Purification and characterization of ADP-ribosylarginine hydrolase from turkey erythrocytes.
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)
Moss
J
J
Laboratory of Cellular Metabolism, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892.
Tsai
S C
SC
Adamik
R
R
Chen
H C
HC
Stanley
S J
SJ
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D002845:Chromatography; D004229:Dithiothreitol / Q000494:pharmacology; D004912:Erythrocytes / Q000201:enzymology*; D005033:Ethylmaleimide / Q000494:pharmacology; D006026:Glycoside Hydrolases; D006867:Hydrolases / Q000037:antagonists & inhibitors / Q000097:blood*; D008274:Magnesium; D009699:N-Glycosyl Hydrolases; D014422:Turkeys
D000246:Adenosine Diphosphate Ribose; D006867:Hydrolases; D006026:Glycoside Hydrolases; D009699:N-Glycosyl Hydrolases; C046422:ADP-ribosylarginine hydrolase; D008274:Magnesium; D005033:Ethylmaleimide; D004229:Dithiothreitol