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)

Preincubation of human peripheral blood polymorphonuclear leukocytes (HPPMN) with recombinant human tumor necrosis factor-alpha (rHuTNF-alpha) enhanced the formylmethionyl-leucylphenylalanine (FMLP)-induced superoxide (O2-.) generation in a concentration- and preincubation time-dependent manner. The enhancement was very high for the FMLP- or opsonized zymosan (OZ)-induced O2-. generation, but was low for arachidonic acid (AA)- and phorbol myristate acetate (PMA)-induced O.2- generation. The rHuTNF-alpha has no effect on the steady state of intracellular calcium ion concentration ([Ca2+]i) nor on the membrane potential of neutrophils. The rHuTNF-alpha-primed FMLP-induced O2-. generation was inhibited by nicotineamide (NA), pertussis toxin (PT), and by the tyrosine kinase (TK) inhibitor, genistein, but was enhanced by the protein kinase C (PKC) inhibitor, H-7 (1-(5-isoquinolinesulfonyl)-3-methyl-piperazine). The inhibitory actions of NA and PT were also observed in in vivo primed guinea pig peritoneal neutrophils (GPtPMN). However, FMLP-induced O2-. generation of GPtPMN was enhanced by genistein, but was inhibited by H-7. These data indicate that TNF-alpha does not induce changes in [Ca2+]i nor in membrane potential of HPPMN, and that TNF-alpha-primed FMLP-induced O.2- generation of HPPMN is coupled with ADP-ribosylation and activation of G-proteins, and that protein kinases, especially TK, seem to exert an important role in the priming action of TNF.
Physiol Chem Phys Med NMR 1992
PMID:Effect of tumor necrosis factor-alpha on the stimulus-coupled responses of neutrophils and their modulation by various inhibitors. 132 5

ADP-ribosylation is a posttranslational modification of proteins by amino acid-specific ADP-ribosyltransferases. Both pertussis toxin and eukaryotic enzymes ADP-ribosylate cysteine residues in proteins and also, it has been suggested, free cysteine. Analysis of the reaction mechanisms of cysteine-specific ADP-ribosyltransferases revealed that free ADP-ribose combined nonenzymatically with cysteine. L- and D-cysteine, L-cysteine methyl ester, and cysteamine reacted with ADP-ribose, but alanine, serine, lysine, arginine, N-acetyl-L-cysteine, 2-mercaptoethanol, dithiothreitol, and glutathione did not. The 1H NMR spectrum of the product, along with the requirement for both free sulfhydryl and amino groups of cysteine, suggested that the reaction produced a thiazolidine linkage. ADP-ribosylthiazolidine was labile to hydroxylamine and mercuric ion, unlike the ADP-ribosylcysteine formed by pertussis toxin and NAD in guanine nucleotide-binding (G-) proteins, which is labile to mercuric ion but stable in hydroxylamine. In the absence of G-proteins but in the presence of NAD and cysteine, pertussis toxin generated a hydroxylamine-sensitive product, suggesting that a free ADP-ribose intermediate, expected to be formed by the NADase activity of the toxin, reacted with cysteine. Chemical analysis, or the use of alternative thiol acceptors lacking a free amine, is necessary to distinguish the enzymatic formation of ADP-ribosylcysteine from nonenzymatic formation of ADP-ribosylthiazolidine, thereby differentiating putative NAD:cysteine ADP-ribosyltransferases from NAD glycohydrolases.
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PMID:Amino acid-specific ADP-ribosylation: structural characterization and chemical differentiation of ADP-ribose-cysteine adducts formed nonenzymatically and in a pertussis toxin-catalyzed reaction. 144 18

The solution conformation of a synthetic peptide of 20 amino acids (P235-254) derived from the calmodulin-binding domain of Bordetella pertussis adenylate cyclase was studied by proton two-dimensional NMR spectroscopy and circular dichroism. Based on the standard techniques we have assigned all the resonances in the NMR spectrum to the corresponding protons of the peptide. Analysis of the secondary chemical shift distribution and of the nuclear Overhauser effect connectivities showed no evidence for a highly populated regular conformation but suggested the tendency to form an alpha-helix around the unique Trp residue. The propensity for a helical structure is in agreement with the results of circular dichroic spectroscopy showing a slight negative band at 222 nm which was cancelled by 6 M guanidine hydrochloride. Increasing amounts of 2,2,2-trifluoroethanol (up to 40%) increase considerably the helical population of the peptide as reflected in the circular dichroic spectra. Analysis of the present results shows that the free peptide P235-254 has the tendency to form a basic amphiphilic helix. The presence of two acid residues, Glu236 and Asp239, on the hydrophilic side of the alpha-helix, which is mainly composed by basic residues, may explain the lower affinity of this peptide for calmodulin as compared with other peptides derived from calmodulin-activated enzymes.
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PMID:NMR and circular dichroic studies on the solution conformation of a synthetic peptide derived from the calmodulin-binding domain of Bordetella pertussis adenylate cyclase. 200 8

A simple method for the simultaneous assay of both substrate utilization and product formation by Bordetella pertussis adenylate cyclase has been developed. This method involves measurement of ATP remaining in the reaction mixture and cyclic 3',5'-AMP (cAMP) formation by 31p-NMR spectroscopy. No separation of the nucleotides is required. The measurement of the rate of cAMP formation compared very well with other methods that require separation of product from the substrate. With this method it has been possible to show calmodulin activation of B. pertussis adenylate cyclase and to demonstrate an inhibition of calmodulin activation by melittin. The inhibition of calmodulin-activated adenylate cyclase by melittin is not permanent and can be overcome by long-term incubation.
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PMID:A simple method for the assay of Bordetella pertussis adenylate cyclase employing 31P nuclear magnetic resonance spectroscopy. 287 63

Gentamicin nucleotidyltransferase-catalyzed reaction of (Sp)-[alpha-17O]dATP with tobramycin produced 2"-(2'-deoxyadenosine 5'-[17O]phosphoryl)tobramycin. The configuration at phosphorus in this product was shown to be Rp by chemical degradation to chiral [17O, 18O]dAMP using a stereochemically defined procedure, and determination of the configuration at phosphorus in this product. Periodate-base treatment of 2"-(2'-deoxyadenosine 5'-[17O]phosphoryl)tobramycin followed by NaBH4 reduction produced (2-glyceryl)-[17O]dAMP, which upon snake venom phosphodiesterase-catalyzed hydrolysis in H(2)18O produced [17O,18O] dAMP. The configuration at phosphorus in this product was shown to be S by enzymatic phosphorylation to [17O,18O]dATP, adenylylcyclase (Bordetella pertussis)-catalyzed cyclization to 3',5'-cyclic [17O,18O]dAMP, and 31P NMR analysis of the ethyl esters. Since snake venom phosphodiesterase-catalyzed hydrolyses proceed with retention of configuration at phosphorus, (Sp)-[17O,18O]dAMP must have been produced from (Rp)-(2-glyceryl)-[17O]dAMP; and since the chemical degradation to the latter compound did not involve cleavage of any bonds to phosphorus, the initial enzymatic product must have been (Rp)-2"-(2'-deoxyadenosine 5'-[17O]phosphoryl)tobramycin. Therefore, nucleotidyl transfer catalyzed by gentamicin nucleotidyl-transferase proceeds with inversion of configuration at phosphorus, and the reaction mechanism involves an uneven number of phosphotransfer steps. Inasmuch as this is an uncomplicated two-substrate group transfer reaction, the mechanism probably involves direct nucleotidyl transfer from the nucleoside triphosphate to the aminoglycoside. The B. pertussis adenylylcyclase reaction was shown to proceed with inversion at phosphorus, as has been established for other adenylylcyclases.
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PMID:Gentamicin nucleotidyltransferase. Stereochemical inversion at phosphorus in enzymatic 2'-deoxyadenylyl transfer to tobramycin. 287 83

This paper reports the solution conformation of a peptide (P196-267) derived from the calmodulin-binding domain of Bordetella pertussis adenylate cyclase. P196-267 corresponding to the protein fragment situated between amino acid residues 196-267 was overproduced by a recombinant Escherichia coli strain. Its affinity for calmodulin is only one order of magnitude lower (Kd = 2.4 nM) than that of the whole bacterial enzyme (Kd = 0.2 nM). The proton resonances of the NMR spectra of P196-267 were assigned using homonuclear two-dimensional techniques (double-quantum-filtered J-correlated spectroscopy, total correlation spectroscopy, and nuclear Overhauser enhancement spectroscopy) and a standard assignment procedure. Analysis of the nuclear Overhauser effect connectivities and the secondary shift distribution of C alpha protons along the sequence allowed us to identify the elements of regular secondary structure. The peptide is flexible in solution, being in equilibrium between random coil and helical structures. Two segments of 11 amino acids (situated between V215 and A225) and 15 amino acids (situated between L233 and A247) populate in a significant proportion the helix conformational state. The two helices can be considerably stabilized in a mixed solvent, trifluoroethanol/water (30/70), suggesting that the corresponding fragment in the intact protein assumes a similar secondary conformation. No elements of tertiary structure organization were detected by the present experiments. The conformational properties of the isolated calmodulin target fragment are discussed in relation with the available NMR and X-ray data on various peptides complexed to calmodulin.
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PMID:Structural characterization by nuclear magnetic resonance spectroscopy of a genetically engineered high-affinity calmodulin-binding peptide derived from Bordetella pertussis adenylate cyclase. 762 28

The immunogenic efficacy of multiple antigen peptides, MAPs, i.e. branched molecules in which multiple copies of a given immunogenic peptide are attached on a scaffold of lysine residues via both alpha and epsilon linkages, has been repeatedly demonstrated, but little is known about the structural arrangement of these peptide constructs. A conformational characterization was therefore performed for a known T cell epitope of the S1 subunit of Pertussis toxin, whose sequence is predicted to form alpha-helix. The peptide DNVLDHLTGR, its N-acetylated and C-amidated analogue and a tetrabranched MAP based on the N-acetylated peptide were prepared and studied by CD and two-dimensional 1H-NMR. No evidence of helical structure was obtained in water for the isolated peptides. In contrast, in triflouroethanol, the isolated epitopes fold into a helical structure spanning the segment Val3-Thr8 in the uncapped molecule and encompassing also the N-terminal region in the capped analogue. The mobile C-terminal region tends to adopt a distorted turn arrangement in both peptides due to the folding of Arg10 guanidinium over the backbone. No distortion of the helix structure was observed for the single-copy epitope in the four-branched MAP molecule in trifluoroethanol: each peptide chain is equivalent within the MAP and shows an even more regular helical pattern than the isolated end-blocked sequence. A slight difference was located at the junction with the lysine scaffold: the peptide bond to epsilon NH was found in a much more extended conformation than the corresponding link to alpha NH. These structural results correlate with in vitro T cell stimulatory activity of the three compounds examined and provide arguments supporting the previous suggestion that MAP tetramers are unlikely to elicit an immune response specific for the synthetic template assembly, a feature necessary to retain the advantage of the polymeric epitope presentation.
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PMID:Conformational study of a short Pertussis toxin T cell epitope incorporated in a multiple antigen peptide template by CD and two-dimensional NMR. Analysis of the structural effects on the activity of synthetic immunogens. 769 60

This paper reports the solution conformation and calmodulin binding of a 43-residue peptide from the calmodulin-binding domain of Bordetella pertussis adenylate cyclase. The peptide (P225-267) was synthesized and 15N-labeled at specific amino acids. It binds calmodulin with an equilibrium dissociation constant of 25 nM. Assignment of the NMR spectrum of the free peptide and analysis of the NOE connectivities and secondary shifts of C alpha protons allowed us to identify a 10-amino acid fragment (Arg237 to Arg246) which is in rapid equilibrium between alpha-helical and irregular structures. Titration experiments showed that at substoichiometric molar ratios the two molecules are in intermediate exchange between free and bound conformations. Using 15N-edited methods we assigned a large part of resonances of the labeled residues in the bound peptide. Analysis of the chemical shift differences between free and bound states shows that the fragment Leu240-Ala257 is the most affected by the interaction. The proton spectra of the calmodulin, in the free and complexed states were extensively assigned using homonuclear experiments. Medium- and long-range NOE patterns are consistent with a largely conserved secondary and tertiary structure. The main changes in chemical shift of calmodulin resonances are grouped in six structural regions both in NH2- and COOH-terminal domains. Intermolecular NOE connectivities indicate that the NH2-terminal of the bound peptide fragment is engulfed in the COOH-terminal domain of calmodulin. The interaction geometry appears to be similar to those previously described for myosin light chain kinase or calmodulin kinase II fragments.
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PMID:Calmodulin binding of a peptide derived from the regulatory domain of Bordetella pertussis adenylate cyclase. 770 46

A 34-amino acid peptide corresponding to residues 532-565 of Bacillus anthracis adenylate cyclase (P532-565), a calmodulin (CaM)-activated enzyme, was synthesized by solid phase method. Although not homologous to any known CaM binding sequence, P532-565 exhibits molecular features characteristic of this class of peptides: a higher proportion of basic and hydrophobic residues, segregated onto the two faces of the alpha-helical structure. Fluorescence measurements and gel retardation analysis showed that P532-565 binds CaM in a Ca(2+)-dependent manner, with a binding energy that represents 80% of the binding energy of the adenylate cyclase-CaM complex. Circular dichroism analysis showed that P532-565 exists in solution as a mixture of random-coil and alpha-helical structures and that trifluoroethanol increases the relative proportion of alpha-helical population. Analysis of proton NMR spectrum in H2O allowed identification of the different amino acid spin systems and complete spectral assignment. The pattern of nuclear Overhauser effect connectivities, intense NN(i,i + 1) and medium range alpha N(i,i + 3) and alpha beta (i,i + 3) indicate the presence of an alpha-helix in the carboxylterminal end (between residues 551 and 563) in fast exchange with extended structures. These data, together with CaM-binding properties of Bordetella pertussis adenylate cyclase, show that despite rather divergent primary structures, the two bacterial enzymes possess similar structural organization of their binding sites for activator protein.
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PMID:Characterization of a synthetic calmodulin-binding peptide derived from Bacillus anthracis adenylate cyclase. 842 Sep 46

Whooping cough, an acute respiratory disease affecting over sixty million infants, can be prevented by vaccination. The vaccine currently used, composed of killed bacterial cells, however, has been associated with many side effects. An improved vaccine against the disease should contain pertussis toxin (PT), a major virulent factor of Bordetella pertussis (B. pertussis). In order to be included in the vaccine, PT needs to be detoxified and the chemical methods used so far are not completely satisfactory, since they give a product with reduced immunogenicity and possible residual toxicity. To avoid this problem, we have used recombinant DNA technologies to clone the PT gene, express it in bacteria, map the B and T cell epitopes of the molecule and identify the amino acids that are important for the enzymatic activity and toxicity. Based on this information, the gene coding for PT was mutated to produce an inactive protein. This genetically modified PT was non toxic, highly immunogenic and able to protect mice from intracerebral challenge with virulent B. pertussis. The mutant was included as a main component of an acellular pertussis vaccine which has been shown in numerous clinical trials to be more safe and immunogenic than the old cellular vaccine.
Physiol Chem Phys Med NMR 1995
PMID:Acellular pertussis vaccine composed of genetically inactivated pertussis toxin. 876 91


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