Sphingosine kinase assay system with fluorescent detection in high performance liquid chromatography.
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 microM of C17-Sph and 30 microg protein of F9-12 cells lysate in 20 min. Sphingosine analog C17-Sph was efficiently phosphorylated by Sphk activity (Km:67.08 microM, 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 microM 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.
Jin
You-Xun
YX
College of Pharmacy, Chungbuk National University, Chongju 361-763, Korea.
Yoo
Hwan-Soo
HS
Kihara
Akio
A
Choi
Chang-Hwan
CH
Oh
Seikwan
S
Moon
Dong-Cheul
DC
Igarashi
Yasuyuki
Y
Lee
Yong-Moon
YM
D000255:Adenosine Triphosphate / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D007202:Indicators and Reagents; D008246:Lysophospholipids / Q000378:metabolism; D051379:Mice; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000032:analysis / Q000037:antagonists & inhibitors*; D013050:Spectrometry, Fluorescence; D013110:Sphingosine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D009764:o-Phthalaldehyde / Q000737:chemistry; D000099157:Sphingosine Kinase
D000255:Adenosine Triphosphate; D000469:Alkaline Phosphatase; D007202:Indicators and Reagents; D008246:Lysophospholipids; D017853:Phosphotransferases (Alcohol Group Acceptor); D013110:Sphingosine; D013755:Tetradecanoylphorbol Acetate; D009764:o-Phthalaldehyde; D000099157:Sphingosine Kinase; C060506:sphingosine 1-phosphate; C061800:N,N-dimethylsphingosine