Assay for spermidine synthase activity by micellar electrokinetic chromatography with laser-induced fluorescence detection

被引：3

作者：

Sano, Masatake ^{[1
]}

Nishino, Ikuko ^{[1
]}

机构：

[1] Shionogi & Co Ltd, Shionogi Res Labs, Fukushima Ku, Osaka 5530002, Japan

来源：

JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES | 2007年 / 845卷 / 01期

关键词：

polyamine; spermidine synthase; spermidine; putrescine; micellar electrokinetic chromatography; laser-induced fluorescence detection; 7-fluoro-4nitrobenzo-2-oxa-1,3-diazone; rat liver;

D O I：

10.1016/j.jchromb.2006.07.052

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

An assay for spermidine synthase (SPDS) activity in rat liver has been developed using micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence (LIF) detection to enable the discovery of SPDS inhibitors. The assay was established by estimating the amount of spermidine (SPD) produced from the putrescine (PUT) present by SPDS. The SPD in an enzyme reaction mixture of homogenized rat liver could directly react with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) as a fluorescence derivatization reagent. The NBD derivatives of SPD and PUT could be separated and detected by MEKC-LIF detection within 15 min. The IC50 value measured for SPDS inhibitor, 4-methylcyclohexylamine, in rat liver by this assay was consistent with published data. Our SPDS assay using MEKC-LIF is simple and allows easy determination of SPDS activity in homogenized samples without troublesome procedures such as preparation of antibody or fluorescence-labeled substrate. The assay should be effective for discovering the SPDS inhibitors using biological samples. (c) 2006 Elsevier B.V. All rights reserved.

引用

页码：80 / 83

页数：4

共 26 条

[1] INHIBITION OF MAMMALIAN SPERMINE SYNTHASE BY N-ALKYLATED-1,3-DIAMINOPROPANE DERIVATIVES INVITRO AND IN CULTURED RAT HEPATOMA-CELLS [J].

BAILLON, JG ;

KOLB, M ;

MAMONT, PS .

EUROPEAN JOURNAL OF BIOCHEMISTRY, 1989, 179 (01) :17-21

[2] Capillary electrophoresis as a versatile tool for the bioanalysis of drugs - a review [J].

Boone, CM ;

Waterval, JCM ;

Lingeman, H ;

Ensing, K ;

Underberg, WJM .

JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS, 1999, 20 (06) :831-863

[3] Production and characterization of a monoclonal antibody specific for the polyamine spermidine and its application in ELISA [J].

Catcheside, JA ;

Stead, AD ;

Rider, CC .

HYBRIDOMA, 1996, 15 (03) :199-204

[4] Development of high-throughput spermidine synthase activity assay using homogeneous time-resolved fluorescence [J].

Enomoto, K ;

Nagasaki, T ;

Yamauchi, A ;

Onoda, J ;

Sakai, K ;

Yoshida, T ;

Maekawa, K ;

Kinoshita, Y ;

Nishino, I ;

Kikuoka, S ;

Fukunaga, T ;

Kawamoto, K ;

Numata, Y ;

Takemoto, H ;

Nagata, K .

ANALYTICAL BIOCHEMISTRY, 2006, 351 (02) :229-240

[5] Determination of polyamines in human prostate by high-performance liquid chromatography with fluorescence detection [J].

Fu, SJ ;

Zou, X ;

Wang, X ;

Liu, XX .

JOURNAL OF CHROMATOGRAPHY B, 1998, 709 (02) :297-300

[6] ASSAY OF THE POLYAMINE SPERMINE BY A MONOCLONAL ANTIBODY-BASED ELISA [J].

GARTHWAITE, I ;

STEAD, AD ;

RIDER, CC .

JOURNAL OF IMMUNOLOGICAL METHODS, 1993, 162 (02) :175-178

[7] Micellar electrokinetic capillary chromatographic separation and fluorescent detection of amino acids derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole [J].

Hu, S ;

Li, PCH .

JOURNAL OF CHROMATOGRAPHY A, 2000, 876 (1-2) :183-191

[8] FLUORIMETRIC DETERMINATION OF SECONDARY AMINO-ACIDS BY 7-FLUORO-4-NITROBENZO-2-OXA-1,3-DIAZOLE [J].

IMAI, K ;

WATANABE, Y .

ANALYTICA CHIMICA ACTA, 1981, 130 (02) :377-383

[9] Determination of peptides by high-performance liquid chromatography with laser-induced fluorescence detection [J].

Kanazawa, H ;

Nagatsuka, T ;

Miyazaki, M ;

Matsushima, Y .

JOURNAL OF CHROMATOGRAPHY A, 1997, 763 (1-2) :23-29

[10] Control of spermidine and spermine levels in rat tissues by trans-4-methylcyclohexylamine, a spermidine-synthase inhibitor [J].

Kobayashi, M ;

Watanabe, T ;

Xu, YJ ;

Tatemori, M ;

Goda, H ;

Niitsu, M ;

Shirahata, A ;

Samejima, K .

BIOLOGICAL & PHARMACEUTICAL BULLETIN, 2005, 28 (04) :569-573

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