Abstract
Phosphatidylserine decarboxylases (PSDs) catalyze the conversion of phosphatidylserine (PS) to phosphatidylethanolamine (PE), a critical step in membrane biogenesis and a potential target for development of antimicrobial and anti-cancer drugs. PSD activity has typically been quantified using radioactive substrates and products. Recently, we described a fluorescence-based assay that measures the PSD reaction using distyrylbenzene-bis-aldehyde (DSB-3), whose reaction with PE produces a fluorescence signal. However, DSB-3 is not widely available and also reacts with PSD's substrate, PS, producing an adduct with lower fluorescence yield than that of PE. Here, we report a new fluorescence-based assay that is specific for PSD and in which the presence of PS causes only negligible background. This new assay uses 1,2-diacetyl benzene/β-mercaptoethanol, which forms a fluorescent iso-indole-mercaptide conjugate with PE. PE detection with this method is very sensitive and comparable with detection by radiochemical methods. Model reactions examining adduct formation with ethanolamine produced stable products of exact masses (m/z) of 342.119 and 264.105. The assay is robust, with a signal/background ratio of 24, and can readily detect formation of 100 pmol of PE produced from Escherichia coli membranes, Candida albicans mitochondria, or HeLa cell mitochondria. PSD activity can easily be quantified by sequential reagent additions in 96- or 384-well plates, making it readily adaptable to high-throughput screening for PSD inhibitors. This new assay now enables straightforward large-scale screening for PSD inhibitors against pathogenic fungi, antibiotic-resistant bacteria, and neoplastic mammalian cells.
Highlights
PE is an essential membrane phospholipid for many Gram-negative prokaryotes and eukaryotes [1,2,3]
We described the first fluorescence assay for Phosphatidylserine decarboxylases (PSDs) catalysis, which can be used for highthroughput screening (HTS), using the bis-aldehyde reagent DSB-3 [19]
Phospholipid metabolism is a fundamental requirement for membrane biogenesis across all biological domains (Archaea, Bacteria, and Eukarya)
Summary
The work of Medici et al [21] provided evidence that a mixture of 1,2-DAB/b-ME readily reacted with primary amines The data are from six independent experiments and are means 6 S.D. D, PSD activities of the E. coli membrane fractions are shown after conducting PSD assays with various amounts of membrane fractions (4.7–14.1 ng/ml) and 0.5 mM PS as the substrate with incubations at 37 °C for 45 min, as described under “Experimental procedures.”. To further verify the accuracy and reliability of the 1,2-DAB/b-ME–based enzyme assay, we monitored PE formation using TLC analysis of lipid components extracted from the PSD enzyme reactions of HeLa mitochondrial fractions.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
