ADP-RIBOSYLARGININE HYDROLASES

ADP-ribosylation is a reversible post-translational modification of pr oteins involving the addition of the ADP-ribose moiety of NAD to an ac ceptor protein or amino acid. NAD:arginine ADP-ribosyltransferase, pur ified from numerous animal tissues, catalyzes the transfer of ADP-ribo se to an arginine residue in proteins. The reverse reaction, catalyzed by ADP-ribosylarginine hydrolase, removes ADP-ribose, regenerating fr ee arginine. An ADP-ribosylarginine hydrolase, purified extensively fr om turkey erythrocytes, was a 39-kDa monomeric protein under denaturin g and non-denaturing conditions, and was activated by Mg2+ and dithiot hreitol. The ADP-ribose moiety was critical for substrate recognition; the enzyme hydrolyzed ADP-ribosylarginine and (2-phospho-ADP-ribosyl) arginine but not phosphoribosylarginine or ribosylarginine. The hydrol ase cDNA was cloned from rat and subsequently from mouse and human bra in. The rat hydrolase gene contained a 1086-base pair open reading fra me, with deduced amino acid sequences identical to those obtained by a mino terminal sequencing of the protein or of HPLC-purified tryptic pe ptides. Deduced amino acid sequences from the mouse and human hydrolas e cDNAs were 94% and 83% identical, respectively to the rat. Anti-rat brain hydrolase polyclonal antibodies reacted with turkey erythrocyte, mouse and bovine brain hydrolase. The rat hydrolase, expressed in E. coli, demonstrated enhanced activity in the presence of Mg2+ and thiol , whereas the recombinant human hydrolase was stimulated by Mg2+ but w as thiol-independent. In the rat and mouse enzymes, there are five cys teines in identical positions; four of the cysteines are conserved in the human hydrolase. Replacement of cysteine 108 in the rat hydrolase (not present in the human enzyme) resulted in a thiol-independent hydr olase without altering specific activity. Rabbit anti-rat brain hydrol ase antibodies reacted on immunoblot with the wild-type rat hydrolase and only weakly with the mutant hydrolase. There was no immunoreactivi ty with either the wild-type or mutant human enzyme. Cysteine 108 in t he rat and mouse hydrolase may be responsible in part for thiol-depend ence as well as antibody recognition. Based on these studies, the mamm alian and avian ADP-ribosylarginine hydrolases exhibit considerable co nservation in structure and function.