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)

Kenimer et al. (J. G. Kenimer, J. Kim, P. G. Probst, C. R. Manclark, D. G. Burstyn, and J. L. Lowell, Hybridoma 8:37-51, 1989) identified three classes of monoclonal antibodies, termed A, B, and C, that recognize the S1 subunit of pertussis toxin. This report presents data demonstrating that class A monoclonal antibodies (3CX4, 6D11C, and 3C4D), which block the ADP-ribosyltransferase activity and recognize the predominant neutralizing epitope on the S1 subunit of the toxin, do not inhibit the NAD-glycohydrolase activity of the toxin. In addition, alkylation of cysteine 41 of the S1 subunit, which may interact with NAD, inactivates the toxin but does not prevent binding by class A antibodies. Taken together, these results support the conclusion that proper alterations of amino acids that interact with NAD should allow for inactivation of the toxin without destruction of the predominant neutralizing epitope. The class A antibodies recognized control but not heat-treated pertussis toxin spotted onto nitrocellulose, indicating that class A antibodies do not recognize denatured S1 subunit. In contrast, a nonneutralizing class C antibody (X2X5) failed to bind to control toxin or S1 subunit in solution and recognized heat-treated pertussis toxin better than control toxin when spotted onto nitrocellulose. Thus, this type of analysis presents a heterogeneous mixture of fully or partially denatured and native S1 proteins and fails to distinguish between neutralizing and nonneutralizing antibodies.
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PMID:Monoclonal antibodies that inhibit ADP-ribosyltransferase but not NAD-glycohydrolase activity of pertussis toxin. 215 82

The S1 subunit (Mr 28,000) of pertussis toxin expresses thiol-dependent enzymatic ADP-ribosyltransferase and NAD-glycohydrolase activities. Site-directed mutagenesis experiments were performed on the codon for Cys-41 of this subunit to investigate the role of this residue in both enzymatic activities. Deletion of Cys-41 caused a decrease in both activities below detectable levels, whereas replacement of this residue by serine, glycine, proline, or asparagine only slightly reduced the activities. The enzymatic activities of these mutants were thiol-independent. The deletion of Ser-40, adjacent to Cys-41, again caused reduction of the enzymatic activities to undetectable levels. Steady-state kinetic experiments showed that the kcat of the mutant protein in which Cys-41 was replaced by glycine was nearly identical to the kcat of the parent version. However, the Km for NAD of the mutant was significantly higher relative to that of the wild type version. These results indicate that the side-chain of Cys-41 is not essential for enzymatic activities and that Cys-41 is not involved in the rate of catalysis but is probably located at or close to the NAD-binding site. The introduction of a negative charge at position 41 through the replacement of Cys-41 by either aspartate or glutamate reduced the enzymatic activities to very low but measurable levels, suggesting a charge-charge repulsive interaction between these residues and possibly one or both of the phosphates of NAD. Cys-41 may therefore be located close to the phosphate subsite of the NAD-binding site.
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PMID:The role of cysteine 41 in the enzymatic activities of the pertussis toxin S1 subunit as investigated by site-directed mutagenesis. 215 32

Limited proteolysis of Pseudomonas aeruginosa exotoxin A by four proteases (chymotrypsin, Staphylococcal serine proteinase, pepsin A and subtilisin) resulted in the formation of polypeptides having a molecular mass of approximately 25 kDa. They possessed both enzymatic activity and residual antigenicity. Their N-terminal sequence analysis showed that the different proteases cleaved exotoxin A in a very restricted area within domain Ib (amino acids 365-404). As a result, the polypeptides contained a large portion (13-34 amino acids) of domain Ib linked to the adjacent C-terminal domain III (amino acids 405-613). The major fragment derived from subtilisin cleavage, at a final yield of 35% (S-fragment; residues 392-613; 24201 Da; pI 4.7) possessed the same level of ADP-ribosyltransferase activity as uncleaved exotoxin A (by mass), and a 37-fold higher NAD-glycohydrolase activity. Polyclonal antibodies from rabbits against exotoxin A completely inhibited the ADP-ribosyltransferase activity of both exotoxin A and the S-fragment, but not the NAD-glycohydrolase activity of the S-fragment. Antibodies against the S-fragment neutralized the ADP-ribosyltransferase activity of exotoxin A. These data determine the primary proteolytic cleavage site of exotoxin A, suggest that some residues in the amino acid sequence 392-404 of exotoxin A seem to have a role in binding or positioning elongation factor 2 (EF-2) and show that antibodies recognize the EF-2-binding site but not the NAD(+)-binding site.
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PMID:Biochemical and immunochemical studies of proteolytic fragments of exotoxin A from Pseudomonas aeruginosa. 217 Jan 23

The A subunit of cholera toxin contains the ADP-ribosyltransferase activity in its major constituent polypeptide A1 (Mr 23,000) which is responsible for the elevation of cAMP typically observed with most mammalian cell types after exposure to the toxin. The primary structure of the A subunit, recently established by sequence analyses, is presented and used as the basis for the secondary structure prediction according to the method of Chou and Fasman. The results indicated the presence of 27% alpha-helix, 25% beta-structure, 12% beta-turn, and 36% random coil. The majority of the beta-structure consisted of six strands located in the NH2-terminal portion of the molecule (residues 33-106) covering one-half of the region corresponding to the A1 polypeptide portion. The beta-sheet domain led immediately into the active site region characterized by the alternating structures of beta-pleated sheet and alpha-helix (residues 95-140) similar to that reported for other NAD+ binding proteins. The presence of this structural feature in the region was confirmed by the use of another predictive method (J. Garnier et al., J. Mol. Biol. 1978, 120, 97-120). In addition, two regions (residues 14-18 and 200-214), previously identified to contain binding sites for the B subunit as evidenced by chemical modification and monoclonal antibody studies, were found to be in alpha-helix configuration.
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PMID:Cholera toxin A subunit: functional sites correlated with regions of secondary structure. 240 74

NAD is hydrolyzed during incubation with isolated renal brush border membranes (BBM). The specific enzymatic mechanisms have not been identified apart from the activity of ADP-ribosyltransferase, which accounts for a very small proportion of the total hydrolysis. In the present study, an NAD-glycohydrolase (NGH) was identified in the renal BBM using the cyanide-addition assay to monitor hydrolysis of NAD at the nicotinamide-ribose bond. The production of nicotinamide and ADP-ribose, the expected reaction products, was determined by thin-layer chromatography. The NGH was enriched ninefold in the BBM fraction and accounted for 36% of the total rate of NAD hydrolysis by BBM enzymes at pH 7.4. Assay of NGH in sealed BBM vesicles subjected to osmotic shock indicated that about 23% of the NGH is exposed on the cytoplasmic surface of the BBM. The enzyme was inhibited by nicotinamide in vitro and also when the nicotinamide was administered in vivo, suggesting, indirectly, that the enzyme may play a role in mediating the effects of nicotinamide on BBM phosphate transport.
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PMID:NAD-glycohydrolase in renal brush border membranes. 241 52

Poly(ADP ribose) polymerase (EC 2.4.2.30) was studied using monoclonal antibodies for three different epitopes on the enzyme. The epitopes were mapped in relation to the functional domains of the protein and the inhibitory properties of the antibodies. The intranuclear and interspecies immunoreactivity of the enzyme was also investigated. The epitope of antibody 2 was mapped to the 17 kDa fragment generated by chymotryptic digestion of the C-terminal 54 kDa NAD-binding domain. Antibody 9 binds to the N-terminal 29 kDa fragment of the DNA binding domain and inhibits the enzyme activity by 80%. This antibody was used to purify poly(ADP ribose) polymerase by immunoaffinity chromatography. The third antibody binds to a central 36 kDa fragment that possesses part of the DNA-binding domain and the automodification domain. This antibody increases the enzymatic activity by 30%. An analysis of the species cross-reactivity of the antibodies was carried out by immunoblot analysis of nuclear proteins. Antibody 10 binding was detected in rat FR3T3 cells, Chinese hamster ovary cells (CHO) and epidermoid carcinoma lung human cells (CALU-1). The other two antibodies are specific for the human and bovine enzymes. Western blot analysis showed the association of poly(ADP ribose) polymerase with residual nuclear material obtained after nuclease treatment and high-salt extraction. Immunofluorescence studies with the three different monoclonals demonstrated that accessibility of the epitopes varies in the nucleus.
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PMID:Structural and functional analysis of poly(ADP ribose) polymerase: an immunological study. 245 68

Treatment of platelets with a prostacyclin analogue, iloprost, decreased the cholera-toxin-induced ADP-ribosylation of membrane-bound Gs alpha (alpha-subunit of G-protein that stimulates adenylate cyclase; 42 kDa protein) and a cytosolic substrate (44 kDa protein) [Molina y Vedia, Reep & Lapetina (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5899-5902]. This decrease is apparently not correlated with a significant change in the quantity of membrane Gs alpha, as detected by two Gs alpha-specific antisera. This finding contrasts with the suggestion in a previous report [Edwards, MacDermot & Wilkins (1987) Br. J. Pharmacol. 90, 501-510], indicating that iloprost caused a loss of Gs alpha from the membrane. Our evidence points to a modification in the ability of the 42 kDa protein to be ADP-ribosylated by cholera toxin. This modification of Gs alpha might be related to its ADP-ribosylation by endogenous ADP-ribosyltransferase activity. Here we present evidence showing that Gs alpha was ADP-ribosylated in platelets that had been electropermeabilized and incubated with [alpha-32P]NAD+. This endogenous ADP-ribosylation of Gs alpha is inhibited by nicotinamide and stimulated by iloprost.
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PMID:The effect of iloprost on the ADP-ribosylation of Gs alpha (the alpha-subunit of Gs). 247 20

A non-histone acceptor protein for hen liver nuclear ADP-ribosyltransferase was purified to an apparently homogeneous state through salt extraction and chromatography on hydroxyapatite, phenyl-Sepharose, carboxy-methyl-cellulose, Sephadex G-75, phenyl 5-PW, mono S and Radial PAK C18. This protein was termed p33. The ADP-ribosylation of p33 was enhanced more than 60-fold by double-stranded DNA. Single-stranded DNA, RNA and poly(L-glutamate), but not deoxyribonucleotide, were partially effective. DNA-dependent ADP-ribosylation was also observed when whole histones were used as acceptor. DNA required for the maximal ADP-ribosylation depended on the dose of the acceptor protein; the optimal mass ratio of DNA to the acceptor protein was 1:1 with both p33 and whole histones. DNA decreased the Km for NAD and concomitantly increased the Vmax value, but did not alter the Km for p33. These results are consistent with the idea that p33 may participate in chromatin processes such as replication or transcription, through modification by nuclear ADP-ribosyltransferase.
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PMID:DNA-dependent mono(ADP-ribosyl)ation of p33, an acceptor protein in hen liver nuclei. 249 38

The gene for human nuclear NAD+ ADP-ribosyltransferase [NAD+:poly(adenosine diphosphate D-ribose) ADP-D-ribosetransferase, EC 2.4.2.30; pADPRT] was localized to chromosome 1 at q41-q42 by in situ hybridization with a pADPRT-specific cDNA probe. Expression of a pADPRT cDNA under control of the lac promoter in Escherichia coli induces the synthesis of a group of related proteins that were immunoreactive with pADPRT antibody and that had catalytic properties very similar to those of the human enzyme. Purification of this enzymatic activity was performed essentially as described for the human enzyme. The Km, pH optimum, optimal reaction temperature, and inhibition by 3-aminobenzamide and 3-methoxybenzamide were found to be similar for the recombinant and the human enzymes. The purified recombinant enzyme consists of two major proteins of Mr 99,000 and Mr 89,000. Both proteins show pADPRT activity in activity gel analysis with [32P]NAD+ as substrate. Microsequencing of these two proteins isolated by denaturing gel electrophoresis and deletion mutagenesis of the pADPRT expression plasmid shows that the Mr 99,000 and Mr 89,000 proteins derive from initiation of translation at internal translational start signals located within the pADPRT cDNA.
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PMID:Human nuclear NAD+ ADP-ribosyltransferase: localization of the gene on chromosome 1q41-q42 and expression of an active human enzyme in Escherichia coli. 249 72

Guanine nucleotide-binding (G) proteins are involved in several transmembrane signaling systems. Choleragen (cholera toxin) activates adenylate cyclase by catalyzing the ADP-ribosylation of Gs alpha, the stimulatory G protein of the cyclase system. This reaction is enhanced by another guanine nucleotide-binding protein termed ADP-ribosylation factor or ARF that was purified from bovine brain membranes [R. A. Kahn and A. G. Gilman, Journal of Biological Chemistry (1986) 261, 7906-7911]. It was recently found that this ARF also increases the NAD:agmatine and NAD:protein ADP-ribosyltransferase, NAD glycohydrolase and auto-ADP-ribosylation activities of the toxin. We have purified and characterized two soluble proteins from bovine brain that act in a similar fashion to enhance choleragen activity in each of these reactions. The membrane and soluble factors are all proteins of approximately 19 kDa that require GTP or GTP analogues for activity and are ADP-ribosylated by the toxin. The ARF proteins apparently interact directly with choleragen in a GTP-dependent fashion to increase its catalytic activity and thus are part of a G protein cascade through which the toxin activates adenylate cyclase. The physiological function of the ARF proteins, as well as their possible relationships to the ras oncogene products and/or the family of G proteins that includes Gs alpha, remains to be determined.
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PMID:Participation of a guanine nucleotide-binding protein cascade in cholera toxin activation of adenylate cyclase. 249 82


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