Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.4.2.30 (PARP)
13,611 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An ADP-ribosyltransferase was purified approximately 500-fold from the supernatant fraction of turkey erythrocytes. The enzyme hydrolyzed [carbonyl-(14)C]NAD to ADP-ribose and [carbonyl-(14)C]nicotinamide at a low rate. Nicotinamide formation from NAD was enhanced by arginine methyl ester > D-arginine approximately L-arginine > guanidine; lysine, histidine, and citrulline were ineffective. Incubation of [adenine-U-(14)C]NAD and arginine methyl ester or arginine with the purified enzyme resulted in the formation of new compounds that contained (14)C, reacted with ninhydrin, and quenched background fluorescence of thin-layer plates viewed in ultraviolet light. Their mobilities on thin-layer chromatograms were indistinguishable from those of ADP-ribosylarginine methyl ester and ADP-ribosylarginine formed during incubation of choleragen with NAD and arginine methyl ester or arginine, respectively [Moss, J. & Vaughan, M. (1977) J. Biol. Chem. 252, 2455-2457]. The purified transferase also catalyzed the incorporation of label from [adenine-(14)C]-NAD into lysozyme, histones and polyarginine. When the (14)C-labeled lysozyme was incubated with snake venom phosphodiesterase, the radioactivity was released and, on thin-layer chromatograms, exhibited a mobility indistinguishable from that of 5'-AMP, as would be expected of an ADP-ribosylated protein, but not of a poly(ADP-ribosylated) product. The purified transferase activated rat brain adenylate cyclase and, as is the case with choleragen, activation was absolutely dependent on NAD. The presence in the avian erythrocyte of a protein that, like choleragen and Escherichia coli heat-labile enterotoxin, apparently activates adenylate cyclase and possesses ADP-ribosyl transferase activity is consistent with the view that the mechanisms through which the bacterial toxins produce pathology are not entirely foreign to vertebrate cells, at least some of which may possess and employ an analogous mechanism for activation of adenylate cyclase.
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PMID:Isolation of an avian erythrocyte protein possessing ADP-ribosyltransferase activity and capable of activating adenylate cyclase. 21 2

Poly(ADP-ribose) polymerase (PARP, EC 2.4.2.30) is a zinc finger DNA-binding protein involved in DNA repair processes in eukaryotes. By deletion and extensive site-directed mutagenesis, its DNA-binding domain fused to the N-terminus of beta-galactosidase was shown to contain a nuclear localization signal (NLS) of the form KRK-X(11)-KKKSKK (residues 207-226). In vitro, both the DNA-binding capacity and the polymerizing activity of PARP are independent of the nuclear location function. Each basic cluster is essential but not sufficient on its own for this function, while both motifs together are. Crucial basic amino acids (K207, R208 and K222) in each of these two motifs are required for nuclear homing. The results presented here support the concept that the human PARP NLS is an autonomous functional element and belongs to the class of bipartite NLSs. We show that the linear distance between the two basic clusters is not crucial. Insertional mutation analysis leading to a partial reversion of the cytoplasmic phenotype displayed by the mutant K222I highlights the crucial positioning of this lysine. The structure-function relationship of the second cluster of basic residues is discussed.
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PMID:The human poly(ADP-ribose) polymerase nuclear localization signal is a bipartite element functionally separate from DNA binding and catalytic activity. 150 17

Previous studies of the S1 subunit of pertussis toxin, an NAD(+)-dependent ADP-ribosyltransferase, suggested that a small amino-terminal region of amino acid sequence similarity to the active fragments of both cholera toxin and Escherichia coli heat-labile enterotoxin represents a region containing critical active-site residues that might be involved in the binding of the substrate NAD+. Other studies of two other bacterial toxins possessing ADP-ribosyltransferase activity, diphtheria toxin and Pseudomonas exotoxin A, have revealed the presence of essential glutamic acid residues vicinal to the active site. To help determine the relevance of these observations to activities of the enterotoxins, the A-subunit gene of the E. coli heat-labile enterotoxin was subjected to site-specific mutagenesis in the region encoding the amino-terminal region of similarity to the S1 subunit of pertussis toxin delineated by residues 6 through 17 and at two glutamic acid residues, 110 and 112, that are conserved in the active domains of all of the heat-labile enterotoxin variants and in cholera toxin. Mutant proteins in which arginine 7 was either deleted or replaced with lysine exhibited undetectable levels of ADP-ribosyltransferase activity. However, limited trypsinolysis of the arginine 7 mutants yielded fragmentation kinetics that were different from that yielded by the wild-type recombinant subunit or the authentic A subunit. In contrast, mutant proteins in which glutamic acid residues at either position 110 or 112 were replaced with aspartic acid responded like the wild-type subunit upon limited trypsinolysis, while exhibiting severely depressed, but detectable, ADP-ribosyltransferase activity. The latter results may indicate that either glutamic acid 110 or glutamic acid 112 of the A subunit of heat-labile enterotoxin is analogous to those active-site glutamic acids identified in several other ADP-ribosylating toxins.
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PMID:Effect of site-directed mutagenic alterations on ADP-ribosyltransferase activity of the A subunit of Escherichia coli heat-labile enterotoxin. 190 25

Cholera and pertussis toxins each contain a subunit with ADP-ribosyltransferase activity, sharing a region of nearly identical amino acid sequence near the NH2 terminus. Previous investigations have shown that substitution of a lysine residue for Arg-9 in the catalytic A subunit of pertussis toxin substantially eliminates its enzyme activity. We now report that substitution of lysine for the position-equivalent Arg-7 of cholera toxin subunit A leads to a similar loss of catalytic activity. This result suggests a correlation of function with structure between the sequence-related cholera and pertussis toxin A subunits and may contribute to the design of a vaccine containing an enzymatically inert analog of cholera toxin.
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PMID:Site-specific mutagenesis of the catalytic subunit of cholera toxin: substituting lysine for arginine 7 causes loss of activity. 193 84

To be capable of selective killing of tumor cells, the non-selective Pseudomonas aeruginosa exotoxin A must have its cell-binding domain inactivated or removed and then be chemically linked to, or genetically fused with, a specific targeting agent. In the present study, epsilon-NH2 groups of lysine residues of the cell-binding domain of exotoxin A were extensively propionylated with N-succinimidyl-3-propionate (NSP). The NSP-treated exotoxin retained its cytocidal ADP-ribosyltransferase activity, but it could no longer bind to, and inhibit the proliferation of, Friend murine erythroleukemia cells. Cytotoxicity (i.e., the ability to inhibit proliferation) for the Friend erythroid cells was restored completely to the NSP-inactivated exotoxin by conjugating it to ADIF, an autocrine factor secreted by chicken erythroleukemia cells which selectively inhibits the differentiation of erythroid cells such as Friend erythroleukemia cells without inhibiting their proliferation.
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PMID:The cytotoxicity of Pseudomonas exotoxin A, inactivated by modification of the cell-binding domain I, is restored when conjugated to an erythroid cell-specific targeting agent. 210 50

Sulfhydryl-alkylating reagents are known to inactivate the NAD glycohydrolase and ADP-ribosyltransferase activities of the S1 subunit of pertussis toxin, a protein which contains two cysteines at positions 41 and 200. It has been proposed that NAD can retard alkylation of one of the two cysteines of this protein (Kaslow, H.R., and Lesikar, D.D. (1987) Biochemistry 26, 4397-4402). We now report that NAD retards the ability of these alkylating reagents to inactivate the S1 subunit. In order to determine which cysteine is protected by NAD, we used site-directed mutagenesis to construct analogs of the toxin with serines at positions 41 and/or 200. Sulfhydryl-alkylating reagents reduced the ADP-ribosyltransferase activity of the analog with a single cysteine at position 41; NAD retarded this inactivation. In contrast, sulfhydryl-alkylating reagents did not inactivate analogs with serine at position 41. An analog with alanine at position 41 possessed substantial ADP-ribosyltransferase activity. We conclude that alkylation of cysteine 41, and not cysteine 200, inactivates the S1 subunit of pertussis toxin, but that the sulfhydryl group of cysteine 41 is not essential for the ADP-ribosyltransferase activity of the toxin. These results suggest that the region near cysteine 41 contributes to features of the S1 subunit important for ADP-ribosyltransferase activity. Using site-directed mutagenesis, we found that changing aspartate 34 to asparagine, arginine 39 to lysine, and glutamine 42 to glutamate had little effect on ADP-ribosyltransferase activity. However, substituting an asparagine for the histidine at position 35 markedly decreased, but did not eliminate, ADP-ribosyltransferase activity. Chou-Fasman analysis predicted no significant modifications in secondary structure of the S1 peptide with the change of histidine 35 to asparagine. Thus, histidine 35 may interact with a substrate of the S1 subunit without being essential for catalysis.
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PMID:Alkylation of cysteine 41, but not cysteine 200, decreases the ADP-ribosyltransferase activity of the S1 subunit of pertussis toxin. 270 95

We investigated the effect on the Ca2+-dependent ATPase activity of ADP-ribosylation of the enzyme from the rabbit skeletal muscle sarcoplasmic reticulum. A reconstituted ADP-ribosylation system of Ca2+-dependent ATPase in which the enzyme and ADP-ribosyltransferase, both were partially purified from the vesicles, and poly L-lysine were contained, was preincubated with 1 mM NAD, and the Ca2+-dependent ATPase activity was assayed. The NAD-dependent suppression of the enzyme activity depended on both the concentration of NAD and preincubation-time for the ADP-ribosylation, and was reversed by adding 20 mM arginine during the preincubation. These results taken together with the findings that Ca2+-dependent ATPase is a major acceptor protein for the modification in rabbit skeletal muscle sarcoplasmic reticulum [Hara et al. (1987) Biochem. Biophys. Res. Commun. 144; 856-862] suggest that Ca2+-transport in the sarcoplasmic reticulum may be regulated through changes in the rate of ADP-ribosylation of Ca2+-dependent ATPase.
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PMID:ADP-ribosylation of Ca2+-dependent ATPase in vitro suppresses the enzyme activity. 296 35

A partially purified protein preparation from rat liver catalyzed the ADP-ribosylation of low molecular weight guanidino compounds and proteins. Agmatine and arginine, previously shown to be effective acceptors for the guanidine-dependent erythrocyte ADP-ribosyltransferase, were used as acceptors by the rat liver enzyme; lysine, histidine, and serine were inactive. The product of the reaction between [adenine-U-14C]NAD and agmatine catalyzed by the rat liver enzyme co-chromatographed with [adenine-U-14C]ADP-ribose-agmatine which was synthesized by the erythrocyte transferase; in parallel assays, formation of this product was associated with stoichiometric release of [carbonyl-14C]nicotinamide from [carbonyl-14C]NAD. In the presence of histones or other proteins and [adenine-U-14C]NAD or [32P]NAD, the rat liver enzyme catalyzed the formation of a radioactive product which was precipitable by trichloroacetic acid. Digestion of the [adenine-U-14C]-labeled precipitate with snake venom phosphodiesterase released a labeled compound identified as 5'-AMP. These data are consistent with the conclusion that a mono-(ADP-ribosyltransferase) is present in rat liver which utilizes guanidino compounds such as arginine as ADP-ribose acceptors. The ADP-ribose-glutamate bond has been shown to exist in rat liver. Since the catalytic sites of each transferase can accommodate and thus ADP-ribosylate only one specific amino acid, a family of site-specific transferases must be present. The availability of multiple site-specific transferases permits the cell to exert further control over ADP-ribosylation.
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PMID:Amino acid-specific ADP-ribosylation. Identification of an arginine-dependent ADP-ribosyltransferase in rat liver. 626 27

Chemical modification of amino groups in the molecule of islet-activating protein (IAP), pertussis toxin, resulted in differential modification of biological activities of the toxin estimated in vivo with rats. Acetamidination of epsilon-amino groups of 50% (or more) of lysine residues in the IAP molecule totally abolished the lymphocytosis-promoting activity, but exerted no effects on the epinephrine-hyperglycemia inhibitory activity, of the toxin. Both activities were abolished by acylation of 50% or more of the amino groups probably due to the destruction of the toxin's quarternary structure. In contrast, the subunit assembly of IAP was maintained after exhaustive acetamidination of its lysine residues. The ADP-ribosyltransferase (or NAD-glycohydrolase) activity of the A-promoter (the biggest subunit) of IAP, which is responsible for the principal action of the toxin, enhancing insulin secretory responses and thereby inhibiting epinephrine hyperglycemia, was not affected by acetamindination of lysine residues. Thus, the A-protomer moiety of IAP is not directly involved in, but the amino groups of lysine residues in other subunits are selectively essential for, the development of the toxin-induced lymphocytosis.
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PMID:Chemical modification of islet-activating protein, pertussis toxin. Essential role of free amino groups in its lymphocytosis-promoting activity. 654 Oct 59

Cholera toxin catalyzed the ADP-ribosylation of a single plasma membrane protein (Mr 55 000) of both RL-PR-C rat hepatocytes and purified rat liver plasma membranes. Labeling of this protein from nicotinamide [2,8-3H]adenine dinucleotide was competitively inhibited by free arginine, but by no other amino acid tested, including lysine. The same protein was ADP-ribosylated from NAD+ endogenously, i.e., in the absence of toxin. This process was, however, not competitively inhibited by added arginine nor by any other amino acid tested lysine. Free ADP-ribose, even in 50-fold molar excess over the nicotinamide [2,8-3H]adenine dinucleotide substrate, did not reduce (by isotope dilution) the endogenous or cholera toxin-catalyzed labeling of the 55 000 dalton membrane protein. It is likely, therefore, that hepatocyte plasma membranes contain an ADP-ribosyltransferase, with a mechanism similar to that of the A subunit of cholera toxin, in that both transfer ADP-ribose to the same membrane protein and in that neither apparently produce free ADP-ribose as an intermediate. It is also clear that the acceptor residue in the 55 000 dalton protein is different for each process. Cholera toxin-catalyzed and endogenous transfer of ADP-ribose to the hepatocyte plasma membrane protein, in contrast to a pigeon erythrocyte system, required no cytosolic factors. The results indicate that ADP-ribosylation in cloned differentiated rat hepatocytes differs from that in pigeon erythrocytes in that the acceptor protein is larger (55 000 compared to 42 000 daltons), cytosolic factors are not required and transfer of ADP-ribose to the acceptor protein occurs endogenously.
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PMID:Endogenous and cholera toxin-catalyzed ADP-ribosylation of a plasma membrane protein by RL-PR-C cloned rat hepatocytes. 722 28


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