Detection of Arginine-ADP-Ribosylated Protein Using Recombinant ADP-Ribosylarginine Hydrolase

We made use of ADP-ribosylarginine hydrolase to detect arginine-ADP-ribosylated proteins. The hydrolase was expressed in Escherichia coli as a protein fused with glutathione S-transferase (GST). The fusion protein GST-ADP-ribosylarginine hydrolase catalyzed the hydrolysis of α-ADP-ribosylarginine to produce ADP-ribose and arginine. Casein ADP-ribosylated with [ 32P]NAD and chicken heterophil arginine-specific ADP-ribosyltransferase served as a substrate for the recombinant ADP-ribosylarginine hydrolase and the released ADP-ribose was determined. Protein ADP-ribosylated by cholera toxin could serve as substrate of the hydrolase but protein ADP-ribiosylated by pertussis toxin, diphtheria toxin, or C 3 enzyme of Clostridium botulinum could not. The hydrolase did not release the radioactivity incorporated into isolated rat liver nuclei incubated with [ 32P]NAD or in bovine brain cytosol incubated with [ 32P]ADP-ribose. In homogenate of mouse heart which contained arginine-specific ADP-ribosyltransferase, labeling of a 55-kDa protein by incubation with [ 32P]NAD was removed by ADP-ribosylarginine hydrolase treatment; hence, the specific hydrolysis of ADP-ribose-arginine bond by GST-ADP-ribosylarginine hydrolase can be used to detect the arginine-ADP-ribosylated proteins in crude preparations. Arginine-ADP-ribosylated proteins in mouse spleen lymphocytes were identified using this method.