Sally J. Stanley

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.

Amino Acid-Specific ADP-Ribosylation: Purification and Properties of an Erythrocyte ADP-Ribosylarginine Hydrolase

There is a class of mono-ADP-ribosyltransferases that are distinguished by their ability to utilize as ADP-ribose acceptors the free amino acid arginine, other simple guanidino compounds, and proteins. Several transferases of this type were identified in and purified from turkey erythrocytes (1–3). The enzymes displayed different physical, kinetic and regulatory properties and were localized to the soluble, membrane and nuclear compartments (1–3). Similar NAD:arginine ADP-ribosyltransferases have been observed in other tissues and organ systems from a variety of species (1–5). The enzymes are found in viruses, bacteria and animal cells (1–7).