A new detection method for arginine-specific ADP-ribosylation of protein -- a combinational use of anti-ADP-ribosylarginine antibody and ADP-ribosylarginine hydrolase.
Arginine-specific ADP-ribosylation is one of the posttranslational modifications of proteins by transferring one ADP-ribose moiety of NAD to arginine residues of target proteins. This modification, catalyzed by ADP-ribosyltransferase (Art), is reversed by ADP-ribosylarginine hydrolase (AAH). In this study, we describe a new method combining an anti-ADP-ribosylarginine antibody (alphaADP-R-Arg Ab) and AAH for detection of the target protein of ADP-ribosylation. We have raised alphaADP-R-Arg Ab with ADP-ribosylated histone and examined the reactivity of the antibody with proteins treated by Art and/or AAH, as well as in situ ADP-ribosylation system with mouse T cells. Our results indicate that the detection of ADP-ribosylated protein with alphaADP-R-Arg Ab and AAH is a useful tool to explore the target proteins of ADP-ribosylation. We applied the method to search endogenously ADP-ribosylated protein in the rat, and detected possible target proteins in the skeletal muscle, which has high Art activity.
Osago
Harumi
H
Department of Biochemistry, Faculty of Medicine, Shimane University, Izumo 693-8501, Japan. biochem1@med.shimane-u.ac.jp
Terashima
Masaharu
M
Hara
Nobumasa
N
Yamada
Kazuo
K
Tsuchiya
Mikako
M
D000246:Adenosine Diphosphate Ribose / Q000032:analysis* / Q000276:immunology / Q000378:metabolism*; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D001120:Arginine / Q000032:analysis* / Q000276:immunology / Q000378:metabolism*; D002645:Chickens; D051379:Mice; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D051381:Rats
D000906:Antibodies; D000246:Adenosine Diphosphate Ribose; D001120:Arginine; D009699:N-Glycosyl Hydrolases; C046422:ADP-ribosylarginine hydrolase