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.

中文翻译：

---

一种改进的高选择性荧光测定法，用于测量磷脂酰丝氨酸脱羧酶活性。


磷脂酰丝氨酸脱羧酶 (PSD) 催化磷脂酰丝氨酸 (PS) 转化为磷脂酰乙醇胺 (PE)，这是膜生物发生的关键步骤，也是抗菌和抗癌药物开发的潜在靶标。 PSD 活性通常使用放射性底物和产品进行量化。最近，我们描述了一种基于荧光的检测方法，使用二苯乙烯基苯双醛 (DSB-3) 测量 PSD 反应，DSB-3 与 PE 的反应会产生荧光信号。然而，DSB-3 并未广泛使用，并且还会与 PSD 的底物 PS 发生反应，产生荧光产量低于 PE 的加合物。在这里，我们报告了一种新的基于荧光的检测方法，该检测方法专门针对 PSD，其中 PS 的存在仅导致可以忽略不计的背景。这种新的检测方法使用 1,2-二乙酰基苯/β-巯基乙醇，它与 PE 形成荧光异吲哚硫醇缀合物。该方法的PE检测非常灵敏，可与放射化学方法的检测相媲美。检查与乙醇胺形成加合物的模型反应产生了精确质量 (m/z) 为 342.119 和 264.105 的稳定产物。该检测方法稳健，信号/背景比为 24，可以轻松检测大肠杆菌膜、白色念珠菌线粒体或 HeLa 细胞线粒体产生的 100 pmol PE 的形成。通过在 96 孔板或 384 孔板中连续添加试剂，可以轻松定量 PSD 活性，使其很容易适用于 PSD 抑制剂的高通量筛选。这种新的检测方法现在可以直接大规模筛选针对病原真菌、抗生素耐药细菌和肿瘤哺乳动物细胞的 PSD 抑制剂。