A rapid fluorescence assay for sphingosine-1-phosphate lyase enzyme activity
A rapid fluorescence assay for sphingosine-1-phosphate lyase enzyme activity
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DOI：
10.1194/jlr.d700010-jlr200
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发表时间：
2007-12-01
影响因子：
6.5
通讯作者：
Saba, Julie D.
中科院分区：
文献类型：
--
作者：
Bandhuvula, Padmavathi;Fyrst, Henrik;Saba, Julie D.
1-磷酸鞘氨醇 (S1P) 裂解酶 (SPL) 催化 S1P 转化为磷酸乙醇胺和十六碳烯醛。这种酶在生理学和疾病中发挥着多种作用，因此可用作疾病标志物和/或药物靶点。不幸的是，目前用于量化 SPL 活性的放射性同位素测定方法并不理想。我们设计了一种使用市售的 v(7-硝基-2-1,3-苯并恶二唑-4-基)-D-赤式 (NBD) 标记荧光底物的测定方法。或者，我们提供了一种从 NBD-鞘氨醇合成底物的方法。酶活性通过跟踪 NBD-醛产物的形成来确定，该产物通过脂质提取与未反应的底物分离，并在使用 C18 柱通过 HPLC 分离后进行定量。荧光 NBD-C18-鞘氨醇内标用于控制提取效率。反应在 20 分钟内呈线性，总蛋白浓度为 20-200 mg/l。荧光测定的灵敏度与放射性测定相当或更好，并且可以轻松检测到低至 8 pmol/mg/min 的 SPL 水平。氨基脲是一种非特异性 SPL 抑制剂，使用标准方法和荧光方法可将体外 SPL 活性降低约 70%。使用乙醇胺磷酸盐和市售的十六碳烯醛来源没有观察到产物抑制。该方法适用于量化多种细胞和组织来源中的 SPL 活性。
Sphingosine-1-phosphate (S1P) lyase (SPL) catalyzes the conversion of S1P to ethanolamine phosphate and hexadecenal. This enzyme plays diverse roles in physiology and disease and, thus, may be useful as a disease marker and/or drug target. Unfortunately, the radioisotope-based assay currently used to quantify SPL activity is suboptimal. We have devised an assay using a commercially available v(7-nitro-2-1,3-benzoxadiazol-4-yl)-D-erythro (NBD)-labeled fluorescent substrate. Alternatively, we provide a method for synthesis of the substrate from NBD-sphingosine. Enzyme activity is determined by following the formation of NBD-aldehyde product, which is isolated from unreacted substrate by lipid extraction and quantified after separation by HPLC using a C18 column. A fluorescent NBD-C18-sphingosine internal standard is used to control for extraction efficiency. The reaction is linear over 20 min and total protein concentrations of 20-200 mg/l. The sensitivity of the fluorescence assay is comparable to or better than that of the radioactive assay, and SPL levels as low as 8 pmol/mg/min were readily detected. Semicarbazide, a nonspecific SPL inhibitor, reduced SPL activity in vitro by similar to 70% using both standard and fluorescence methods. Product inhibition was not observed using ethanolamine phosphate and a commercially available source of hexadecenal. This method is suitable for quantifying SPL activity in a variety of cell and tissue sources.