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)

Thiols such as cysteine and dithiothreitol are substrates for the ADP-ribosyltransferase activity of pertussis toxin. When cysteine was incubated with NAD+ and toxin at pH 7.5, a product containing ADP-ribose and cysteine (presumably ADP-ribosylcysteine) was isolated by high-performance liquid chromatography, and characterized by its composition and release of AMP with phosphodiesterase. Cysteine has a Km of 105 mM at saturating NAD+ concentration. The ability of thiols to act as a substrate is one explanation for the very high concentrations (250 mM or greater) that have been observed to enhance the apparent NAD glycohydrolase activity of the toxin.
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PMID:Thiol reagents are substrates for the ADP-ribosyltransferase activity of pertussis toxin. 313 46

We attempted to characterize ADP-ribose-amino acid bonds formed by various bacterial toxins. The ADP-ribose-arginine bond formed by botulinum C2 toxin in actin was cleaved with a half-life of about 2 h by treatment with hydroxylamine (0.5 M). In contrast, the ADP-ribose-cysteine bond formed by pertussis toxin in transducin and the ADP-ribose-amino acid linkage formed by botulinum ADP-ribosyltransferase C3 in platelet cytosolic proteins were not affected by hydroxylamine. HgCl2 cleaved the ADP-ribose-amino acid bond formed by pertussis toxin in transducin but not those formed by botulinum C2 toxin or botulinum ADP-ribosyltransferase C3 in actin and platelet cytosolic proteins, respectively. NaOH (0.5 M) cleaved the ADP-ribose-amino acid bonds formed by botulinum C2 toxin and pertussis toxin but not the one formed by botulinum ADP-ribosyltransferase C3. The data indicate that the ADP-ribose bond formed by botulinum ADP-ribosyltransferase C3 differs from those formed by the known bacterial ADP-ribosylating toxins.
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PMID:Different types of ADP-ribose protein bonds formed by botulinum C2 toxin, botulinum ADP-ribosyltransferase C3 and pertussis toxin. 314 Aug 13

We have constructed three different truncated versions of diphtheria toxin (a 535-amino-acid polypeptide) which correspond to the N-terminal 290, 377, and 485 amino acids of the toxin. These lengths include one, three, and all four of the putative membrane-spanning sequences of the toxin which are thought to play a role in the translocation of fragment A into cells. Each of these three genes has been modified at its 3' end to code for a C-terminal cysteine (to allow for disulfide linkage of a targeting ligand) or a gene fusion with alpha-melanocyte-stimulating hormone. We have also substituted the native diphtheria tox promoter (ptox) with the lambda pR promoter in an effort to overexpress these proteins. The truncated genes are expressed in Escherichia coli from both the tox promoter in a constitutive fashion and from the pR promoter by using the heat-inducible cI857 repressor. The clones produce proteins which react with anti-diphtheria toxin serum, which migrate at the anticipated Mr on Western blots, and which have ADP-ribosyltransferase activity. Constitutive synthesis from ptox leads to severe proteolytic degradation even in a protease-deficient strain. High-level expression from the pR promoter in the same lon htpR strain allows the full-length polypeptides to accumulate but also stops the growth of the cells. It appears that removal of as few as 50 amino acids from the C-terminus of diphtheria toxin alters its conformation, making it a target for proteases and causing overexpression lethality in the host cells.
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PMID:Cloning and expression in Escherichia coli of three fragments of diphtheria toxin truncated within fragment B. 354 95

A novel enzymatic activity, i.e., the catalysis of the formation of ADP-ribosylcysteine, was found in the cytosol of human erythrocytes. The NAD:cysteine ADP-ribosyltransferase was partially purified by sequential chromatographic steps on phenyl-Sepharose, phosphocellulose, and Sepharose CL-6B. The enzyme has an apparent molecular weight of 27,000 +/- 3,000, as determined by gel permeation. The formation of ADP-ribosylcysteine was associated with the stoichiometric release of nicotinamide from NAD. The enzyme was found to be highly specific toward cysteine and cysteine methyl ester as ADP-ribose acceptors. S-Benzoyl-L-cysteine, cystine, histidine, glutamic acid, arginine, arginine methyl ester, and agmatine were ineffective as acceptors for this enzyme.
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PMID:An NAD:cysteine ADP-ribosyltransferase is present in human erythrocytes. 359 54

An NAD+:cysteine ADP-ribosyltransferase activity was purified from bovine erythrocytes on the assumption that, like pertussis toxin, the enzyme would exhibit a cysteine-dependent NAD+ glycohydrolase activity. A three-step purification procedure was developed involving (1) precipitation with 40% (NH4)2SO4, (2) binding to a cysteine-Sepharose affinity column, and (3) binding to an NAD+ affinity column. PAGE showed a single band of M(r) 45,000. The enzyme had been purified 47,000-fold and had a specific activity of 1900 nmol nicotinamide released/min per mg. A study of the kinetic properties of this enzyme showed saturation kinetics for cysteine (Km = 4.0 mM). The ability of this enzyme to ADP-ribosylate protein was investigated using re-sealed inverted bovine erythrocyte ghosts. Incubation of the purified enzyme with erythrocyte ghosts and [adenylate-32P]NAD+ led to the enhanced dose-dependent labelling of several proteins, a doublet of high M(r) and proteins of M(r) 60,000, 55,000 and 29,000, identified by autoradiography of separated proteins on SDS/PAGE. The enzyme-catalysed labelling of the major component at M(r) 55,000 was blocked by pre-treatment of the erythrocyte ghosts with N-ethymaleimide, a sulphydryl alkylating agent, and the label was released by mercuric ion, but not by hydroxylamine. These experiments suggested that a cysteine residue on the target protein had been mono-ADP-ribosylated. This supposition was further supported by identification of the mercf1p4ion-released radiolabelled product as ADP-ribose by HPLC, and the observation that free ADP-ribose was unable to modify the membrane target protein directly.
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PMID:The purification of a cysteine-dependent NAD+ glycohydrolase activity from bovine erythrocytes and evidence that it exhibits a novel ADP-ribosyltransferase activity. 757 29

Nitric oxide (NO) has been suggested to act as a regulator of endogenous intracellular ADP-ribosylation, based on radiolabelling of proteins in tissue homogenates incubated with [32P]NAD and NO. After the NO-stimulated modification was replicated in a defined system containing only the purified acceptor protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the hypothesis of NO-stimulation of an endogenous ADP-ribosyltransferase became moot. The NO-stimulated, NAD-dependent modification of GAPDH was recently characterized as covalent binding of the whole NAD molecule to the enzyme, not ADP-ribosylation. With this result, along with the knowledge that GAPDH is stoichiometrically S-nitrosylated, the role of NO in protein modification with NAD may be viewed as the conferring of an unexpected chemical reactivity upon GAPDH, possibly due to nitrosylation of a cysteine in the enzyme active site.
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PMID:Nitric oxide and NAD-dependent protein modification. 789 64

Mono-ADP-ribosylation is a protein modification that occurs at a number of different amino acids, dictated by the specificity of the individual ADP-ribosyltransferases. A specific cysteine in several guanine nucleotide-binding regulatory proteins is ADP-ribosylated by the bacterial protein pertussis toxin. Recent purification of an ADP-ribosylcysteine hydrolase and NAD:cysteine ADP-ribosyltransferase, and detection of ADP-ribose-cysteine linkages in tissue samples has raised hope that an endogenous regulatory cysteine-specific ADP-ribosylation pathway exists. A current goal is the identification of such a pathway for ADP-ribosylation of cysteine within animal cells. Interpretation of the data in this field has been complicated by recent reports that revealed several unforeseen chemical reactions of NAD and its metabolites with free cysteine and cysteine in proteins. This mini-review covers the latest understanding of the ADP-ribosylation reactions associated with cysteine, and provides a set of criteria for future research to establish positively the existence of an endogenous cysteine-specific mono-ADP-ribosyltransferase.
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PMID:Enzymatic and nonenzymatic ADP-ribosylation of cysteine. 789 67

A cDNA spanning the entire coding region for poly(ADP-ribose) polymerase (PARP) of Sarcophaga peregrina was isolated and the nucleotide sequence was determined. The longest open reading frame encodes a polypeptide of 996 amino acid residues with a molecular mass of 113,033 Da. The similarities to the human PARP in amino acid sequence were relatively low in the DNA-binding and auto-modification domains, but very high in the C-terminal catalytic domain: identity of amino acids is 34% in the N-terminal DNA-binding domain (residues 1-369), 27% in the auto-modification domain (residues 370-507), and 56% in the C-terminal NAD-binding domain (residues 508-996). Two zinc-fingers (C-X2-C-X28-H-X2-C and C-X2-C-X31-H-X2-C)2 and a basic region in the N-terminal DNA-binding domain recognized in other PARP are conserved. Downstream of the basic region, another cysteine-rich motif (C-X2-C-X13-C-X9-C), a putative zinc-finger, was found to be well conserved in the PARP of Sarcophaga, Drosophila and human. A leucine-zipper motif (L-X6-L-X6-L-X6-L) which was found in the auto-modification domain of Drosophila PARP, is disrupted in the Sarcophaga enzyme: the second leucine is replaced by proline, and the third leucine by valine. Full-length cDNA for Sarcophaga PARP was cloned into an expression plasmid and expressed in Escherichia coli. A lysate of E. coli cells containing expressed protein reacted with antibody against Sarcophaga PARP, and PARP activity was detected. Thus, we conclude that isolated cDNA encodes a functional Sarcophaga PARP cDNA.
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PMID:Cloning and functional expression of poly(ADP-ribose) polymerase cDNA from Sarcophaga peregrina. 812 21

Incubation of lysate from human polymorphonucleated neutrophils and human platelets with [32P]NAD resulted in the labeling of a 42-kDa protein. Phosphodiesterase (Crotalus durissus) released 5'-AMP from the radiolabeled protein. The 42-kDa protein was identified as actin by binding to DNAse-I, two-dimensional gel electrophoresis and partial proteolysis. The rate of ADP-ribosylation was greater with [32P]ADP-ribose than with [32P]NAD, indicating a non-enzymic modification. ADP-ribose also modified actin in the actin-DNAase-I complex, but denatured actin was not modified by ADP-ribose. Only cytoplasmic beta/gamma-actin isoforms were non-enzymically ADP-ribosylated but not muscle alpha-actin. The acceptor amino acid was identified as a cysteine residue whereas the bacterial ADP-ribosyltransferase C. perfringens iota toxin catalyzes incorporation of ADP-ribose to Arg177 of actin. Alkylation of cysteine residues of actin with N-ethylmaleimide prevented subsequent non-enzymic ADP-ribosylation but not the toxin catalyzed modification. Non-enzymically ADP-ribosylated actin was further modified by C. perfringens iota toxin. The F-actin stabilizing mycotoxin phalloidin blocked the non-enzymatic ADP-ribosylation and, conversely, ADP-ribosylation inhibited the phalloidin-induced polymerization of ADP-ribosylated actin. The data indicate that cytoplasmic actin is non-enzymically ADP-ribosylated by ADP-ribose at a cysteine residue to inhibit actin polymerization.
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PMID:Cysteine-specific ADP-ribosylation of actin. 818 61

Recent evidence suggests that mammalian cysteine proteases related to Caenorhabditis elegans CED-3 are key components of mammalian programmed cell death or apoptosis. We have shown recently that the CPP32 and Mch2 alpha cysteine proteases cleave the apoptotic markers poly(ADP-ribose) polymerase (PARP) and lamins, respectively. Here we report the cloning of a new Ced-3/interleukin 1 beta-converting enzyme-related gene, designated Mch3, that encodes a protein with the highest degree of homology to CPP32 compared to other family members. An alternatively spliced isoform, named Mch3 beta, was also identified. Bacterially expressed recombinant Mch3 has intrinsic autocatalytic/autoactivation activity. The specific activity of Mch3 alpha toward the peptide substrate DEVD-7-amino-4-methylcoumarin and PARP resembles that of CPP32. Like interleukin 1 beta-converting enzyme and CPP32, the active Mch3 alpha is made of two subunits derived from a precursor (proMch3 alpha). It was of interest that recombinant CPP32-p17 subunit can form an active heteromeric enzyme complex with recombinant Mch3 alpha-p12 subunit and vice versa, as determined by the ability of the heteromeric complexes to induce apoptosis in Sf9 cells. These data suggest that proMch3 alpha and proCPP32 can interact to form an active Mch3 alpha/CPP32 heteromeric complex. We also provide evidence that CPP32 can efficiently cleave proMch3 alpha, but not the opposite, suggesting that Mch3 alpha activation in vivo may depend in part on CPP32 activity. The high degree of conservation in structure and specific activity and the coexistence of Mch3 alpha and CPP32 in the same cell suggests that the PARP cleavage activity observed during apoptosis cannot solely be attributed to CPP32 but could also be an activity of Mch3 alpha.
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PMID:Mch3, a novel human apoptotic cysteine protease highly related to CPP32. 852 91


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