Molecular and immunological characterization of ADP-ribosylarginine hydrolases.
Mono-ADP-ribosylation is a reversible modification of proteins with NAD:arginine ADP-ribosyltransferases and ADP-ribosylarginine hydrolases catalyzing the forward and reverse reactions, respectively. Hydrolase activities were present in a variety of animal species, with the highest specific activities found in rat and mouse brain, spleen, and testis. Rat and mouse hydrolases were dithiothreitol- and Mg(2+)-dependent, whereas the bovine and guinea pig enzymes were dithiothreitol-independent. A rat brain hydrolase was purified approximately 20,000-fold and represented the major approximately 39-kDa protein on denaturing gels. Immunoaffinity-purified rabbit polyclonal antibodies reacted with 39-kDa proteins from turkey erythrocytes and rat, mouse, and calf brains. A rat brain cDNA library was screened using oligonucleotide and polymerase chain reaction-generated cDNA probes. Inserts from two overlapping clones yielded a composite sequence that included a 1086-base pair open reading frame, which contained amino acid sequences found in the purified hydrolase. A hydrolase fusion protein, synthesized in Escherichia coli, reacted with anti-39-kDa polyclonal antibodies and exhibited Mg(2+)- and dithiothreitol-dependent hydrolase activity. A coding region cDNA hybridized readily to a 1.7-kilobase band in rat and mouse poly(A)+ RNA, but poorly to bovine, chicken, rabbit, and human poly(A)+ RNA. The immunological and molecular biological data are consistent with partial conservation of hydrolase structure across animal species.
Moss
J
J
Laboratory of Cellular Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Stanley
S J
SJ
Nightingale
M S
MS
Murtagh
J J
JJ
Monaco
L
L
Mishima
K
K
Chen
H C
HC
Williamson
K C
KC
Tsai
S C
SC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology; D002413:Cations, Divalent; D002417:Cattle; D004247:DNA / Q000235:genetics; D004229:Dithiothreitol / Q000378:metabolism; D006026:Glycoside Hydrolases; D006168:Guinea Pigs; D006867:Hydrolases / Q000235:genetics* / Q000276:immunology; D008274:Magnesium / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases; D009693:Nucleic Acid Hybridization; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D012313:RNA / Q000235:genetics; D051381:Rats; D013045:Species Specificity; D014018:Tissue Distribution; D014422:Turkeys
D002413:Cations, Divalent; D012313:RNA; D004247:DNA; D006867:Hydrolases; D006026:Glycoside Hydrolases; D009699:N-Glycosyl Hydrolases; C046422:ADP-ribosylarginine hydrolase; D008274:Magnesium; D004229:Dithiothreitol