A novel fluorescence assay for measuring phosphatidylserine decarboxylase catalysis
Phosphatidylserine decarboxylases (PSDs) are central enzymes in phospholipid metabolism that produce phosphatidylethanolamine (PE) in bacteria, protists, plants, and animals. We developed a fluorescence-based assay for selectively monitoring production of PE in reactions using a maltose-binding prot...
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| Hauptverfasser: | , |
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| Dokumenttyp: | Article (Journal) |
| Sprache: | Englisch |
| Veröffentlicht: |
[2018]
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| In: |
The journal of biological chemistry
Year: 2018, Jahrgang: 293, Heft: 5, Pages: 1493-1503 |
| ISSN: | 1083-351X |
| DOI: | 10.1074/jbc.RA117.000525 |
| Online-Zugang: | Verlag, Volltext: https://doi.org/10.1074/jbc.RA117.000525 Verlag, Volltext: http://www.jbc.org/content/293/5/1493 
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| Verfasserangaben: | Jae-Yeon Choi, Yulia V. Surovtseva, Sam M. Van Sickle, Jan Kumpf, Uwe H.F. Bunz, Choukri Ben Mamoun, and Dennis R. Voelker |
MARC
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| 520 | |a Phosphatidylserine decarboxylases (PSDs) are central enzymes in phospholipid metabolism that produce phosphatidylethanolamine (PE) in bacteria, protists, plants, and animals. We developed a fluorescence-based assay for selectively monitoring production of PE in reactions using a maltose-binding protein fusion with Plasmodium knowlesi PSD (MBP-His6-Δ34PkPSD) as the enzyme. The PE detection by fluorescence (λex = 403 nm, λem = 508 nm) occurred after the lipid reacted with a water-soluble distyrylbenzene-bis-aldehyde (DSB-3), and provided strong discrimination against the phosphatidylserine substrate. The reaction conditions were optimized for enzyme, substrate, product, and DSB-3 concentrations with the purified enzyme and also tested with crude extracts and membrane fractions from bacteria and yeast. The assay is readily amenable to application in 96- and 384-well microtiter plates and should prove useful for high-throughput screening for inhibitors of PSD enzymes across diverse phyla. | ||
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| 650 | 4 | |a phosphatidylserine decarboxylase | |
| 650 | 4 | |a phospholipid | |
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