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J Biol Chem. 2018 Feb 2;293(5):1493-1503. doi: 10.1074/jbc.RA117.000525. Epub 2017 Dec 15.

A novel fluorescence assay for measuring phosphatidylserine decarboxylase catalysis.

Author information

1
From the Basic Science Section, Department of Medicine, National Jewish Health, Denver, Colorado 80206.
2
the Yale Center for Molecular Discovery, West Haven, Connecticut 06516.
3
the Organisch-Chemisches Institut, Ruprecht-Karls-Universitat, Heidelberg, Im Neuenheimer Feld 270, 69120 Heidelberg, Germany.
4
the Department of Internal Medicine, Section of Infectious Diseases, Yale School of Medicine, New Haven, Connecticut 06520, and.
5
From the Basic Science Section, Department of Medicine, National Jewish Health, Denver, Colorado 80206, voelkerd@njhealth.org.

Abstract

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.

KEYWORDS:

fluorescence; inhibitor; inhibitor screening; membrane biogenesis; phosphatidylserine decarboxylase; phospholipid

PMID:
29247006
PMCID:
PMC5798280
DOI:
10.1074/jbc.RA117.000525
[Indexed for MEDLINE]
Free PMC Article

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