SPHINGOSINE KINASE FROM SWISS 3T3 FIBROBLASTS - A CONVENIENT ASSAY FOR THE MEASUREMENT OF INTRACELLULAR LEVELS OF FREE SPHINGOID BASES
SPHINGOSINE KINASE FROM SWISS 3T3 FIBROBLASTS - A CONVENIENT ASSAY FOR THE MEASUREMENT OF INTRACELLULAR LEVELS OF FREE SPHINGOID BASES
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DOI：
10.1006/abio.1994.1589
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发表时间：
1994-12-01
影响因子：
2.9
通讯作者：
SPIEGEL, S
中科院分区：
文献类型：
--
作者：
OLIVERA, A;ROSENTHAL, J;SPIEGEL, S
鞘氨醇碱对多种细胞过程都很重要。本文介绍了一种简单，快速的酶法定量细胞脂质提取物中游离长链鞘氨醇碱的质量水平的发展。该试验基于Swiss 3T3成纤维细胞的鞘氨醇激酶磷酸化鞘氨醇碱（主要是鞘氨醇）的能力。为了定量测定鞘氨醇或其他长链鞘氨醇碱的细胞水平，有必要通过超声处理溶解细胞脂质，并在含有至少50 nmol磷脂的脂质提取物存在下生成标准曲线。对测定条件进行了优化，以允许在25至1000 pmol的宽范围内定量鞘氨醇。使用这种方法，我们能够获得可重复的测量游离鞘氨醇碱在各种类型的细胞，并检测增加细胞内鞘氨醇碱水平治疗后与外源性鞘氨醇或刺激与胎牛血清。(C)1994年出版社出版。
Sphingoid bases are important for diverse cellular processes. This paper describes the development of a simple and rapid enzymatic method to quantify mass levels of free long-chain sphingoid bases in cellular lipid extracts. The assay is based on the ability of sphingosine kinase from Swiss 3T3 fibroblasts to phosphorylate sphingoid bases, predominantly sphingosine. To quantitatively determine cellular levels bf sphingosine or other long-chain sphingoid bases, it was necessary to solubilize cellular lipids by sonication and to generate standard curves in the presence of lipid extracts containing at least 50 nmol of phospholipids. The assay conditions were optimized to allow quantification of sphingosine over a broad range from 25 to 1000 pmol. Using this method we were able to obtain reproducible measurements of free sphingoid bases in various cell types and to detect increases in intracellular sphingoid base levels after treatment with exogenous sphingosine or stimulation with fetal bovine serum. (C) 1994 Academic Press, Inc.