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A continuous fluorometric assay for gamma-glutamyltranspeptidase
I N White1, N Razvi, R M Lawrence
1MRC Toxicology Unit, University of Leicester, United Kingdom.
Analytical Biochemistry
|January 1, 1996
Summary
A new fluorescent substrate allows sensitive detection of gamma-glutamyltranspeptidase (GGT) enzyme activity. This method accurately measures GGT levels in cells and can detect changes, such as those induced by tamoxifen.
Area of Science:
- Biochemistry
- Enzymology
- Fluorometry
Background:
- Gamma-glutamyltranspeptidase (GGT) is a key enzyme involved in various physiological and pathological processes.
- Accurate and sensitive methods for GGT activity determination are crucial for research and diagnostics.
- Existing methods may require complex procedures or lack sensitivity for low-level activity detection.
Purpose of the Study:
- To synthesize and characterize a novel fluorescent substrate, gamma-glutamyl-7-amino-4-(trifluoromethyl)coumarin.
- To utilize this substrate for a sensitive and continuous fluorometric assay of gamma-glutamyltranspeptidase (GGT) activity.
- To evaluate the applicability of the assay in biological samples, including isolated hepatocytes, and assess its response to modulators.
Main Methods:
- Synthesis of gamma-glutamyl-7-amino-4-(trifluoromethyl)coumarin.
- Fluorometric analysis of the substrate and its reaction product (7-amino-4-(trifluoromethyl)coumarin) at specific wavelengths (Ex: 400 nm, Em: 490 nm).
- Assay development for GGT activity at optimal pH (8.4) using purified enzyme and isolated rat hepatocytes, including inhibition studies with acivicine and assessment after tamoxifen treatment.
Main Results:
- The synthesized substrate yielded a fluorescent product, 7-amino-4-(trifluoromethyl)coumarin, detectable at neutral pH.
- A linear relationship was observed between fluorescence intensity and GGT concentration (10-300 pmol/3 ml).
- The assay allowed continuous monitoring of GGT activity, accurate estimation of initial reaction rates, and detection of low GGT levels in hepatocytes. Tamoxifen pretreatment led to a fourfold increase in GGT activity.
Conclusions:
- The novel fluorescent substrate provides a convenient and sensitive method for determining gamma-glutamyltranspeptidase activity.
- The assay is suitable for continuous monitoring and accurate rate determination without complex workup.
- The method is applicable for studying GGT activity in cellular systems and its modulation by pharmacological agents.