A high-performance liquid chromatography-based fluorometric method for assaying serine hydroxymethyltransferase toward serine formation

被引：5
|
作者：
Wei, Zhaoyang [1 ]
Roje, Sanja [1 ]
机构：
[1] Washington State Univ, Inst Biol Chem, Pullman, WA 99164 USA
来源：
ANALYTICAL BIOCHEMISTRY | 2011年 / 409卷 / 01期
基金：
美国国家科学基金会;
关键词：
ONE-CARBON METABOLISM; HIGHER-PLANTS;
D O I：
10.1016/j.ab.2010.10.004
中图分类号：
Q5 [生物化学];
学科分类号：
071010 ; 081704 ;
摘要：
A high-performance liquid chromatography (HPLC)-based fluorometric method for measuring serine hydroxymethyltransferase (SHMT) activity toward formation of serine and (6S)-H(4)PteGlu(n) has been developed. In this method, serine formed by SHMT activity is reacted with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) to form the fluorescent adduct NBD-serine. The fluorescent assay components are then separated by reversed-phase chromatography, and NBD-serine is quantified by comparison with standards. This method was used to determine the K-m and k(cat) values for 5,10-CH2-H(4)PteGlu(5) of an SHMT from Arabidopsis thaliana. These data represent the first determination of kinetic parameters for (6S)-5,10-CH2-H(4)PteGlu(5) for an SHMT from any organism. (C) 2010 Elsevier Inc. All rights reserved.
引用
收藏
页码：156 / 158
页数：3
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