A rapid radioassay for sphingosine kinase
A rapid radioassay for sphingosine kinase
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DOI：
10.1016/j.ab.2004.10.032
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发表时间：
2005-02-01
影响因子：
2.9
通讯作者：
Karliner, JS
中科院分区：
文献类型：
--
作者：
Vessey, DA;Kelley, M;Karliner, JS
建立了一种基于溶剂萃取的鞘氨醇激酶(Skase)活性的放射分析方法。该方法利用[H-3]鞘氨醇底物，并区别提取[H-3]鞘氨醇-1-磷酸产物。提取的放射性主要是[H-3]鞘氨醇-1-磷酸，[H-3]鞘氨醇的污染不到1%。当测定可溶性细胞部分中的Skase活性时，标记的鞘氨醇-1-磷酸产物的提取效率可重复性为78%，这允许简单的反计算来修正22%的提取损失。经过较小的修改，该方法也是一种可重复性的方法，用于测定亚细胞膜部分中的Skase活性。该方法比薄层层析和高效液相色谱方法快速得多，使得在短时间内进行大量的分析成为可能。用该方法对大鼠心肌酶进行了完整的双底物动力学分析，证明了该方法的实用性。由爱思唯尔公司出版。
A solvent-extraction-based radioassay for measuring sphingosine kinase (SKase) activity has been developed. The assay utilizes [H-3]sphingosine substrate and differentially extracts the [H-3]sphingosine-1-phosphate product. The extracted radioactivity is demonstrated to be primarily [H-3]sphingosine-1-phosphate with less than 1% contamination by [H-3]sphingosine. When assaying SKase activity in the soluble cell fraction, the extraction efficiency of the labeled sphingosine-1-phosphate product is a reproducible 78%, which allows for a simple back calculation to correct for the 22% extraction loss. With minor modification, the assay is also a reproducible procedure for determining SKase activity in subcellular membrane fractions. The assay is far more rapid than thin-layer chromatography and high-performance liquid chromatography methods, which makes it possible to do a large number of assays in a short period of time. The utility of the assay is demonstrated by using it to conduct a complete bisubstrate kinetic analysis of rat heart SKase. Published by Elsevier Inc.