Larew J S, Peterson J E, Graves D J
Biochemistry and Biophysics Department, Iowa State University, Ames 50011.
J Biol Chem. 1991 Jan 5;266(1):52-7.
A high performance liquid chromatographic method has been developed for the assay of arginine-specific ADP-ribosyl transferases. The assay utilizes L-arginine methyl ester (LAME) as the acceptor substrate. ADP-ribosylated-LAME is separated from the reaction mixture using a C-8 reversed-phase column. Before injection, the assay mixture is derivatized with an orthophthaldialdehyde/2-mercaptoethanol reagent. Fluorescence detection of the orthophthaldialdehyde-derivatized product provides excellent sensitivity and a limit of detection of less than 100 fmol. The kinetic mechanism of two arginine-specific ADP-ribosyltransferases, cholera toxin A subunit and an endogenous transferase from rabbit skeletal muscle, were both determined to be random sequential. The kinetic studies utilized 3-aminobenzamide and NG-monomethylarginine as competitive inhibitors for NAD and LAME, respectively. Cholera toxin was reported to have Km values of 5.6 and 39 mM for NAD and LAME, respectively. Km values of 0.56 and 1.2 mM were determined for NAD and LAME, respectively, using the transferase from rabbit skeletal muscle.
已开发出一种高效液相色谱法用于检测精氨酸特异性ADP-核糖基转移酶。该检测方法使用L-精氨酸甲酯（LAME）作为受体底物。使用C-8反相柱从反应混合物中分离出ADP-核糖基化的LAME。在进样前，检测混合物用邻苯二甲醛/2-巯基乙醇试剂进行衍生化。对邻苯二甲醛衍生化产物进行荧光检测，具有出色的灵敏度，检测限小于100飞摩尔。两种精氨酸特异性ADP-核糖基转移酶，即霍乱毒素A亚基和来自兔骨骼肌的内源性转移酶的动力学机制均被确定为随机顺序。动力学研究分别使用3-氨基苯甲酰胺和NG-单甲基精氨酸作为NAD和LAME的竞争性抑制剂。据报道，霍乱毒素对NAD和LAME的Km值分别为5.6和39 mM。使用来自兔骨骼肌的转移酶，分别测定NAD和LAME的Km值为0.56和1.2 mM。
