ADP-ribosylarginine hydrolases.

Abstract

ADP-ribosylation is a reversible post-translational modification of proteins involving the addition of the ADP-ribose moiety of NAD to an acceptor protein or amino acid. NAD:arginine ADP-ribosyltransferase, purified from numerous animal tissues, catalyzes the transfer of ADP-ribose to an arginine residue in proteins. The reverse reaction, catalyzed by ADP-ribosylarginine hydrolase, removes ADP-ribose, regenerating free arginine. An ADP-ribosylarginine hydrolase, purified extensively from turkey erythrocytes, was a 39-kDa monomeric protein under denaturing and non-denaturing conditions, and was activated by Mg2+ and dithiothreitol. 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 hydrolase cDNA was cloned from rat and subsequently from mouse and human brain. The rat hydrolase gene contained a 1086-base pair open reading frame, with deduced amino acid sequences identical to those obtained by amino terminal sequencing of the protein or of HPLC-purified tryptic peptides. Deduced amino acid sequences from the mouse and human hydrolase 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 was thiol-independent. In the rat and mouse enzymes, there are five cysteines in identical positions; four of the cysteines are conserved in the human hydrolase.(ABSTRACT TRUNCATED AT 250 WORDS)