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Related Experiment Videos

A microtiter-based fluorescence assay for (1,3)-beta-glucan synthases

E Shedletzky1, C Unger, D P Delmer

  • 1Department of Plant Sciences, Hebrew University of Jerusalem, Israel.

Analytical Biochemistry
|June 15, 1997
PubMed
Summary

A new, inexpensive assay for UDP-Glc:(1,3)-beta-glucan synthase uses nonradioactive substrates and aniline blue detection. This method is safe, reproducible, and ideal for high-throughput screening of enzyme inhibitors.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Plant and Fungal Biology

Background:

  • UDP-Glc:(1,3)-beta-glucan synthase is crucial for fungal cell wall and plant callose synthesis.
  • Existing assays often rely on expensive or hazardous radioactive substrates.
  • A need exists for cost-effective, safe, and high-throughput methods for enzyme activity assessment.

Purpose of the Study:

  • To develop a novel, cost-effective, high-throughput assay for UDP-Glc:(1,3)-beta-glucan synthase.
  • To enable sensitive detection of enzyme activity using nonradioactive reagents.

Main Methods:

  • A microtiter plate-based assay was designed using nonradioactive UDP-glucose as a substrate.
  • The produced nonradioactive glucan was quantified via a fluorescent complex with aniline blue.

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  • Enzyme activity was measured by fluorescence intensity.

    • Main Results:

    • The assay demonstrated a >100-fold cost reduction compared to traditional radioactive methods.
    • High reproducibility and sensitivity, comparable to radioactive assays, were achieved.
    • The assay eliminated the need for filtration and washing steps, enhancing safety and efficiency.

    Conclusions:

    • This novel assay provides a significantly cheaper, safer, and more efficient alternative for studying UDP-Glc:(1,3)-beta-glucan synthase.
    • The assay is highly suitable for high-throughput screening of inhibitors targeting this enzyme.
    • The method facilitates broader research into glucan synthesis in fungi and plants.