Purification and characterization of ADP-ribosylarginine hydrolase from turkey erythrocytes

Abstract

ADP-ribosylation of arginine appears to be a reversible modification of proteins with NAD: arginine ADP-ribosyltransferases and ADP-ribosylarginine hydrolases catalyzing the opposing arms of the ADP-ribosylation cycle. ADP-ribosylarginine hydrolases have been purified extensively (greater than 90%) (150,000-250,000-fold) from the soluble fraction of turkey erythrocytes by DE-52, phenyl-Sepharose, hydroxylapatite, Ultrogel AcA 54, and Mono Q chromatography. Mobilities of the hydrolase on gel permeation columns and on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions are consistent with an active monomeric species of approximately 39 kDa. Insertion of an organomercurial agarose chromatographic step prior to Ultrogel AcA 54 resulted in the isolation of a hydrolase exhibiting approximately 35-fold greater sensitivity to dithiothreitol (Ka,sensitive = 41 +/- 16.7 microM, n = 4; Ka,resistant = 1.44 +/- 0.12 mM, n = 3). A similar dithiothreitol-sensitive hydrolase was generated by exposure of the purified resistant enzyme to HgCl2. At 30 degrees C, both thiol-sensitive (HS) and thiol-resistant (HR) hydrolases were relatively resistant to N-ethylmaleimide (NEM); incubation with dithiothreitol prior to NEM resulted in complete inactivation. Both HS and HR required Mg2+ and thiol for enzymatic activity. Mg2+ stabilized both HS and HR against thermal inactivation in the absence and presence of thiol. A purified NAD:arginine ADP-ribosyltransferase, in the presence of NAD, inactivated both HS and HR; Mg2+ and to a greater extent Mg2+ plus dithiothreitol protected both HS and HR from NAD- and transferase-dependent inactivation. Thus, activation of the hydrolase enhanced its resistance to inactivation by transferase.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydrolysis of O-Acetyl-ADP-ribose Isomers by ADP-ribosylhydrolase 3

“…Mono-ADP-ribosylation is a post-translational modification, in which the ADP-ribose (ADPr) 5 moiety of NAD is transferred to an acceptor protein (1). This modification serves as the mechanism by which several bacterial toxins (e.g.…”

“…Mammalian cells also produce endogenous ADP-ribosyltransferases that catalyze reactions similar to the bacterial toxins, specifically, the ADPribosylation of arginine residues in proteins (4). In addition, mammalian cells possess hydrolases that cleave the ADPr-protein linkage, releasing ADPr and regenerating the unmodified protein (5,6). An ADP-ribosyl(arginine) hydrolase, termed ARH1, catalyzes in a stereospecific manner, hydrolysis of the ␣-linkage of arginine-ribose found in ADP-ribosyl(arginine)-protein to ADPr and (arginine)-protein (7,8), consistent with the regulation of ADP-ribosyl(arginine)-protein levels by opposing activities of transferases and hydrolases, participating in an ADP-ribosylation cycle (4,9).…”

Enhanced Sensitivity to Cholera Toxin in ADP-Ribosylarginine Hydrolase-Deficient Mice

“…In mammalian cells, ADP-ribosylation appears to be a reversible posttranslational modification of proteins (23,30,43). An ADP-ribosylarginine hydrolase (ADPRH) that cleaves the ribosylarginine linkage, generating the unmodified protein (23,25,27), can complete a potentially regulatory ADP-ribosylation cycle. Such a cycle is thus far best documented in the photosynthetic bacterium Rhodospirillum rubrum, where it controls the activity of dinitrogenase reductase, a key enzyme in nitrogen fixation (20).…”

Genomic organization and promoter analysis of the mouse ADP-ribosylarginine hydrolase gene

“…Taken together, these data strongly suggest a significant role for the existence of ADP-ribosylation cycles in regulatory pathways involving G proteins or nonmuscle actin. 39 kDa ADP-ribosylarginine hydrolases (ADPRH) have been purified from avian and mammalian tissues (Moss et al, 1985(Moss et al, , 1988(Moss et al, , 1992. Rabbit anti-rat brain hydrolase polyclonal antibodies cross-react with turkey erythrocyte and with calf, mouse, and rat brain hydrolases, consistent with partial crossspecies conservation of hydrolase structure (Moss et al, 1992).…”

“…ADP-ribosyltransferases catalyze the forward reaction, the stereospecific ADPribosylation of arginine, whereas ADP-ribosylarginine or ADP-ribosylcysteine hydrolases catalyze the reverse reaction, cleavage of the ribose-amino acid linkage (Williamson and Moss, 1990;Okazaki and Moss, 1998). ADP-ribosylarginine hydrolases, which release ADP-ribose from ADPribosylated (arginine) proteins, have been identified in bacterial, avian, and mammalian cells (Moss et al, 1985(Moss et al, , 1986(Moss et al, , 1988(Moss et al, , 1992Lowery and Ludden, 1990). The regulatory role of an ADP-ribosylation cycle has been best documented in the photosynthetic bacterium Rhodospirillum rubrum where it regulates dinitrogenase reductase, a key enzyme in nitrogen fixation (Lowery and Ludden, 1990).…”