Development of a simple and robust assay to screen for inhibitors of sphingosine kinases
Development of a simple and robust assay to screen for inhibitors of sphingosine kinases
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DOI：
10.1089/adt.2006.049
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发表时间：
2007-04-01
影响因子：
1.8
通讯作者：
Reinemer, Peter
中科院分区：
文献类型：
--
作者：
Togame, Hiroko;Dodo, Reiko;Reinemer, Peter
鞘氨醇激酶（SPHKs）催化鞘脂代谢产物鞘氨醇I-磷酸（SIP）的形成，在细胞内外发挥重要作用。常规地，SPHK活性已经使用放射性同位素薄层色谱法（TLC）和放射自显影法检测产物SIP来测定。在这里，我们描述了一种简单而稳健的体外SPHK测定方法，该方法采用384孔格式，不需要任何分离步骤，如提取和TLC。该测定基于P-33-磷酸从[γ-P-33]ATP转移到鞘氨醇，随后使用AquaBind（TM）板（Asahi Techno Glass，Tokyo，Japan）检测[P-33]SIP。用该测定法测定的酶和抑制特性与先前报道的在常规TLC测定法中测定的值良好一致。D-β-鞘氨醇和ATP的K值分别为17.5 μ M和19.2 μ M。ADP和N，N-二甲基鞘氨醇均能抑制该激酶反应，其50%抑制浓度分别为410 μ M和450 μ M。所建立的测定形式易于适应自动化筛选平台，其特征在于。高信号背景比、小变化和优异的Z因子。
Sphingosine kinases (SPHKs) catalyze the formation of the bioactive sphingolipid metabolite sphingosine I-phosphate (SIP), which plays important roles in a wide variety of intraand extracellular functions. Conventionally, SPHK activity has been determined using radioisotope thin layer chromatography (TLC) and.autoradiography to detect the product SIP. Here we describe the development of a simple and robust in vitro SPHK assay in 384-well format with no requirement for any separation steps such as extraction and TLC. The assay is based on P-33-phosphate transfer from [gamma-P-33]ATP to sphingosine and subsequent detection of the [P-33]SIP using AquaBind(TM) plates (Asahi Techno Glass, Tokyo, Japan). Enzymatic and inhibition characteristics determined with this assay are in good agreement with previously reported values determined in the conventional TLC assay. K values for D-erythro-sphingosine and ATP were determined to be 17.5 mu M and 19.2 mu M, respectively. The kinase reaction could be inhibited by ADP and NN-dimethylsphingosine with a 50% inhibitory concentration of 410 mu M and 450 mu M, respectively. The established assay format was easily adapted to an automated screening platform and is characterized by. a high signal-to-background ratio, small variation, and excellent Z factors.