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Nonradioactive assay for cellular dimethylallyl diphosphate.
A J Fisher1, T N Rosenstiel, M C Shirk
1Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, USA.
Analytical Biochemistry
|May 18, 2001
Summary
A new, nonradioactive method accurately measures cellular dimethylallyl diphosphate (DMAPP), a key isoprenoid metabolism intermediate. This technique simplifies analysis by directly converting DMAPP to isoprene for gas chromatography detection.
Area of Science:
- Biochemistry
- Metabolomics
- Plant Physiology
Background:
- Isoprenoid metabolism is crucial in all living organisms.
- Dimethylallyl diphosphate (DMAPP) is a central intermediate in isoprenoid biosynthesis.
- Accurate measurement of DMAPP is essential for understanding metabolic pathways.
Purpose of the Study:
- To develop a sensitive, nonradioactive assay for quantifying cellular DMAPP levels.
- To establish a method suitable for diverse biological samples, including plant leaves and microorganisms.
- To validate the assay's reliability and ease of use, minimizing sample preparation steps.
Main Methods:
- Acid hydrolysis of DMAPP to isoprene within sealed reaction vials.
- Headspace gas chromatography with reduction gas detection for isoprene analysis.
- Direct acidification of cell samples, eliminating the need for metabolite extraction and purification.
Main Results:
- Quantified DMAPP levels in Populus deltoides leaves (23-80 nmol/g fresh weight) and in Bacillus subtilis and Saccharomyces cerevisiae (pmol/OD or pmol/mg dry weight).
- Demonstrated that DMAPP is the primary metabolite yielding isoprene upon acid hydrolysis in leaves.
- Validated the method's applicability across different cell types and isolated organelles.
Conclusions:
- The developed assay provides a sensitive and efficient means to measure cellular DMAPP.
- This nonradioactive method simplifies the study of isoprenoid metabolism in various biological systems.
- The assay's direct sample processing enhances its utility and reduces potential errors.
