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 alpha-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-ribosylated 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 [(32)P]NAD or in bovine brain cytosol incubated with [(32)P]ADP-ribose. In homogenate of mouse heart which contained arginine-specific ADP-ribosyltransferase, labeling of a 55-kDa protein by incubation with [(32)P]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 crude preparations. Arginine-ADP-ribosylated proteins in mouse spleen lymphocytes were identified using this method.
Ohno
T
T
Department of Biochemistry, Shimane Medical University, Japan.
Tsuchiya
M
M
Osago
H
H
Hara
N
N
Jidoi
J
J
Shimoyama
M
M
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000378:metabolism*; D001483:Base Sequence; D004926:Escherichia coli; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D051379:Mice; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases; D009928:Organ Specificity; D011065:Poly(ADP-ribose) Polymerases / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D011506:Proteins; D011993:Recombinant Fusion Proteins; D000246:Adenosine Diphosphate Ribose; D001120:Arginine; D011065:Poly(ADP-ribose) Polymerases; D006026:Glycoside Hydrolases; D009699:N-Glycosyl Hydrolases; C046422:ADP-ribosylarginine hydrolase