Maria S. Nightingale

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.

INTERSPECIES RELATIONSHIPS AMONG ADP-RIBOSYLATION FACTORS (ARFS) : EVIDENCE OF EVOLUTIONARY PRESSURE TO MAINTAIN INDIVIDUAL IDENTITIES

ADP-ribosylation factors (ARFs) are ∼20-kDa guanine nucleotide-binding proteins that are allosteric activators of the NAD:arginine ADP-ribosyltransferase activity of cholera toxin and appear to play a role in intracellular vesicular trafficking. Although the physiological roles of these proteins have not been defined, it has been presumed that each has a specific intracellular function. To obtain genetic evidence that each ARF is under evolutionary pressure to maintain its structure, and presumably function, rat ARF cDNA clones were isolated and their nucleotide and deduced amino acid sequences were compared to those of other mammalian ARFs. Deduced amino acid sequences for rat ARFs 1, 2, 3, 5 and 6 were identical to those of the known cognate human and bovine ARFs; rat ARF4 was 96% identical to human ARF4. Nucleotide sequences of both the untranslated as well as the coding regions were highly conserved. These results indicate that the ARF proteins are, as a family, extraordinarily well conserved across mammalian species. The unusually high degree of conservation of the untranslated regions is consistent with these regions having important regulatory roles and that individual ARFs contain structurally unique elements required for specific functions.