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Related Experiment Videos

Assay for ADP-ribosyl cyclase by reverse-phase high-performance liquid chromatography.

K Schweitzer1, G W Mayr, A H Guse

  • 1University Hospital Hamburg-Eppendorf, Institute for Medical Biochemistry and Molecular Biology, Division of Cellular Signal Transduction, University of Hamburg, Martinistrasse 52, D-20246 Hamburg, Germany.

Analytical Biochemistry
|December 4, 2001
PubMed
Summary

Researchers developed a sensitive enzymatic assay to detect ADP-ribosyl cyclase activity in T cells. This method overcomes challenges in nucleotide extraction, enabling precise quantification of enzymes involved in calcium signaling.

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Area of Science:

  • Immunology
  • Cell Biology
  • Biochemistry

Background:

  • Cyclic ADP-ribose (cADPR) is a crucial second messenger regulating Ca(2+) signaling in T cells.
  • Understanding ADP-ribosyl cyclase activity is vital for T cell function research.
  • Existing methods for enzyme detection face challenges with nucleotide stability during extraction.

Purpose of the Study:

  • To develop a sensitive and specific enzymatic assay for identifying ADP-ribosyl cyclases in T cells.
  • To overcome the instability of 1,N(6)-etheno-cADPR in perchloric acid extraction.
  • To quantify ADP-ribosyl cyclase and NAD(+)-glycohydrolase activities in subcellular fractions of T cells.

Main Methods:

  • Developed a novel enzymatic assay using 1,N(6)-etheno-NAD(+) as a substrate.

Related Experiment Videos

  • Replaced perchloric acid extraction with a filtration method to enhance 1,N(6)-etheno-cADPR stability.
  • Produced standard compounds (1,N(6)-etheno-cADPR, 1,N(6)-etheno-ADPR, 1,N(6)-etheno-AMP) using purified enzymes.
  • Applied the assay to subcellular fractions of human Jurkat T cells for activity analysis.

    • Main Results:

    • Successfully developed a sensitive and specific enzymatic assay for ADP-ribosyl cyclases.
    • The filtration method effectively preserved the stability of 1,N(6)-etheno-cADPR.
    • Detected and precisely quantified ADP-ribosyl cyclase and NAD(+)-glycohydrolase activities in various subcellular fractions.
    • Evidence suggests the presence of distinct isoenzymes within T cells.

    Conclusions:

    • The new assay provides a robust tool for studying ADP-ribosyl cyclases and NAD(+)-glycohydrolases in T cells.
    • The findings indicate differential localization and potential roles of enzyme isoenzymes in T cell signaling.
    • This methodology facilitates further investigation into cADPR-mediated calcium signaling pathways.