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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
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.
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.
- 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.