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)

The effects of cholera toxin, a secretory product of Vibrio cholerae, result from ADP-ribosylation of the stimulatory guanine nucleotide-binding (Gs) protein of the adenylyl cyclase system. Cholera toxin A subunit (CTA) also uses agmatine, a simple guanidino compound, several proteins unrelated to Gs, and CTA itself as alternative ADP-ribose acceptors. The effects of toxin occur in the jejunum presumably at body core temperature. With agmatine as a model substrate, the optimal temperature for CTA-catalyzed ADP-ribosylation was 25-30 degrees C, and that for CTA-catalyzed auto-ADP-ribosylation was 20-25 degrees C. Both activities were significantly less at 37 degrees C, reflecting lower initial velocities, not heat-inactivation of the toxin. All the transferase activities of CTA are enhanced by ADP-ribosylation factors (ARFs), approximately 20-kDa guanine nucleotide-binding proteins that are ubiquitous in mammalian cells. Phospholipids and a soluble brain ARF, in a GTP-dependent manner, activated toxin NAD:agmatine ADP-ribosyltransferase activity; their simultaneous effect was maximal at physiological temperatures (approximately 37 degrees C). At lower temperatures, the stimulation by ARF was much less. There were similar effects on other toxin-catalyzed reactions, notably, the ADP-ribosylation of Gs alpha and the hydrolysis of NAD. Thus, host factors, such as ARF and phospholipid, synergistically increase cholera toxin activity at 37 degrees C and may be important in toxin action in the mammalian gut.
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PMID:Effects of temperature on ADP-ribosylation factor stimulation of cholera toxin activity. 842 66

ADP-ribosylation factors (ARFs) are a family of approximately 20-kDa guanine nucleotide-binding proteins that stimulate the ADP-ribosyltransferase activities of cholera toxin in vitro and function in protein trafficking in vivo. The six cloned mammalian ARFs can be grouped into three classes based on size and sequence identity. ARF 2 is a class I ARF, whose approximately 2.6-kilobase mRNA exhibits species and tissue selective expression and is developmentally regulated in rat brain. Here we report the sequence, structure, and functional promoter region of the bovine ARF 2 gene, which was facilitated by constructing a composite cDNA. The ARF 2 cDNA, constructed from a partial cDNA clone and polymerase chain reaction-amplified fragments from reverse-transcribed poly(A)+ RNA, was approximately 2270 base pairs (bp) (minus the poly(A) tail). In the 3'-untranslated region, there are two potential polyadenylation signals, ATTAAA and AATAAA, at positions 1064 and 2232, respectively, and two ATTTA motifs, believed to signal mRNA degradation, at positions 2115 and 2165. The ARF 2 gene, represented in three overlapping genomic clones, spans approximately 20 kilobase pairs with five exons and four introns. Consensus sequences for guanine nucleotide-binding and GTP hydrolysis are in separate exons, except for the NKXD sequence, which is divided by intron 4. There are multiple transcriptional initiation sites. Transient transfection of embryonic trachea cells with deletion constructs defined the functional promoter region to be within 400 bp upstream of the most 5' site of transcription initiation. This 400-bp region lacks a TATA-like sequence but contains six inverted CCAAT boxes, four potential Sp1-binding sites, and a potential AP-2-binding site. Although the pattern of expression of ARF 2 is unique among the ARFs, the structures of the class I ARF genes are conserved among its members and across species.
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PMID:Characterization of the gene for ADP-ribosylation factor (ARF) 2, a developmentally regulated, selectively expressed member of the ARF family of approximately 20-kDa guanine nucleotide-binding proteins. 844 65

Cholera toxin and Escherichia coli heat-labile enterotoxin (LT) exert their effects on cells through ADP-ribosylation of guanine nucleotide-binding proteins. Both toxins consist of one A subunit, which is an ADP-ribosyltransferase, and five B (or binding) subunits. Their enzymatic activities are latent; activation requires reduction and proteolysis, resulting in a catalytically active A1 protein and a much smaller A2 protein. These ADP-ribosyltransferases are activated by GTP-dependent 20-kDa ADP-ribosylation factors or ARFs. To determine if proteolysis plus reduction is required for appearance of the ARF allosteric site as well as for catalytic activity, an inactive mutant of LT, LT(E112K), with replacement of glutamate by lysine at position 112 of its A subunit, was utilized as a competitor in cholera toxin ADP-ribosyltransferase assays containing limiting amounts of ARF. LT(E112K) required trypsinization and reduction to become a potent, concentration-dependent inhibitor. Inhibition was reversed by increasing concentrations of ARF. Reduction or trypsinization alone did not generate an inhibitory form of LT(E112K). These studies are consistent with the conclusion that the ARF site is not expressed in the latent toxin. Both trypsinization and reduction are required for expression of a functional ARF binding site as well as for catalytic activity.
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PMID:Interaction of ADP-ribosylation factor with Escherichia coli enterotoxin that contains an inactivating lysine 112 substitution. 845 9

ADP-ribosylation factors (ARFs), a family of approximately 20-kDa guanine nucleotide-binding proteins that activate cholera toxin ADP-ribosyltransferase in vitro, have been implicated in intracellular protein trafficking and are thought to cycle between cytosolic and membrane compartments. Although isolated predominantly as soluble proteins, ARFs associate with membranes and phospholipids in a GTP-dependent manner. In contrast to other small GTP-binding proteins, ARFs are NH2 terminally myristoylated. Using a bacterial expression system, recombinant myristoylated and non-myristoylated human ARF5 were produced to investigate the role of myristoylation in its association with Golgi. The recombinant ARFs (myristoylated and non-myristoylated) exhibited similar biochemical activity as measured by GTP binding and in vitro activation of cholera toxin. Myristoylated ARF5, however, demonstrated a temperature- and GTP-dependent association with Golgi membranes, whereas non-myristoylated ARF did not bind to Golgi under any of the experimental conditions. These data indicate that myristoylation is necessary, although not sufficient, for membrane attachment, but is not necessary for activation of cholera toxin.
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PMID:Effect of myristoylation on GTP-dependent binding of ADP-ribosylation factor to Golgi. 846 39

Clones referred to as ARD 1 were isolated from human and rat cDNA libraries. ARD 1 genes encode a putative 64-kDa protein that contains an 18-kDa ADP-ribosylation factor (ARF) domain at the carboxyl terminus and is much larger than the other monomeric approximately 20-kDa guanine nucleotide-binding ARF proteins thus far identified. ARD 1 mRNAs of 3.7 and 4.1 kilobases were detected in all rat tissues as well as in mouse and rabbit brain, human fibroblasts, and human neuroblastoma cells but not in HL-60 cells. Based on sequence identities, ARD 1 is highly conserved between rat and human. The ARF domain of ARD 1 contains the consensus sequences believed to be involved in guanine nucleotide binding, which are conserved in the ARFs and other GTP-binding proteins. Recombinant ARD 1 or the ARF domain of ARD 1, which lacks the 15 amino acids corresponding to the amino-terminal regions of ARFs stimulated, in a GTP-dependent manner, cholera toxin ADP-ribosyltransferase activity in the presence of 0.3% Tween 20. It had no effect in the presence of SDS, dimyristoylphosphatidylcholine/cholate, or cardiolipin. These observations are consistent with the conclusion that the amino-terminal region of ARF proteins is not required for activation of cholera toxin. In addition, the characteristic features of ARF proteins may be found as domains of larger mammalian proteins.
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PMID:ARD 1, a 64-kDa guanine nucleotide-binding protein with a carboxyl-terminal ADP-ribosylation factor domain. 847 24

Vibrio cholerae O1, No. 31, a strain isolated from a patient with mild diarrhea, produced mainly the unnicked cholera toxin. The amount of toxin that had accumulated in the cells was approximately 200 times lower than that secreted into the culture medium. When the unnicked toxin was purified by three successive column chromatographies and then extracted from the polyacrylamide gel, the unnicked toxin showed two bands corresponding to the A and B subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the A1 fragment was detected by trypsinization. Biological and enzymatic activities of the purified toxin with trypsinization were identical to those of cholera toxin from V. cholerae 569B as seen in the rabbit skin permeability test and the NAD:agmatine ADP-ribosyltransferase assay. DNA sequences of the A and B subunits were identical to those of the A- and B-subunit genes from the El Tor 2125 and classical 0395 strains, respectively. These data suggest that the wild V. cholerae strain, No. 31, produces a toxin identical to toxins previously reported in the literature and secretes it without accumulation in the cell, as is the case with other strains. However, strain No. 31's ability to nick the toxin is diminished compared with such abilities of other strains.
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PMID:A classical strain of Vibrio cholerae with diminished ability to process the proteolytically sensitive site in the A subunit of cholera toxin. 864 66

We made use of ADP-ribosylarginine hydrolase to detect arginine-ADP- ribosylated proteins. The hydrolase was expressed in Escherichia coli as a protein fused with glutathione S-transferase (GST). The fusion protein GST-ADP-ribosylarginine hydrolase catalyzed the hydrolysis of alpha-ADP-ribosylarginine to produce ADP-ribose and arginine. Casein ADP-ribosylated with [32P]NAD and chicken heterophil arginine-specific ADP-ribosyltransferase served as a substrate for the recombinant ADP-ribosylarginine hydrolase and the released ADP-ribose was determined. Protein ADP-ribosylated by cholera toxin could serve as substrate of the hydrolase but protein ADP-ribosylated by pertussis toxin, diphtheria toxin, or C(3) enzyme of Clostridium botulinum could not. The hydrolase did not release the radioactivity incorporated into isolated rat liver nuclei incubated with [(32)P]NAD or in bovine brain cytosol incubated with [(32)P]ADP-ribose. In homogenate of mouse heart which contained arginine-specific ADP-ribosyltransferase, labeling of a 55-kDa protein by incubation with [(32)P]NAD was removed by ADP-ribosylarginine hydrolase treatment; hence, the specific hydrolysis of ADP-ribose-arginine bond by GST-ADP-ribosylarginine hydrolase can be used to detect the arginine-ADP-ribosylated proteins in crude preparations. Arginine--ADP-ribosylated proteins in crude preparations. Arginine-ADP-ribosylated proteins in mouse spleen lymphocytes were identified using this method.
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PMID:Detection of arginine-ADP-ribosylated protein using recombinant ADP-ribosylarginine hydrolase. 867 89

Mono-ADP-ribosylation is a post-translational modification of proteins in which the ADP-ribose moiety of NAD is transferred to proteins and is responsible for the toxicity of some bacterial toxins (e.g. cholera toxin and pertussis toxin). NAD:arginine ADP-ribosyltransferases cloned from human and rabbit skeletal muscle and from mouse lymphoma (Yac-1) cells are glycosylphosphatidylinositol-anchored and have similar enzymatic and physical properties; transferases cloned from chicken heterophils and red cells have signal peptides and may be secreted. We report here the cloning and characterization of an ADP-ribosyltransferase (Yac-2), also from Yac-1 lymphoma cells, that differs in properties from the previously identified eukaryotic transferases. The nucleotide and deduced amino acid sequences of the Yac-1 and Yac-2 transferases are 58 and 33% identical, respectively. The Yac-2 protein is membrane-bound but, unlike the Yac-1 enzyme, appears not to be glycosylphosphatidylinositol-anchored. The Yac-1 and Yac-2 enzymes, expressed as glutathione S-transferase fusion proteins in Escherichia coli, were used to compare their ADP-ribosyltransferase and NAD glycohydrolase activities. Using agmatine as the ADP-ribose acceptor, the Yac-1 enzyme was predominantly an ADP-ribosyltransferase, whereas the transferase and NAD glycohydrolase activities of the recombinant Yac-2 protein were equivalent. The deduced amino acid sequence of the Yac-2 transferase contained consensus regions common to several bacterial toxin and mammalian transferases and NAD glycohydrolases, consistent with the hypothesis that there is a common mechanism of NAD binding and catalysis among ADP-ribosyltransferases.
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PMID:Cloning and characterization of a novel membrane-associated lymphocyte NAD:arginine ADP-ribosyltransferase. 870 12

ADP-ribosylation factors (ARFs) are approximately20-kDa guanine nucleotide-binding proteins that participate in vesicular transport in the Golgi and other intracellular compartments and stimulate cholera toxin ADP-ribosyltransferase activity. Both GTP binding and hydrolysis are necessary for its physiological functions, although purified mammalian ARF lacks detectable GTPase activity. An ARF GTPase-activating protein (GAP) was purified >15,000-fold from rat spleen cytosol using (NH4)2SO4 precipitation and chromatography on Ultrogel AcA 34, DEAE-Sephacel, heparin-Sepharose, hydroxylapatite, and Ultrogel AcA 44. In fractions ( approximately100-kDa proteins) from Ultrogel AcA 44, a major protein band of approximately50 kDa on SDS-polyacrylamide gel electrophoresis correlated with GAP activity, consistent with it being a homodimer, thus differing from an ARF GAP purified from rat liver (Makler, V., Cukierman, E., Rotman, M., Admon, A., and Cassel, D. (1995) J. Biol. Chem. 270, 5232-5237). Purified spleen GAP accelerated hydrolysis of GTP bound to recombinant ARF1, ARF3, ARF5, and ARF6; no effect of NH2-terminal myristoylation was observed. ARF GAP also activated GTP hydrolysis by ARL1, which is 56% identical in amino acid sequence to ARF1, but lacks ARF activity. ARD1 is a 64-kDa guanine nucleotide-binding protein that contains an 18-kDa ARF domain at its carboxyl terminus; the ARF domain lacks the amino-terminal alpha-helix found in native ARF and hence is similar to the amino-terminal truncated mutant Delta13ARF1. Both the ARF domain of ARD1 and Delta13ARF1 were poor substrates for ARF GAP. The non-ARF1 domain of ARD1 enhanced the GTPase activity of the ARF domain, but not that of the ARF proteins and Delta13ARF1, i.e. it lacks the relatively broad substrate specificity exhibited by ARF GAP.
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PMID:Characterization of a GTPase-activating protein that stimulates GTP hydrolysis by both ADP-ribosylation factor (ARF) and ARF-like proteins. Comparison to the ARD1 gap domain. 879 35

ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins that are allosteric activators of the NAD:arginine ADP-ribosyltransferase activity of cholera toxin and appear to play a role in intracellular vesicular trafficking. Although the physiological roles of these proteins have not been defined, it has been presumed that each has a specific intracellular function. To obtain genetic evidence that each ARF is under evolutionary pressure to maintain its structure, and presumably function, rat ARF cDNA clones were isolated and their nucleotide and deduced amino acid sequences were compared to those of other mammalian ARFs. Deduced amino acid sequences for rat ARFs 1, 2, 3, 5 and 6 were identical to those of the known cognate human and bovine ARFs; rat ARF4 was 96% identical to human ARF4. Nucleotide sequences of both the untranslated as well as the coding regions were highly conserved. These results indicate that the ARF proteins are, as a family, extraordinarily well conserved across mammalian species. The unusually high degree of conservation of the untranslated regions is consistent with these regions having important regulatory roles and that individual ARFs contain structurally unique elements required for specific functions.
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PMID:Interspecies relationships among ADP-ribosylation factors (ARFs): evidence of evolutionary pressure to maintain individual identities. 881 5


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