistic studies of HAT enzymes.
  • To compare the performance of the new assays with existing radioactive methods.

Main Methods:

  • Development of two continuous, nonradioactive assays utilizing coupled enzyme systems.

Related Experiment Videos

  • Measurement of CoASH production spectrophotometrically at 340 nm via NADH formation.
  • Determination of steady-state rate constants using acetyl-CoA and a synthetic H3 histone peptide substrate.

    • Main Results:

    • The newly developed continuous assays accurately measured GCN5 HAT activity.
    • Rate constants obtained from the two coupled assays were consistent, validating the methods.
    • Comparison with a radioactive filter-binding assay revealed significant differences in measured rate constants.

    Conclusions:

    • The developed continuous, nonradioactive assays provide a robust and validated method for studying HAT activity.
    • These assays offer advantages over traditional radioactive methods, addressing technical limitations.
    • The CoASH production-based methodology is broadly applicable to all HAT enzymes.