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Sphingosine kinase assay system with fluorescent detection in high performance liquid chromatography

Title
Sphingosine kinase assay system with fluorescent detection in high performance liquid chromatography
Authors
Jin Y.-X.Yoo H.-S.Kihara A.Choi C.-H.Oh S.Moon D.-C.Igarashi Y.Lee Y.-M.
Ewha Authors
오세관
SCOPUS Author ID
오세관scopus
Issue Date
2006
Journal Title
Archives of Pharmacal Research
ISSN
0253-6269JCR Link
Citation
vol. 29, no. 11, pp. 1049 - 1054
Indexed
SCIE; SCOPUS; KCI WOS scopus
Abstract
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; 100 μM of C 17-Sph and 30 μg protein of F9-12 cells lysate in 20 min. Sphingosine analog C17-Sph was efficiently phosphorylated by Sphk activity (Km :67.08 μM, Vmax :1507.5 pmol/min/mg). New product C17-S1P 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 20 μM 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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의학전문대학원 > 의학과 > Journal papers
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