Mono-ADP-ribosylation, a post-translational modification in which the ADP-ribose moiety of NAD is transferred to an acceptor protein, is catalyzed by a family of amino acid-specific ADP-ribosyltransferases. ADP-ribosyltransferase 5 (ART5), a murine transferase originally isolated from Yac-1 lymphoma cells, differed in properties from previously identified eukaryotic transferases in that it exhibited significant NAD glycohydrolase (NADase) activity. To investigate the mechanism of regulation of transferase and NADase activities, ART5 was synthesized as a FLAG fusion protein in Escherichia coli. Agmatine was used as the ADP-ribose acceptor to quantify transferase activity. ART5 was found to be primarily an NADase at 10 M NAD, whereas at higher NAD concentrations (1 mM), after some delay, transferase activity increased, whereas NADase activity fell. This change in catalytic activity was correlated with auto-ADP-ribosylation and occurred in a time-and NAD concentration-dependent manner. Based on the change in mobility of auto-ADP-ribosylated ART5 by SDS-polyacrylamide gel electrophoresis, the modification appeared to be stoichiometric and resulted in the addition of at least two ADP-ribose moieties. Auto-ADP-ribosylated ART5 isolated after incubation with NAD was primarily a transferase. These findings suggest that auto-ADP-ribosylation of ART5 was stoichiometric, resulted in at least two modifications and converted ART5 from an NADase to a transferase, and could be one mechanism for regulating enzyme activity.Mono-ADP-ribosylation is a post-translational modification of proteins catalyzed by enzymes that transfer the ADP-ribose moiety of NAD to specific amino acids in protein acceptors (1, 2). The best characterized mono-ADP-ribosylation reactions are those catalyzed by bacterial toxin ADP-ribosyltransferases such as cholera (3), diphtheria (4), and pertussis (5) toxins, which alter critical metabolic and regulatory pathways. For example, cholera toxin ADP-ribosylates an arginine in the ␣-subunit of the stimulatory heterotrimeric guanine nucleotidebinding protein (G protein), resulting in the activation of adenylyl cyclase and an increase in intracellular cyclic AMP (3).Mono-ADP-ribosyltransferase activity specific for arginine has been detected in numerous animal tissues (2, 6 -14). Transferases have been cloned from rabbit (7) and human (8) skeletal muscle, chicken polymorphonuclear granulocytes (9) and nucleoblasts (10), and mouse lymphoma cell lines Yac-1 (12, 13) and SL12 (16). Based on immunological, biochemical, and sequence analysis, it appears that the transferase, termed ART1, is glycosylphosphatidylinositol (GPI) 1 -anchored to the cell surface (8, 12). Consistent with its extracellular location, a GPIlinked muscle transferase in C2C12 mouse myotubes ADPribosylates integrin ␣7 (17). Inhibitor studies suggest that the muscle transferase may participate in the regulation of myogenesis (18).GPI-anchored transferases were found also in mouse cytotoxic T lymphocytes (CTL) and some murine T cell lymphoma and h...