A novel fluorescence assay for measuring phosphatidylserine decarboxylase catalysis.
J Biol Chem
; 293(5): 1493-1503, 2018 02 02.
Article
em En
| MEDLINE
| ID: mdl-29247006
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.
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1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Fosfatidiletanolaminas
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Carboxiliases
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Proteínas de Protozoários
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Plasmodium knowlesi
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Proteínas Ligantes de Maltose
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Fluorescência
Idioma:
En
Revista:
J Biol Chem
Ano de publicação:
2018
Tipo de documento:
Article