Development of a high-performance liquid chromatography assay method and characterization of adenosine diphosphate-ribosylarginine hydrolase in skeletal muscle.

A new HPLC assay method for the enzyme that cleaves the bond between ADP-ribose and arginine was developed by using ADP-ribose-4-dimethylaminoazobenzene-4-sulfonyl arginine methyl ester (ADP-R-DABS-AME) as a substrate analog. Because of the difference of hydrophobicity, ADP-R-DABS-AME and DABS-AME elute as well-separated peaks on the C-18 reversed-phase HPLC column, which is monitored at 475 nm. Because ADP-R-DABS-AME gives two peaks of alpha and beta anomers on the C-18 HPLC column, the stereospecificity of ADP-ribosylarginine hydrolase can be easily determined. By using this new assay method, the existence of ADP-ribosylarginine hydrolase was identified in cultured chicken muscle cells. This enzyme activity was also found in rat and rabbit skeletal muscle. The muscle hydrolase cleaves the alpha anomer of ADP-R-DABS-AME specifically and requires magnesium and dithiothreitol for its full activation. By using this HPLC assay method, ADP-ribosylarginine hydrolase activity was also found in the crude extracts from the rat heart, liver, kidney, and brain.