Pertussis toxin catalyzed ADP-ribosylation of the guanyl nucleotide binding protein transducin was stimulated by adenine nucleotide and either phospholipids or detergents. To determine the sites of action of these agents, their effects were examined on the transducin-independent NAD glycohydrolase activity. Toxin-catalyzed NAD hydrolysis was increased synergistically by ATP and detergents or phospholipids; the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) was more effective than the nonionic detergent Triton X-100 greater than lysophosphatidylcholine greater than phosphatidylcholine. The A0.5 for ATP in the presence of CHAPS was 2.6 microM; significantly higher concentrations of ATP were required for maximal activation in the presence of cholate or lysophosphatidylcholine. In CHAPS, NAD hydrolysis was enhanced by ATP greater than ADP greater than AMP greater than adenosine; ATP was more effective than MgATP or the nonhydrolyzable analogue adenyl-5'-yl imidodiphosphate. GTP and guanyl-5'-yl imidodiphosphate were less active than the corresponding adenine nucleotides. Activity in the presence of CHAPS and ATP was almost completely dependent on dithiothreitol; the A0.5 for dithiothreitol was significantly decreased by CHAPS alone and, to a greater extent, by CHAPS and ATP. To determine the site of action of ATP, CHAPS, and dithiothreitol, the enzymatic (S1) and binding components (B oligomer) were resolved by chromatography. The purified S1 subunit catalyzed the dithiothreitol-dependent hydrolysis of NAD; activity was enhanced by CHAPS but not ATP. The studies are consistent with the conclusion that adenine nucleotides, dithiothreitol, and CHAPS act on the toxin itself rather than on the substrate; adenine nucleotides appear to be involved in the activation of toxin but not the isolated catalytic unit.
An ADP-ribosylarginine hydrolase, which catalyzes the degradation of ADP-ribosyl[14C]arginine to ADP-ribose plus arginine, was separated by ion exchange, hydrophobic, and gel permation chromatography from NAD:arginine ADP-ribosyltransferases, which are responsible for the stereospecific formation of alpha-ADP-ribosylarginine. As determined by NMR, the specific substrate for the hydrolase was alpha-ADP-ribosylarginine, the product of the transferase reaction. The ADP-ribose moiety was critical for substrate recognition; (phosphoribosyl) [14C]arginine and ribosyl[14C]arginine were poor substrates and did not significantly inhibit ADP-ribosyl[14C]arginine degradation. In contrast, ADP-ribose was a potent inhibitor of the hydrolase and significantly more active than ADP greater than AMP greater than adenosine. In addition to ADP-ribosyl[14C]arginine, both ADP-ribosyl[14C]guanidine and (2'-phospho-ADP-ribosyl)[14C]arginine were also substrates; at pH greater than 7, ADP-ribosyl[14C]guanidine was degraded more readily than the [14C]arginine derivative. Neither arginine, guanidine, nor agmatine, an arginine analogue, was an effective hydrolase inhibitor. Thus, it appears that the ADP-ribosyl moiety but not the arginine group is critical for substrate recognition. Although the hydrolase requires thiol for activity, dithiothreitol accelerated loss of activity during incubation at 37 degrees C. Stability was enhanced by Mg2+, which is also necessary for optimal enzymatic activity. The findings in this paper are consistent with the conclusion that different enzymes catalyze ADP-ribosylarginine synthesis and degradation. Furthermore, since the hydrolase and transferases possess a compatible stereospecificity and substrate specificity, it would appear that the two enzymatic activities may serve as opposing arms in an ADP-ribosylation cycle.
Enzymes have been identified in animal tissues that catalyze the mono(ADP-ribosyl)ation of arginine and proteins. Since these NAD:arginine ADP-ribosyltransferases under physiological conditions do not appear to catalyze the degradation of the product ADP-ribose-arginine, the possibility was investigated that a different family of enzymes exists that cleaves the ADP-ribose-arginine linkage. An ADP-ribosylation is a covalent modification catalyzed by viral, bacterial, and animal enzymes (1, 2); the ADP-ribose moiety of NAD is transferred to either an acceptor protein or amino acid or to another ADP-ribose moiety to yield an acceptor-linked monomer or polymer (1, 2). These synthetic reactions are catalyzed by two classes of enzymes: (i) mono(ADP-ribose)transferases, which catalyze the mono(ADP-ribosyl)ation of proteins, and (ii) poly(ADPribose) synthetases, which catalyze the initial ADP-ribosylation of protein and subsequent chain elongation (3-6). The importance of the mono(ADP-ribosyl)ation reaction has been best documented for bacterial toxins that exert their effects on cells by modifying critical enzymes (7-13). Choleragen (cholera toxin) and Escherichia coli heat-labile enterotoxin, agents involved in the pathogenesis of cholera and traveler's diarrhea, respectively, modify a stimulatory GTP-binding protein of the adenylate cyclase system, leading to an increase in cellular cyclic AMP (9, 12, 13). Pertussis toxin (islet-activating protein) ADP-ribosylates and inactivates an inhibitory component of adenylate cyclase (10-13). Diphtheria toxin and Pseudomonas exotoxin A modify elongation factor II, thereby inhibiting protein synthesis and causing cell death (7, 8); an ADP-ribosyltransferase endogenous to animal cells is believed to catalyze a reaction similar to diphtheria toxin (14). Although the metabolic role of poly(ADP-ribose) synthetase has not been defined, it is closely linked to chromatin-associated events (1, 6).Among the ADP-ribosyltransferases endogenous to animal cells are those that catalyze the formation of ADP-ribosearginine (15-19). These enzymes, which also modify proteins, presumably using a guanidino moiety as an ADP-ribose acceptor, have been identified and purified from turkey erythrocytes, chicken liver, and rabbit muscle (15)(16)(17)(18)(19). A family of NAD:arginine ADP-ribosyltransferases has been identified in turkey erythrocytes, where different transferases are localized in the nucleus, cytosol, and membrane fractions (16,17,20). These transferases differ in physical, kinetic, and regulatory properties (20). The bacterial toxins, choleragen and E. coli heat-labile enterotoxin, also possess NAD:arginine ADP-ribosyltransferase activity (21,22); toxin-catalyzed activation of the adenylate cyclase system is believed to result from modification of an arginine residue on a cyclase regulatory protein (23).The mechanism for the reversal ofthe mono(ADP-ribosyl)-ation reaction has not been elucidated fully. The pyrophosphate moiety of protein-bound ADP-ribose is cleaved by a phospho...
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)
We evaluate the factors associated with an officer’s decision to search a driver or vehicle after a routine traffic stop, and we compare the accuracy of these searches by looking at the share leading to arrest. Racial disparities in search rates by race and gender of driver are similar for all types of officers; all tend to search Black male drivers at higher rates than any other demographic. White male officers have higher search rates for all types of drivers. Further, they conduct the greatest share of “fruitless searches” (those not leading to arrest), and these searches are particularly targeted on those drivers with the greatest number of cumulative disadvantages.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.