Determination of cellular aminopropyltransferase activity using precolumn fluorescent etheno-derivatization with high-performance liquid chromatography.
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.
Laboratory of Bio-analytical Chemistry, Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, Josai University, Saitama, Japan.
Kenichi
K
Yamazaki
Yoshihiko
Y
Ikeguchi
Takuya
T
Niwa
Kaoru
K
Hayashi
Takahiro
T
Iwaki
Ikumi
I
Ishii
Masaru
M
Niitsu
Anthony E
AE
Pegg
Akira
A
Shirahata
D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation; D005470:Fluorometry; D056945:Hep G2 Cells; D006801:Humans; D011073:Polyamines / Q000737:chemistry / Q000378:metabolism; D011436:Spermidine Synthase / Q000032:analysis* / Q000378:metabolism
D011073:Polyamines; D011436:Spermidine Synthase