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Related Experiment Videos

A nonradioactive, high throughput assay for chitin synthase activity.

Héctor A Lucero1, Michael J Kuranda, Dorota A Bulik

  • 1Department of Molecular and Cell Biology, Goldman School of Dental Medicine, Boston University Medical Center, Boston, MA 02118, USA. hlucero@bu.edu

Analytical Biochemistry
|May 23, 2002
PubMed
Summary

This study presents a novel, nonradioactive assay for quantifying chitin synthase activity in yeast. The high-throughput method accurately measures chitin production, offering a sensitive alternative to traditional assays for studying yeast enzymes.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Yeast Genetics

Background:

  • Chitin synthases are crucial enzymes for fungal cell wall synthesis.
  • Existing radioactive assays for chitin synthase activity are cumbersome and pose safety concerns.
  • High-throughput, nonradioactive assays are needed for efficient screening of antimicrobial agents targeting chitin synthesis.

Purpose of the Study:

  • To adapt a patented assay for quantitative enzymatic measurement of individual yeast chitin synthase isozymes.
  • To develop a sensitive, high-throughput, and nonradioactive method for chitin synthase activity assessment.
  • To differentiate and quantify chitin synthase activities (Chs1, Chs2, Chs3) in Saccharomyces cerevisiae.

Main Methods:

  • Modification of a patented assay utilizing wheat germ agglutinin (WGA) for chitin binding.

Related Experiment Videos

  • Immobilization of synthesized chitin onto a WGA-coated surface.
  • Detection of chitin using a horseradish peroxidase-WGA conjugate and measurement of absorbance at 600 nm.
  • Quantification of chitin based on a standard curve using acid-solubilized chitin.

    • Main Results:

    • The assay demonstrated high sensitivity (lower limit of detection ~50 ng chitin) and low dispersion (<10%).
    • The 96-well microtiter plate format allows for high-throughput screening.
    • Differential assay of chitin synthase activities (Chs1, Chs2, Chs3) in yeast cell-free extracts was successfully performed.
    • Chitin synthase 3 (Chs3) activity was found to be approximately sixfold higher in plasma membrane fractions compared to chitosomal fractions.

    Conclusions:

    • The developed WGA-based assay is a sensitive, robust, and high-throughput alternative to radioactive methods for quantifying chitin synthase activity.
    • This assay facilitates the differential analysis of specific chitin synthase isozymes in yeast.
    • The findings highlight distinct localization and activity levels of Chs3 in different cellular compartments of Saccharomyces cerevisiae.