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Determination of cellular aminopropyltransferase activity using precolumn fluorescent etheno-derivatization with high-performance liquid chromatography. 2012

Kenichi Yamazaki, and Yoshihiko Ikeguchi, and Takuya Niwa, and Kaoru Hayashi, and Takahiro Iwaki, and Ikumi Ishii, and Masaru Niitsu, and Anthony E Pegg, and Akira Shirahata
Laboratory of Bio-analytical Chemistry, Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, Josai University, Saitama, Japan.

Polyamines such as spermidine (Spd) and spermine (Spm), produced by aminopropyltransferase (Apt), play roles in cell growth and differentiation. A sensitive and simple fluorometric high-performance liquid chromatographic determination for Apt activity of spermidine synthase (Spdsyn) and spermine synthase (Spmsyn) was developed in order to examine cellular functions of polyamine synthesis. The derivatization procedure for methylthioadenosine (MTA) produced from decarboxylated S-adenosylmethionine by Apt was the reaction with 2-chloroacetaldehyde to give fluorescent 1, N(6)-etheno methylthioadenosine. The reaction conditions for derivatization were optimized. A calibration curve was established, ranging from 0.01 to 25 pmol. Quantification of derivatized MTA was confirmed to be identical to Spd or Spm production. The developed method determined Spdsyn and Spmsyn activities in HepG2 cells treated with oleic acid as a cellular lipid accumulation model.

UI MeSH Term Description Entries
D011073 Polyamines Amine compounds that consist of carbon chains or rings containing two or more primary amino groups. Polyamine
D011436 Spermidine Synthase An enzyme that catalyzes the transfer of the propylamine moiety from 5'-deoxy-5'-S-(3-methylthiopropylamine)sulfonium adenosine to putrescine in the biosynthesis of spermidine. The enzyme has a molecular weight of approximately 73,000 kDa and is composed of two subunits of equal size. Aminopropyltransferase,Propylamine Transferase,Spermidine Synthetase,Putrescine Aminopropyltransferase,Aminopropyltransferase, Putrescine,Synthase, Spermidine,Synthetase, Spermidine,Transferase, Propylamine
D002851 Chromatography, High Pressure Liquid Liquid chromatographic techniques which feature high inlet pressures, high sensitivity, and high speed. Chromatography, High Performance Liquid,Chromatography, High Speed Liquid,Chromatography, Liquid, High Pressure,HPLC,High Performance Liquid Chromatography,High-Performance Liquid Chromatography,UPLC,Ultra Performance Liquid Chromatography,Chromatography, High-Performance Liquid,High-Performance Liquid Chromatographies,Liquid Chromatography, High-Performance
D004789 Enzyme Activation Conversion of an inactive form of an enzyme to one possessing metabolic activity. It includes 1, activation by ions (activators); 2, activation by cofactors (coenzymes); and 3, conversion of an enzyme precursor (proenzyme or zymogen) to an active enzyme. Activation, Enzyme,Activations, Enzyme,Enzyme Activations
D005470 Fluorometry An analytical method for detecting and measuring FLUORESCENCE in compounds or targets such as cells, proteins, or nucleotides, or targets previously labeled with FLUORESCENCE AGENTS. Fluorimetry,Fluorometric Analysis,Analysis, Fluorometric
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D056945 Hep G2 Cells A human liver tumor cell line used to study a variety of liver-specific metabolic functions. Cell Line, Hep G2,Cell Line, Hepatoblastoma G2,Hep G2 Cell Line,HepG2 Cells,Hepatoblastoma G2 Cell Line,Cell, Hep G2,Cell, HepG2,Cells, Hep G2,Cells, HepG2,Hep G2 Cell,HepG2 Cell

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