Enzymatic quantification of sphingosine in the picomole range in cultured cells.
Enzymatic quantification of sphingosine in the picomole range in cultured cells.
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DOI：
10.1016/0003-2697(89)90186-3
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发表时间：
1989-11
影响因子：
2.9
通讯作者：
P. V. Van Veldhoven;W. Bishop;R. Bell
中科院分区：
文献类型：
--
作者：
P. V. Van Veldhoven;W. Bishop;R. Bell
建立了一种定量测定细胞脂质提取物中游离鞘氨醇质量水平的酶促方法。该测定是基于神经酰胺被大肠杆菌二酰基甘油激酶磷酸化的观察结果。虽然鞘氨醇不被酶识别，但它可以通过与己酸酐酰化而转化为底物。使用混合胶束测定，先前报道的质量定量的二酰基甘油，短链神经酰胺（N-C6-鞘氨醇），通过酰化产生的，定量磷酸化为N-C6-[32 P]鞘氨醇磷酸。该测定允许在25至5000 pmol的宽范围内定量鞘氨醇。当该测定应用于标准化合物时，反应产物的反相薄层色谱足以分离长链神经酰胺和N-C6-鞘氨醇的磷酸化衍生物。然而，生物样品提取物中其他脂质（主要是单烷基甘油，也是二酰基甘油激酶的底物）的存在会干扰并需要额外的纯化步骤。设计的最有效的纯化步骤是阴离子和阳离子交换色谱的组合。使用该测定定量不同培养细胞中游离鞘氨醇碱的质量水平。水平在8至20 pmol/106细胞之间变化。当归一化为磷脂时，鞘氨醇水平在0.01和0.04摩尔%之间变化。在L929细胞中发现最低水平，而来自Twitch小鼠的Schwann细胞中含有最高水平。这些水平显著高于来自正常小鼠的雪旺细胞。
An enzymatic method to quantify the mass levels of free sphingosine in cellular lipid extracts was developed. The assay is based upon the observation that ceramide is phosphorylated by Escherichia coli diacylglycerol kinase. Although sphingosine is not recognized by the enzyme, it can be converted to a substrate by acylation with hexanoic anhydride. Using a mixed micellar assay, previously reported for the mass quantification of diacylglycerol, the short-chain ceramide (N-C6-sphingosine), generated by acylation, is quantitatively phosphorylated to N-C6-[32P]sphingosine phosphate. This assay allows quantification of sphingosine over a broad range from 25 to 5000 pmol. When this assay was applied to standard compounds, reverse-phase thinlayer chromatography of the reaction products was adequate to separate the phosphorylated derivatives of long-chain ceramide and N-C6-sphingosine. However, the presence of other lipids in extracts from biological samples (mainly monoalkylglycerols which are also a substrate for the diacylglycerol kinase) interfered and necessitated an additional purification step. The most efficient purification step devised was a combination of anion- and cation-exchange chromatography. The mass levels of free sphingoid bases in different cultured cells were quantified using this assay. Levels varied between 8 to 20 pmol/106cells. When normalized to phospholipids, sphingosine levels varied between 0.01 and 0.04 mol%. The lowest levels were found in L929 cells, while Schwann cells derived from Twitcher mice contained the highest levels. These levels were significantly higher than those of Schwann cells derived from normal mice.