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

You Xun Jin, Hwan Soo Yoo, Akio Kihara, Chang Hwan Choi, Seikwan Oh, Dong Cheul Moon, Yasuyuki Igarashi, Yong Moon Lee

Research output: Contribution to journalArticlepeer-review

10 Scopus citations

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.

Original languageEnglish
Pages (from-to)1049-1054
Number of pages6
JournalArchives of Pharmacal Research
Volume29
Issue number11
DOIs
StatePublished - 30 Nov 2006

Keywords

  • Activity
  • HPLC
  • Sphingosine 1-phosphate
  • Sphingosine kinase

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