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

doi:10.1007/BF02969290

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

Department of Molecular Medicine

Research output: Contribution to journal › Article › peer-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 (K_m :67.08 μM, V_max :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 language	English
Pages (from-to)	1049-1054
Number of pages	6
Journal	Archives of Pharmacal Research
Volume	29
Issue number	11
DOIs	https://doi.org/10.1007/BF02969290
State	Published - 30 Nov 2006

Bibliographical note

Funding Information:
This work was supported by the research grant of the Chungbuk National University in 2004.

Keywords

Activity
HPLC
Sphingosine 1-phosphate
Sphingosine kinase

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1007/BF02969290

Cite this

@article{4d4afc149c3540f5858ac96885a85272,

title = "Sphingosine kinase assay system with fluorescent detection in high performance liquid chromatography",

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.",

keywords = "Activity, HPLC, Sphingosine 1-phosphate, Sphingosine kinase",

author = "Jin, \{You Xun\} and Yoo, \{Hwan Soo\} and Akio Kihara and Choi, \{Chang Hwan\} and Seikwan Oh and Moon, \{Dong Cheul\} and Yasuyuki Igarashi and Lee, \{Yong Moon\}",

note = "Funding Information: This work was supported by the research grant of the Chungbuk National University in 2004.",

year = "2006",

month = nov,

day = "30",

doi = "10.1007/BF02969290",

language = "English",

volume = "29",

pages = "1049--1054",

journal = "Archives of Pharmacal Research",

issn = "0253-6269",

publisher = "Pharmaceutical Society of Korea",

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}

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T1 - Sphingosine kinase assay system with fluorescent detection in high performance liquid chromatography

AU - Jin, You Xun

AU - Yoo, Hwan Soo

AU - Kihara, Akio

AU - Choi, Chang Hwan

AU - Oh, Seikwan

AU - Moon, Dong Cheul

AU - Igarashi, Yasuyuki

AU - Lee, Yong Moon

N1 - Funding Information: This work was supported by the research grant of the Chungbuk National University in 2004.

PY - 2006/11/30

Y1 - 2006/11/30

N2 - 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.

AB - 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.

KW - Activity

KW - HPLC

KW - Sphingosine 1-phosphate

KW - Sphingosine kinase

UR - https://www.scopus.com/pages/publications/33845356305

U2 - 10.1007/BF02969290

DO - 10.1007/BF02969290

M3 - Article

C2 - 17146975

AN - SCOPUS:33845356305

SN - 0253-6269

VL - 29

SP - 1049

EP - 1054

JO - Archives of Pharmacal Research

JF - Archives of Pharmacal Research

IS - 11

ER -

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

Abstract

Bibliographical note

Keywords

UN SDGs

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