UMLS:C0043167 - FACTA Search

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Query: UMLS:C0043167 (pertussis)
19,595 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The relationship between the structure of the A subunit of pertussis toxin and its function was analyzed. Limited tryptic digestion of the A subunit converted the protein to two stable fragments (Mr = 20,000 and 18,000). Antibodies raised to synthetic peptides homologous to regions in the A subunit were used to map these fragments. Both fragments were shown to contain the NH2-terminal portion but not the COOH-terminal portion of the A subunit. While these fragments exhibited NAD glycohydrolase activity, they were unable to reassociate with the B oligomer of the toxin. Thus the COOH-terminal portion of the A subunit does not contain the residues which are required for the NAD glycohydrolase activity of the toxin. However, this region of the molecule may be important for maintaining the oligomeric structure of the toxin. These results suggest that the A subunit of pertussis toxin is similar in structure to the A subunit of cholera toxin. In addition, antibodies raised to a synthetic peptide identical to residues 6-17 of the A subunit of pertussis toxin will bind to the A subunit of cholera toxin.
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PMID:Structural characterization of pertussis toxin A subunit. 332 46

Pertussis toxin (islet-activating protein) activates adenylate cyclase in susceptible cells by ADP-ribosylating an inhibitory component of the cyclase system. This toxin, assayed in a cell-free system in the presence of high concentrations of thiol, catalyzed the hydrolysis of NAD to ADP-ribose and nicotinamide. This NAD glycohydrolase activity co-chromatographed on Sephacryl G-200 in 6.5 M urea, pH 3.2, 0.1 M glycine with the ADP-ribosyltransferase activity of the toxin, as monitored by the transfer of [32P]ADP-ribose from [32P]NAD to a 41,000-Da protein in NG108-15 neuroblastoma X glioma hybrid cells. In the absence of thiol, the native holotoxin was enzymatically inactive. Following addition of 250 mM dithiothreitol to the assay, maximal enzymatic activity was evident after a delay of approximately 1 h; with 20 mM thiol, the delay was longer. The Km for NAD with the fully activated enzyme was 25 microM; the Km did not appear to vary with the extent of activation. Thiol was necessary in a cell-free system to demonstrate NAD glycohydrolase activity. When extensively washed membranes were used as a source of 41,000-Da substrate, thiol was necessary to observe ADP-ribosylation in some cases (human erythrocytes) and significantly stimulated activity in others (NG108-15 cells). In contrast to the bacterial toxins choleragen and Escherichia coli heat-labile enterotoxin that ADP-ribosylate stimulatory components of the cyclase system, pertussis toxin did not transfer ADP-ribose to low molecular weight guanidino compounds, such as arginine or agmatine.
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PMID:Activation by thiol of the latent NAD glycohydrolase and ADP-ribosyltransferase activities of Bordetella pertussis toxin (islet-activating protein). 631 27

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

Retinoic acid-induced differentiation of human leukemic HL-60 cells is accompanied with the early induction of an ecto-enzyme of NAD+ glycohydrolase (NADase), which has recently been identified as human leukocyte cell surface antigen CD38 [Kontani, K. et al. (1993) J. Biol. Chem. 268, 16895-16898]. The terminal cell differentiation attendant upon the cell growth arrest was, but the early induction of CD38 NADase activity was not, inhibited by prior treatment of HL-60 cells with pertussis toxin, which catalyzed ADP-ribosylation of the membrane-bound alpha beta gamma-trimeric GTP-binding proteins. The prior treatment was, however, not essential for the toxin-induced inhibition of the cell differentiation; the inhibition by the addition of pertussis toxin was still observed even after retinoic acid-induced expression of CD38 antigen. This suggested that a pertussis toxin-sensitive mechanism was involved in a late process of cell differentiation. Indeed, HL-60 cells appeared to secrete a differentiation-supporting factor in response to retinoic acid, since the cell differentiation was accelerated and potentiated upon culture of the cells in a conditioned medium prepared from retinoic acid-treated cells. The action of the differentiation-supporting factor was destroyed by heating and markedly attenuated in pertussis toxin-pretreated HL-60 cells. Thus, the whole process of the retinoic acid-induced cell differentiation appeared to consist of two distinguishable periods in terms of sensitivity to pertussis toxin; the toxin-insensitive early period characterized by the induction of CD38 NADase activity and the toxin-sensitive late period in which the secretion of a differentiation-supporting factor might be involved.
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PMID:Involvement of pertussis toxin-sensitive mechanism in retinoic acid-induced differentiation of human leukemic HL-60 cells. 777 89

Pertussis toxin and adenylate cyclase toxin both contribute to the pathogenesis of whooping cough. Production of these proteins is controlled by the bvg locus, which is inactive at 25 degrees C, but at 37 degrees C produces a Vir+ phenotype. In view of the temperature dependence of virulence factor synthesis, the effects of temperature and host factors on their action were examined. The NAD glycohydrolase activity of the S1 subunit of pertussis toxin was enhanced by CHAPS, a zwitterionic detergent, with a temperature optimum of approximately 35 degrees C. Similar temperature optima for the ADP-ribosylation by pertussis toxin of transducin and recombinant Go alpha were observed. Since the temperature--activity relationship of S1 differed from that of S1 in activated holotoxin, and since S1 in activated holotoxin was more stable at 42 degrees C than was S1, it appears that S1 associated with the B oligomer components may, in fact, be an active species. Bordetella pertussis adenylate cyclase is activated by a host factor, calmodulin. In the absence of calmodulin, the temperature optimum for enzymatic activity was approximately 25 degrees C, whereas in its presence it was approximately 35 degrees C. Thus, the temperature optima for pertussis and adenylate cyclase toxins, virulence factors whose production is increased through the bvg locus at physiological temperatures, are either at or near these temperatures when stimulated by host factors.
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PMID:Effect of temperature and host factors on the activities of pertussis toxin and Bordetella adenylate cyclase. 780 92

Pseudomonas aeruginosa exotoxin A (ETA) is a member of the family of bacterial ADP-ribosylating toxins which use NAD+ as the ADP-ribose donor. By analogy to diphtheria and pertussis toxins, the His440 residue of ETA has been proposed to be one of the critical residues within the active site of the toxin. In this study the role of the His440 residue was explored through site-directed mutagenesis which resulted in the production of ETA proteins containing Ala, Asn, and Phe substitutions at the 440 position. The His440-substituted ETA proteins were purified and analyzed. All substitutions at the 440 site displayed severely reduced ADP-ribosylation activity (> 1000-fold). However, NAD glycohydrolase activity remained intact and in the case of ETAH440N actually increased 10-fold. NAD+ binding is not affected by substitutions at the 440 site as indicated by similar Km values for the ETA variants tested. Conformational integrity of the mutant toxins appears to be largely unaffected as assessed by analysis with a conformation-sensitive monoclonal antibody as well as sensitivity to proteinase digestion. In view of the location of His440 residue within or close to the proposed NAD(+)-binding site, these results suggest that His440 may be a catalytic residue involved in the transfer of the ADP-ribose moiety to the EF-2 substrate.
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PMID:Active site mutations of Pseudomonas aeruginosa exotoxin A. Analysis of the His440 residue. 782 95

Molecular modeling of the S1 subunit (S1) of pertussis toxin with other ADP-ribosylating bacterial exotoxins predicted that histidine 35 (His-35) would residue within the active site of S1. Recombinant derivatives of S1 (rS1 and the C180 peptide) which contained either a H35Q or H35P mutation were analyzed to determine the role of His-35 in ADP-ribosylation. C180 peptide is a recombinant peptide composed of the amino-terminal 180 amino acids of S1. Under linear velocity conditions, C180H35Q possessed 2% of wild type C180 peptide activity and C180H35P possessed no detectable activity in the ADP-ribosylation of transducin. The H35Q mutation did not change the affinity of recombinant peptides for NAD or two targets for ADP-ribosylation, transducin, or alpha i3C20, but did lower the kcat in the NAD glycohydrolase and ADP-ribosyltransferase reactions. Neither the H35Q nor H35P mutation reduced the ability of recombinant proteins to be photocross-linked with NAD which was consistent with the His-35 mutations not reducing the affinity for NAD. These data indicate that His-35 does not reduce the affinity of S1 for NAD or transducin, but functions as a catalytic residue in the ADP-ribosylation reaction possibly in a hydrogen bonding capacity.
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PMID:Role of histidine 35 of the S1 subunit of pertussis toxin in the ADP-ribosylation of transducin. 814 93

Molecular modeling and alignment of the primary amino acid sequence of the S1 subunit (S1) of pertussis toxin (PT) with other members of the family of ADP-ribosylating bacterial exotoxins predicted that tyrosine-98 (Y98) of S1 was a conserved residue among these exotoxins. To extend our understanding of the relationship between pertussis toxin and the other ADP-ribosylating exotoxins, we defined the function of Y98 of S1. Using site-directed mutagenesis, Y98 of S1 was substituted with alanine (Y98A), leucine (Y98L), histidine (Y98H), and phenylalanine (Y98F). Mutations were analyzed in the C180 peptide and C219 peptide, recombinant derivatives of S1 which contain the first 180 and 219 amino-terminal residues of S1, respectively. Periplasmic extracts containing the Y98n peptides expressed similar specific activities for the ADP-ribosylation of transducin (Gt) as the periplasmic extract containing wild-type peptides. Mutations at Y98 influenced the subcellular localization of the respective Y98n peptide. The majority of the wild-type Y98 and Y98F peptides localized to the periplasmic extract, while the majority of Y98A and Y98L peptides were associated with the insoluble bacterial outer membrane. Purified C180Y98A and C180Y98F and partially purified C180Y98H peptides possessed similar specific activities for the ADP-ribosylation of Gt as the wild-type C180 peptide. KmNAD and kcat for C180Y98A and C180Y98F in the NAD glycohydrolase reaction were similar to the wild-type C180 peptide. These data show that the R group of Y98 does not participate in the ADP-ribosylation of Gt, but appears to contribute to the proper folding of S1.
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PMID:Biochemical analysis of mutations at tyrosine-98 of the S1 subunit of pertussis toxin. 831 78

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

Pertussis toxin from Bordatella pertussis catalyzes the ADP ribosylation of several G-proteins, using NAD+ as a substrate. In the absence of an acceptor protein, the toxin acts as a NAD+ glycohydrolase. Pertussis toxin is one of the virulent factors for whooping cough and therefore a target for site-specific inhibitors based on the transition state structure. A family of kinetic isotope effects was determined for the hydrolysis reaction, using NAD+ labeled with 3H, 14C, and 15N as substrates. Primary isotope effects were 1.021 +/- 0.001 for [1'N-14C]NAD+ and 1.021 +/- 0.004 for [1N-15N]NAD+, and the double-primary effect of [1'N-14C,1N-15N]NAD+ was 1.049 +/- 0.004. Secondary kinetic isotope effects were 1.207 +/- 0.010 for the [1'N-3H]-, 1.144 +/- 0.005 for the [2'N-3H]-, 0.989 +/- 0.001 for the [4'N-3H]-, and 1.019 +/- 0.004 for the [5'N-3H]NAD+, respectively. Commitment to catalysis was excluded by isotope trapping experiments, and the experimental kinetic isotope effects were independent of pH. The measured isotope effects are therefore intrinsic. The isotope effects are remarkable because they indicate an oxocarbenium-like ribose ring at the transition state but a stiffer than expected vibrational environment for C1' at the reaction center. On the basis of these isotope effects, a bond order vibrational analysis was performed to locate a transition state structure consistent with the isotope effects. The kinetic isotope effects predict a residual bond order to the nicotinamide leaving group of 0.11, corresponding to a distance of 2.14 A. Participation of the water nucleophile is weak, consistent either with an S(N)1-like transition state with no water interaction or with the water oxygen no closer than 3.5 A from the reaction center. The positive charge of the ribose oxocarbenium is stabilized by delocalization between the C1'-O4' and C1'-C2' bonds. The enzyme contacts restrict the vibrational environment of the reaction coordinate requiring increased bonding force constants for the enzyme-stabilized transition state. NAD+ analogues with the nicotinamide ribose replaced by an iminoribitol ring, mimicking the flattened ribose ring of the transition state, are expected to be transition state inhibitors.
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PMID:Kinetic isotope effect characterization of the transition state for oxidized nicotinamide adenine dinucleotide hydrolysis by pertussis toxin. 910 61

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