Research Spotlight: An Improved and Highly Selective Fluorescence Assay for Measuring Phosphatidylserine Decarboxylase Activity

Posted on October 05, 2021

Fluorescence Image For Fb And Li

Hopefully, you’ve had the opportunity to read our interview with Dr. Dennis Voelker. If you haven’t, SPOILER ALERT! In the interview, Dr. Voelker describes the significance that a recently developed fluorescence assay has in future diagnostic and therapeutic research. He says “The field of lipid biochemistry is ripe for expansion into the realm of screening for- and identifying- novel inhibitors of essential enzymes for application as new drugs for treating all types of microbial infections, including viruses, bacteria and fungi.” Keep reading to learn more about this highly selective fluorescence assay for measuring phosphatidylserine decarboxylase activity!

Why is phosphatidylserine decarboxylase (PSD) activity important to measure?

The primary responsibility of PSDs is to convert the phosphatidylserine (PS) to phosphatidylethanolamine (PE). PE is an essential component in Gram-negative prokaryotes and eukaryotes. Typically, it is either the most abundant or second most abundant phospholipid in cell membranes. In some prokaryotes like E. coli PSD is the only source of PE. In eukaryotes such as yeast and mammalian systems, there are redundant pathways for PE pools to form. In these systems, redundancy does not necessarily mean that PSD production of PE is unnecessary, and in many cases, alternative pathways cannot fully satisfy all organelles with PE complements. For example, deletion of the gene encoding mitochondrial PSD results in auxotrophy, respiratory deficiency, and mitochondrial instability in yeast, and embryonic lethality in mice. PSD also has been shown to play a key role in cancer development.

The importance of this enzyme is apparent but even so, an inhibitor of this family of enzymes has not yet been developed. One major challenge associated with the development of PSD inhibitors is the time-consuming nature of assays available for screening inhibitors of the enzyme. The research in this article sought to identify a new method with more widely available reagents for detection of PSD catalysis and then characterize the properties and selectivity of this new method while also testing the feasibility of applying the new method to crude preparation of the enzymes in bacteria, fungi, and neoplastic cells.

A Newly Developed, Highly Selective Fluorescence Assay for Detection of PSD Activity

The newly developed method is much less cumbersome than the previous methods for detecting PSD activity. For context, one previous method required the incubation of membranes in detergent micelles containing radioactive material. The method described in this paper uses the sequential addition of reagents to a single well of multi-well plates to develop strong fluorescence signals that accurately report enzyme activity. The described method is also highly selective and does not create fluorescent adducts with PS like other previously reported methods. This method also excludes PE formed from the precursors (i.e. lyso-PE, Etn, phosphor-Etn, or CDP-Etn) because the reagent makes a fluorescent adduct with the precursors as well as the PE meaning that there will be no increase of fluorescence for non-PSD pathways. Furthermore, the new method has an increased dynamic range for detecting PSD inhibition in screening assays noted by the signal/background ratio of 24 compared to the previously reported ratio of 3.5. The newly developed assay provides a simple screening method for inhibitors of PSDs from pathogenic yeasts, bacteria, neoplastic cells, and tissues.

Thank you, Dr. Voelker, for being a friend and customer of Avanti! We are proud that our lipids are being put to use in your research. Don’t forget to check out our Interview with Dr. Voelker at the link below and click HERE to read the full research article!