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Related Experiment Videos

Colorimetric assay for cellular transglutaminase.

W M Jeon1, K N Lee, P J Birckbichler

  • 1Biomedical Division, Samuel Roberts Noble Foundation, Inc., Ardmore, Oklahoma 73402.

Analytical Biochemistry
|October 1, 1989
PubMed
Summary

A new colorimetric assay quantifies cellular transglutaminase activity. This rapid, sensitive method uses biotinylated substrates and a nonisotopic detection system for accurate enzyme measurement.

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Area of Science:

  • Biochemistry
  • Enzymology

Background:

  • Transglutaminases are crucial enzymes involved in various cellular processes.
  • Accurate measurement of transglutaminase activity is essential for understanding cellular function and disease.
  • Existing methods, such as radiometric assays, can be time-consuming and involve radioactivity.

Purpose of the Study:

  • To develop a novel, rapid, and sensitive colorimetric assay for quantifying cellular transglutaminase activity.
  • To validate the assay's performance against established methods.
  • To establish the assay's utility for both purified enzymes and crude cellular preparations.

Main Methods:

  • A colorimetric assay was developed utilizing 5-(biotinamido)pentylamine as a substrate for transglutaminase.
  • Biotinylated N,N-dimethylcasein products were immobilized on polyvinylidine difluoride membranes.

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  • Detection involved streptavidin-beta-galactosidase conjugation and measurement of p-nitrophenol absorbance at 405 nm.

    • Main Results:

    • The assay demonstrated a strong correlation (gamma = 0.922) with the radiometric filter paper method.
    • High sensitivity was achieved, detecting as little as 20 ng of purified guinea pig liver transglutaminase.
    • The assay effectively quantified activity in crude fibroblast cytosol protein (10 micrograms).

    Conclusions:

    • A robust, nonisotopic colorimetric assay for cellular transglutaminase has been successfully developed.
    • This assay provides a rapid, sensitive, and reliable alternative to existing methods.
    • The assay is suitable for diverse applications, including research with crude cellular extracts and purified enzymes.