A fluorescence-based assay for measuring polyamine biosynthesis aminopropyl transferase-mediated catalysis

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Polyamines are vital polycationic molecules involved in numerous cellular processes.
• Aminopropyl transferases (APTs) are key enzymes in polyamine biosynthesis and represent therapeutic targets.
• A significant hurdle in APT research is the absence of high-throughput activity assays.

Purpose of the Study:

• To develop and validate the first fluorescence-based assay for measuring APT activity.
• To enable high-throughput screening of potential therapeutic compounds targeting APTs.

Main Methods:

• Development of the diacetyl benzene (DAB)-APT assay using 1,2-DAB to detect polyamines.
• Validation of the assay using APT enzymes from Saccharomyces cerevisiae and Plasmodium falciparum.
• Confirmation of assay results using mass spectrometry and thin-layer chromatography (TLC).

Main Results:

• The DAB-APT assay measures APT activity via fluorescent conjugates formed between 1,2-DAB and polyamines (putrescine, spermidine, spermine).
• Fluorescence intensity correlates with polyamine carbon chain length.
• Mass spectrometry identified the fluorescent adducts as substituted 1,3-dimethyl isoindoles.

Conclusions:

• The novel DAB-APT assay is optimized for high-throughput screening of chemical libraries.
• This assay provides a powerful tool for advancing research and drug discovery in infectious diseases, oncology, and neurobiology.