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KMID : 0043320060290111049
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Archives of Pharmacal Research
2006 Volume.29 No. 11 p.1049 ~ p.1054
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Sphingosine Kinase Assay System with Fluorescent Detection in High Performance Liquid Chromatography
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Jin You-Xun
Yoo Hwan-Soo
Kihara Akio
Choi Chang-Hwan
Oh Seik-Wan
Moon Dong-Cheul
Igarashi Yasuyuki
Lee Yong-Moon
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Abstract
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Activation of Sphingosine kinase (Sphk) increases a bioactive lipid, sphingosine 1-phosphate (S1P) and has been observed in a variety of cancer cells. Therefore, inhibition of Sphk activity was an important target for the development of anticancer drugs. As a searching tool for Sphk inhibitor, we developed fluorescent Sphk activity assay combined with high performance liquid chromatography (HPLC). Previously we established murine teraticarcinoma mutant F9-12 cells which lack S1P lyase and stably express Sphk1. By using F9-12 cells, optimal assay conditions were established as follows; protein of F9-12 cells lysate in 20 min. Sphingosine analog was efficiently phosphorylated by Sphk activity (). New product was separated from S1P in reversed-phase HPLC. In optimized conditions, 300 nM of phorbol 12-myristate 13-acetate (PMA) increased Sphk activity approximately twice while of N,N-dimethylsphingosine (DMS) reduced 70% of Sphk activity in F9-12 cells lysate. In conclusion, we established non-radioactive but convenient Sphk assay system by using HPLC and F9-12 cells.
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KEYWORD
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Sphingosine 1-Phosphate, HPLC, Activity, Sphingosine kinase
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