Determination of the kinetic mechanism of arginine-specific ADP-ribosyltransferases using a high performance liquid chromatographic assay

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.