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Determining Diguanylate Cyclase Activity (Radioactive Assay).

Barbara I Kazmierczak1,2

  • 1Department of Medicine, Yale University, 333 Cedar St., New Haven, CT, 06520-8022, USA. barbara.kazmierczak@yale.edu.

Methods in Molecular Biology (Clifton, N.J.)
|September 11, 2017
PubMed
Summary

Researchers developed a sensitive radioactive assay to confirm diguanylate cyclase (DGC) enzyme activity. This method aids in validating bioinformatics predictions by easily separating DGC substrates and products.

Keywords:
Cyclic-di-GMPDiguanylate cyclaseThin-layer chromatography

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Bioinformatics tools can predict sequence motifs linked to diguanylate cyclases (DGCs).
  • Experimental validation of DGC enzymatic activity is crucial but often challenging.
  • Existing methods may lack the sensitivity or ease of use for rapid DGC activity assessment.

Purpose of the Study:

  • To describe a sensitive radioactive assay for quantifying diguanylate cyclase (DGC) activity.
  • To provide a protocol for the experimental demonstration of DGC enzymatic function.
  • To facilitate the validation of DGCs identified through bioinformatics analysis.

Main Methods:

  • Development of a radioactive assay utilizing a labeled substrate.
  • Employing thin-layer chromatography (TLC) for rapid separation of substrate and product.
  • Quantification of DGC activity based on the conversion of substrate to product.

Main Results:

  • The radioactive assay demonstrates high sensitivity in detecting DGC activity.
  • Thin-layer chromatography allows for quick and straightforward separation and analysis.
  • The protocol effectively confirms the enzymatic function of diguanylate cyclases.

Conclusions:

  • This radioactive TLC assay offers a sensitive and practical method for measuring DGC activity.
  • It serves as a valuable tool for validating bioinformatics predictions of DGC function.
  • The assay facilitates biochemical characterization of diguanylate cyclases.