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

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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.",

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doi = "10.1007/BF02969290",

language = "English",

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pages = "1049--1054",

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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

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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

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Keywords

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