Enzymatic measurement of sphingosine 1-phosphate
Enzymatic measurement of sphingosine 1-phosphate
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DOI：
10.1006/abio.1999.4157
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发表时间：
1999-07-15
影响因子：
2.9
通讯作者：
Spiegel, S
中科院分区：
文献类型：
--
作者：
Edsall, LC;Spiegel, S
1-磷酸鞘氨醇(SPP)是一种鞘磷脂代谢产物，具有双重功能，既是G蛋白偶联受体家族的第二信使，又是G蛋白偶联受体家族的配体。本文描述了一种快速的酶促法定量测定血清、哺乳动物组织和培养细胞中SPP的质量水平。该方法利用碱性脂类提取物选择性地将SPP与包括鞘氨醇在内的其他磷脂和鞘磷脂分离。提取的SPP经碱性磷酸酶处理后可有效转化为鞘氨醇。然后使用重组的鞘氨醇激酶和[Gamma-P-32]ATP将由此形成的鞘氨醇定量磷酸化为[P-32]SPP。通过这个程序，我们能够在0.25pmol到2.5nmol的大范围内获得可重复性的SPP测量结果。在大鼠的不同组织中，SPP的含量在0.5到6pmol/mg湿重之间变化。最低的含量仅在心脏和睾丸中发现，而脑中的含量最高。该方法易于测定不同培养细胞类型中存在的微量SPP。细胞提取物中SPP的含量与细胞数成正比，变化范围为0.04~2pmol/10(6)个细胞。同时对鞘氨醇水平的测量表明，在大多数细胞和组织中，其浓度明显高于SPP。此外，通过这一检测，我们能够测量外源性鞘氨醇或神经生长因子刺激鞘氨醇激酶活性处理后大鼠嗜铬细胞瘤PC12细胞内SPP水平的增加。(C)1999年学术出版社。
Sphingosine 1-phosphate (SPP) is a sphingolipid metabolite which has novel dual actions acting as both an intracellular second messenger and a ligand for a family of G protein-coupled receptors. This paper describes a rapid enzymatic method to quantify mass levels of SPP in serum, mammalian tissues, and cultured cells. The assay utilizes an alkaline lipid extraction to selectively separate SPP from other phospholipids and sphingolipids, including sphingosine. Extracted SPP is efficiently converted to sphingosine by alkaline phosphatase treatment. Sphingosine thus formed is then quantitatively phosphorylated to [P-32]SPP using recombinant sphingosine kinase and [gamma-P-32]ATP. With this procedure we were able to obtain reproducible measurements of SPP over a broad range from 0.25 pmol to 2.5 nmol. In various rat tissues, levels of SPP varied between 0.5 and 6 pmol/mg wet wt. The lowest levels mere found in heart and testes, while brain contained the highest levels. The method was adapted easily to measure minute amounts of SPP present in various cultured cell types. The amount of SPP in cell extracts was proportional to the cell number and varied between 0.04 and 2 pmol/10(6) cells. Concurrent measurements of sphingosine levels revealed that its concentration was significantly higher than SPP in most cells and tissues. Furthermore, with this assay we were able to measure increases in intracellular SPP levels in rat pheochromocytoma PC12 cells after treatment with exogenous sphingosine or with nerve growth factor which stimulates sphingosine kinase activity. (C) 1999 Academic Press.