Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0043167 (pertussis)
 19,595 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bordetella pertussis produces a calmodulin-stimulated adenylyl cyclase that invades animal cells and raises intracellular cAMP levels [Confer, D. L., & Eaton, J. W. (1982) Science 217, 948-950; Shattuck, R. L., & Storm, D. R. (1985) Biochemistry 24, 6323-6328]. The mechanism for invasion of animal cells by this enzyme has not been defined, but there is considerable evidence that it does not enter by receptor-mediated endocytosis [Gordon, V. M., Leppla, S. H., & Hewlett, E. L. (1988) Infect. Immun. 56, 1066-1069; Donovan, M. G., & Storm, D. R. (1990) J. Cell. Physiol. 145, 444-449]. In this study, the importance of high-affinity calmodulin (CaM) binding for entry of the enzyme into neuroblastoma cells was evaluated using a mutant enzyme that has significantly lower affinity for calmodulin than the wild-type enzyme. Oligonucleotide-directed site-specific mutagenesis was used to create a point mutant at a critical tryptophan residue (Trp-242) within the proposed CaM binding domain of the B. pertussis adenylyl cyclase. Substitution of Trp-242 with Glu lowered the apparent affinity of the enzyme for calmodulin by 250-fold; however, the maximal enzyme activity in the presence of saturating calmodulin was equivalent to the wild-type enzyme. The Glu-242 mutant adenylyl cyclase was returned to B. pertussis by homologous recombination, and the enzyme produced by this strain was examined for invasion of neuroblastoma cells. Although the mutant enzyme stimulated the production of intracellular cAMP in neuroblastoma cells, the rate of cAMP accumulation was at least 10-fold lower than that caused by the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:High-affinity calmodulin binding is required for the rapid entry of Bordetella pertussis adenylyl cyclase into neuroblastoma cells. 139 Jun 75

Adenylate cyclase (AC) toxin from Bordetella pertussis interacts with and enters eukaryotic cells to catalyze the production of supraphysiologic levels of cyclic AMP. Although the calmodulin-activated enzymatic activity (ability to convert ATP to cyclic AMP in a cell-free assay) of this molecule is calcium independent, its toxin activity (ability to increase cyclic AMP levels in intact target cells) requires extracellular calcium. Toxin activity as a function of calcium concentration is biphasic, with no intoxication occurring in the absence of calcium, low level intoxication (200-300 pmol of cyclic AMP/mg of Jurkat cell protein) occurring with free calcium concentrations between 100 nM and 100 microM and a 10-fold increase in AC toxin activity at free calcium concentrations above 300 microM. The molecule exhibits a conformational change when free calcium concentrations exceed 100 microM as demonstrated by shift in intrinsic tryptophan fluorescence, an alteration in binding of one anti-AC monoclonal antibody, protection of a fragment from trypsin-mediated proteolysis, and a structural modification as illustrated by electron microscopy. Thus, it appears that an increase in the ambient calcium concentration to a critical point and the ensuing interaction of the toxin with calcium induces a conformational change which is necessary for its insertion into the target cell and for delivery of its catalytic domain to the cell interior.
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PMID:Adenylate cyclase toxin from Bordetella pertussis. Conformational change associated with toxin activity. 189 34

The conformation of native pertussis toxin has been investigated by secondary structure prediction and by circular dichroism, fluorescence and second-derivative ultraviolet absorption spectroscopy. The far-ultraviolet circular dichroic spectrum is characteristic of a protein of high beta-sheet and low alpha-helix content. This is also shown by an analysis of the circular dichroic spectrum with the Contin programme which indicates that the toxin possesses 53% beta-sheet, 10% alpha-helix and 37% beta-turn/loop secondary structure. Second-derivative ultraviolet absorption spectroscopy suggests that 34 tyrosine residues are solvent-exposed and quenching of tryptophan fluorescence emission has shown that 4 tryptophan residues are accessible to iodide ions. One of these tryptophans appears to be in close proximity to a positively charged side-chain, since only 3 tryptophans are accessible to caesium ion fluorescence quenching. When excited at 280 nm, the emission spectrum contains a significant contribution from tyrosine fluorescence, which may be a consequence of the high proportion (55%) of surface-exposed tyrosines. No changes in the circular dichroic spectra of the toxin were found in the presence of the substrate NAD. However, NAD did quench both tyrosine and tryptophan fluorescence emission but did not change the shape of the emission spectrum, or the accessibility of the tryptophans to either the ionic fluorescence quenchers or the neutral quencher acrylamide.
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PMID:A spectroscopic and conformational study of pertussis toxin. 205 Jan 51

A truncated, 432 residue long, Bordetella pertussis adenylate cyclase expressed in Escherichia coli was analyzed for intrinsic fluorescence properties. The two tryptophans (Trp69 and Trp242) of adenylate cyclase, each situated in close proximity to residues important for catalysis or binding of calmodulin (CaM), produced overlapping fluorescence emission bands upon excitation at 295 nm. CaM, alone or in association with low concentrations of urea, induced important modifications in the spectra of adenylate cyclase such as shifts of the maxima and change in the shape of the bands. From these changes and from the fluorescence spectrum of a modified form of adenylate cyclase, in which a valine residue was substituted for Trp242, it was deduced that, upon binding of CaM to the wild-type adenylate cyclase, only the environment of Trp242 was affected. The fluorescence maximum of this residue, which is more exposed to the solvent than Trp69 in the absence of CaM, is shifted by 13 nm to shorter wavelength upon interaction of protein with its activator. Trypsin cleaved adenylate cyclase into two fragments, one carrying the catalytic domain, and the second carrying the CaM-binding domain (Ladant et al., 1989). The isolated peptides conserved most of the environment around their single tryptophan residues, as in the intact adenylate cyclase, which suggests that the two domains of truncated B. pertussis adenylate cyclase also conserved most of their three-dimensional structure in the isolated forms.
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PMID:Intrinsic fluorescence of a truncated Bordetella pertussis adenylate cyclase expressed in Escherichia coli. 226 68

The enzymatic ADP-ribosyltransferase activity associated with the S1 subunit of pertussis toxin is considered to be responsible for its biological effects. Although pertussis toxin has no significant homology to other ADP-ribosylating toxins such as diphtheria toxin and Pseudomonas aeruginosa exotoxin A, the results presented in this paper show that, as for diphtheria toxin and exotoxin A, tryptophan and glutamic acid residues are essential for the enzymatic activities of pertussis toxin. Moreover, a structural motif can be identified around the critical glutamic acid residue. Chemical modification or site-directed deletion or replacement of Trp-26 abolishes ADP-ribosyltransferase and the associated NAD glycohydrolase activities. Both enzymatic activities are also abolished when Glu-129 is deleted or replaced by aspartic acid. Mutations at the Glu-106 position do not significantly reduce the enzymatic activities of the S1 subunit. The mutations do not affect the ability of the different S1 forms to be recognized by a variety of monoclonal antibodies, including neutralizing antibodies. Pertussis toxin containing a deletion or replacement of Trp-26, Glu-129, or both in the S1 subunit should thus be devoid of toxic activities without losing its reactivity with protective antibodies and, therefore, could be safely included in new generation vaccines against whooping cough.
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PMID:Identification of amino acid residues essential for the enzymatic activities of pertussis toxin. 247 88

The structural gene of the S-1 subunit of pertussis toxin (rS-1) and the catalytic C180 peptide of the S-1 subunit (C180 peptide) were independently subcloned downstream of the tac promoter in Escherichia coli. Both constructions included DNA encoding for the predicted leader sequence of the S-1 subunit which was inserted between the tac promoter and the structural gene. E. coli containing the plasmids encoding for rS-1 and C180 peptide produced a peptide that reacted with anti-pertussis toxin antibody and had a molecular weight corresponding to that of the cloned gene; some degradation of rS-1 was observed. Extracts of E. coli containing plasmids encoding for rS-1 and the C180 peptide possessed ADP-ribosyltransferase activity. Subcellular fractionation showed that both rS-1 and the C180 peptide were present in the periplasm, indicating that E. coli recognized the pertussis toxin peptide leader sequence. The protein sequence of the amino terminus of the C180 peptide was identical to that of authentic S-1 subunit produced by Bordetella pertussis, which showed that E. coli leader peptidase correctly processed the pertussis toxin peptide leader sequence. Two single amino acid substitutions at residue 26 (C180I-26) and residue 139 (C180S-139) which were previously shown to reduce ADP-ribosyltransferase activity were introduced into the C180 peptide. C180I-26 possessed approximately 1% of the NAD-glycohydrolase activity of the C180 peptide, suggesting that tryptophan 26 functions in the interaction of NAD with the C180 peptide. In contrast, C180S-139 possessed essentially the same level of NAD-glycohydrolase activity as the C180 peptide, suggesting that glutamic acid 139 does not function in the interaction of NAD but plays a role in a later step in the ADP-ribosyltransferase reaction.
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PMID:Expression and secretion of the S-1 subunit and C180 peptide of pertussis toxin in Escherichia coli. 254 19

A gene library of Bordetella pertussis DNA was constructed in Escherichia coli using the broad-host-range cosmid vector pLAFR1. The average insert size was 24.9 kb. From 500 members of the gene library, clones were identified which complemented trpE, glnA and Thr- mutations in E. coli but none which complemented trpD, trpC, trpB, trpA, proA or Leu- mutations. Four clones were identified which complemented trpE in E. coli. Anthranilate synthase activity was detected in a trpE strain only when it harboured a plasmid from one of these clones; activity was repressed when tryptophan was included in the growth medium. Two clones were identified which complemented glnA of E. coli. A recombinant plasmid from one of these clones also restored some of the nitrogen acquisition functions of glnG and glnL in E. coli. Expression of several B. pertussis virulence-associated products (haemolysin, heat-labile toxin, adenylate cyclase, filamentous haemagglutinin, and the cell-envelope polypeptide of Mr 30,000) was not detected in 500 independent clones. However, by transferring the recombinant plasmids to a mutant of B. pertussis deficient in haemolysin and adenylate cyclase, a plasmid was identified which restored both these activities.
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PMID:Complementation of mutations in Escherichia coli and Bordetella pertussis by B. pertussis DNA cloned in a broad-host-range cosmid vector. 288 29

Overlapping 10-amino-acid peptides, which consecutively span the amino acid sequence of the S3 subunit of pertussis toxin, were synthesised on polyethylene pins and screened for their ability to bind the glycoprotein fetuin. Fetuin binding was localised to a single peptide comprising amino acids 46-55. A free peptide, (E)S3c, of longer sequence (S3 amino acids 44-58) was also found to bind alpha-1-acid glycoprotein, mixed brain gangliosides and fetuin. (E)S3c also recognised asialofetuin but with a lower apparent affinity relative to fetuin. The single tryptophan residue of the peptide yielded a fluorescence-emission maximum of 355 nm. In the presence of either ganglioside or the phospholipid L-alpha-lysolecithin, but not N-acetylneuramin-lactose or lactosylceramide, the emission intensity of (E)S3c was enhanced and the emission maximum blue-shifted to 340 nm by ganglioside, or to 345 nm by L-alpha-lysolecithin. Monosialogangliosides, disialogangliosides, and trisialogangliosides, when fluorescence-titrated, were each found to bind the peptide with a similar dissociation constant of 4.4 +/- 2.8 microM. These findings demonstrate that region 44-58 of the pertussis-toxin S3 subunit is likely to be involved in the recognition of both glycosylated and phospholipid constituents of target-cell membranes.
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PMID:Localisation of a receptor-recognition domain on the S3 subunit of pertussis toxin by peptide mapping. 767 39

Cultured rat retinal pigment epithelium cells are shown to contain serotonergic, 5-HT2, receptors associated with phosphoinositide turnover and mobilization of intracellular calcium. Serotonin at a concentration of 10 microM induced a 2.5-fold increase in [3H]-inositol phosphates (more than 75% is in the form of [3H]-inositol-1-phosphate) accumulation within 30 min in cells preincubated in [3H]-myo-inositol and exposed to 5 mM lithium chloride. The EC50 value of serotonin was approx. 0.9 microM and the saturation concentration was 100 microM. Serotonin analogues like tryptamine, 5-methoxytryptamine, alpha-methyl-serotonin and the 5-HT2 agonists quipazine and DOI (1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane) all stimulated InsPs accumulation to some degree. Carbachol, noradrenaline, isoproterenol, dopamine, tryptophan, 5-hydroxytryptophan, 8-hydroxy-2(di-n-propyl-amino) tetralin, 2-methyl-serotonin and NECA (5'-[N-ethyl]-carboxamidoadenosine) were inactive. The serotonin-induced response was blocked most effectively by ketanserin and methysergide but not by 5-HT3 or 5-HT1 antagonists. The serotonin response was attenuated by the active phorbol ester, 4 beta-phorbol 12-myristate 13-acetate and this was attenuated by the non-selective protein kinase C inhibitor, staurosporine. Pertussis toxin failed to influence the serotonin-mediated phosphoinositide turnover. Addition of serotonin to cultures loaded with Fura-2 showed a transient increase in calcium concentrations in most of the cells. This change in calcium was independent of external calcium and the serotonin response was attenuated by ketanserin but not by the 5-HT3 antagonist granisetron.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Serotonergic, 5-HT2, receptor-mediated phosphoinositide turnover and mobilization of calcium in cultured rat retinal pigment epithelium cells. 827 84

The adk gene from the Gram-negative pathogen Bordetella pertussis was cloned by complementing the thermosensitive Escherichia coli adk strain CR341T28. B. pertussis adenylate kinase is a 218-amino-acid protein that has high similarity with adenylate kinase from Escherichia coli and Hemophilus influenzae (57%). A distinct characteristic of enzyme from B. pertussis, not found in other bacterial adenylate kinases, is the presence of a tryptophan residue at position 185. Although distant from the catalytic site, this single tryptophan serves as a convenient probe for monitoring the binding of nucleotide substrates or analogs to the enzyme. Differential scanning calorimetry and equilibrium unfolding experiments in guanidine.HCl indicate similar stabilities for adenylate kinase from B. pertussis and E. coli. An extensive comparison between physico-chemical properties of adenylate kinase from B. pertussis and the enzyme from E. coli showed that the kinetic and structural properties of the two enzymes are very similar. However, infrared spectroscopy has allowed to identify small but significant differences in the secondary structure of the two proteins.
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PMID:Structural and physico-chemical characteristics of Bordetella pertussis adenylate kinase, a tryptophan-containing enzyme. 828 44


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