Molecular and immunological characterization of ADP-ribosylarginine hydrolases.

Abstract

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 Mg2+-dependent, whereas the bovine and guinea pig enzymes were dithiothreitol-independent.A rat brain hydrolase was purified -20,000-fold and represented the major -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 reactiongenerated cDNA probes.Inserts from two overlapping clones yielded a composite sequence that included a 108%-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 M8+-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.ADP-ribosylation is a post-translational modification of proteins, which participates in normal eukaryotic cell physiology and in the pathogenesis of disease (1-6).Several bac-