Highly Selective Fluorescence Assay for Phosphatidylserine Decarboxylase (PSD) Activity Measurement

Currently, the realm of lipid biochemistry is mature enough to be applied to the screening and identification of novel inhibitors of essential enzymes which can be used for treating all types of microvial infections, including viruses, bacteria and fungi. That is to say, there is a kind of highly selective fluorescence assay that can be used to measure phosphatidylserine decarboxylase (PSD) activity.

Primarily, the function of PSD was to convert the phosphatidylserine (PS) to phosphatidylethanolamine (PE). PE, an essential component in Gram-negative prokaryotes and eukaryotes, is normally the most or second most abundant phospholipid in cell membranes. PSD is the only source of PE in some prokaryotes like E. coli. Besides, there are redundant pathways for the formation of PE pool in eukaryotes, such as yeast and mammalian systems. The redundancy does not mean that it is unnecessary for PSD to produce PE. On various occasions, alternative pathways cannot fully satisfy all rganelles with PE complements. For instance, deletion of the gene encoding mitochondrial PSD will lead to auxotrophy, respiratory deficiency, and mitochondrial instability in yeast, as well as embryonic lethality in mice. PSD has also been proven to play an important role in cancer development.

Obviously, PSD is an important enzyme, but there is no inhibitor of this enzyme that has been developed. The major challenge of developing the PSD inhibitor is the time-consuming factor of the assays used to screen inhibitors of the enzyme. To solve the problem, researchers found out a new method to detect the PSD catalysis with more widely used reagents. Then, they characterized the properties and selectivity of the method and test the feasibility of applying the method to production of the enzymes in bacteria, fungi, and neoplastic cells.

Compared to the previous method to detect PSD activity, the new method is easier to use. The previous method requires the membranes to be incubated in detergent micelles containing radioactive material, while the new method is used to report the enzyme activity by developing strong fluorescence signals using the sequential addition of reagents to a single well of multi-well plates. The new method is highly selective and does not create fluorescent adducts with PS. The new method also excludes PE fromed from the precursors, such as lyso-PE, Etn, phosphor-Etn and CDP-Etn, as the fluorescent adduct is formed by the reagent, precursors and PE which means the fluorescence for non-PSD pathways is not increased. In addition, the dynamic range of the new method for detecting PSD inhibition in screening assay is larger with the signal/background ratio of 24. To sum up, the researchers developed a highly selective fluorescent assay to screen the inhibitors of PSD from pathogenic yeasts, bacteria, neoplastic cells and tissues.

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