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:4.6.1.1 (adenylate cyclase)
19,190 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bordetella pertussis adenylate cyclase (AC) toxin is a calmodulin-activated adenylate cyclase enzyme which has the capacity to enter eukaryotic target cells and catalyze the conversion of endogenous ATP into cyclic AMP. In this work, the AC holotoxin molecule is identified and isolated. It is a single polypeptide of apparent 216 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Monoclonal antibodies which immunoprecipitate AC activity from extracts of wild type B. pertussis (BP338) react with this 216-kDa band on Western blots, and it is absent from a transposon Tn5 mutant (BP348) specifically lacking AC toxin. Isolation of the 216-kDa protein to greater than 85% purity by hydrophobic chromatography, preparative sucrose gradient centrifugation, and affinity chromatography using either calmodulin-Sepharose or monoclonal antibody coupled to Sepharose 4B yields stepwise increases in AC toxin potency, to a maximum of 88.3 mumol of cAMP/mg of target cell protein/mg of toxin. Electroelution of the 216-kDa band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis yields a preparation with both AC enzyme and toxin activities. These data indicate that this protein represents the AC holotoxin molecule.
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PMID:Adenylate cyclase toxin from Bordetella pertussis. Identification and purification of the holotoxin molecule. 255 37

Bordetella pertussis produces a calmodulin-dependent adenylate cyclase (AC) which acts as a toxin capable of penetrating eukaryotic cells and generating high levels of intracellular cyclic AMP. Transfer of target cells into B. pertussis AC-free medium leads to a rapid decay in the intracellular AC activity, implying that the invasive enzyme is unstable in the host cytoplasm. We report here that treatment of human lymphocytes with a glycolysis inhibitor and an uncoupler of oxidative phosphorylation completely blocked the intracellular inactivation of B. pertussis AC. Lymphocyte lysates inactivated all forms of B. pertussis AC in the presence of exogenous ATP. This inactivation was associated with degradation of an 125I-labelled 200 kDa form of B. pertussis AC. It appears that ATP is required for the proteolytic pathway, but not as an energy source, since non-hydrolysable ATP analogues supported inactivation and complete degradation of the enzyme. The possibility that binding of ATP to B. pertussis AC renders it susceptible to degradation by the host cell protease is discussed.
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PMID:Bordetella pertussis adenylate cyclase inactivation by the host cell. 255 87

Bordetella pertussis produces a calmodulin-sensitive adenylate cyclase (AC) which is an essential virulence factor in mammalian pertussis. Here we report the purification and characterization of the toxic form of the enzyme, which penetrates eukaryotic cells and generates high levels of intracellular cAMP. This form was purified from an extract of B.pertussis strain carrying a recombinant plasmid which over-produced both enzymatic and toxic activities of the enzyme. Western blot analysis of the extract using anti-B.pertussis AC antibodies detected only one protein of 200 kd. However, gel filtration of the extract resolved two peaks of enzymatic activity. The first peak of aggregated material contained greater than 70% of the total enzymatic activity, and the second peak contained the majority of the toxic activity. Purification of the enzyme from both peaks yielded proteins of 200 kd, with similar biochemical and immunological properties. Yet only the enzyme purified from the second peak could penetrate human lymphocyte and catalyse the formation of intracellular cAMP. B.pertussis AC gene expressed in Escherichia coli produced a calmodulin-dependent enzyme of 200 kd, which lacked lymphocyte penetration capacity. It is proposed that a post-translational modification that occurs in B.pertussis but not in E.coli confers upon the 200 kd protein of B.pertussis AC the toxic properties.
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PMID:Bordetella pertussis adenylate cyclase: purification and characterization of the toxic form of the enzyme. 255 85

Bordetella pertussis, the pathogen responsible for whooping cough, releases a soluble calmodulin-sensitive adenylate cyclase into its culture medium. Several investigators have shown that the partially purified adenylate cyclase is capable of entering animal cells and elevating 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]. However, the mechanism for entry of the catalytic subunit of the adenylate cyclase into animal cells is unknown. Recently, it was determined that the purified catalytic subunit of the enzyme is unable to enter animal cells [Masure, H. R., Oldenburg, D. J., Donovan, M. G., Shattuck, R. L., & Storm, D. R. (1988) J. Biol. Chem. 263, 6933-6940]. On the basis of these data and other observations, we hypothesized that the culture medium of B. pertussis contains one or more additional polypeptides which facilitate entry of the adenylate cyclase catalytic subunit into animal cells. In this study, we report that a cell-invasive preparation of B. pertussis adenylate cyclase was rendered noninvasive after passage through a wheat germ lectin-agarose column. A fraction was eluted from the wheat germ lectin-agarose column with N-acetyl-D-glucosamine. This fraction, when combined with the noninvasive adenylate cyclase, was able to restore the ability of the adenylate cyclase preparation to enter neuroblastoma cells and increase intracellular cAMP levels. Furthermore, the fraction eluted from the wheat germ lectin-agarose column was found to be trypsin and chymotrypsin sensitive, suggesting that this material was proteinaceous.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Isolation of a protein fraction from Bordetella pertussis that facilitates entry of the calmodulin-sensitive adenylate cyclase into animal cells. 255 96

Two independent isolates of a Bordetella pertussis repeated DNA unit were sequenced and shown to be an insertion sequence element with five nucleotide differences between the two copies. The sequences were 1053 bp in length with near-perfect terminal inverted repeats of 28 bp, had three open reading frames, and were each flanked by short direct repeats. The two insertion sequences showed considerable homology to two other B. pertussis repeated DNA sequences reported recently: IS481 and a 530 bp repeated DNA unit. The B. pertussis insertion sequence would appear to comprise a group of closely related sequences differing mainly in flanking direct repeats and the terminal inverted repeats. The two isolates reported here, which were from the adenylate cyclase and agglutinogen 2 regions of the genome, were numbered IS48lvl and IS48lv2 respectively.
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PMID:Analysis of separate isolates of Bordetella pertussis repeated DNA sequences. 255 51

Bordetella pertussis produces an adenylate cyclase which is a toxin. The enzyme penetrates eukaryotic cells and, upon activation by host calmodulin, generates high levels of intracellular cAMP; as a result bactericidal functions of immune effector cells are considerably impaired. The toxin is composed of a single polypeptide that possesses both the catalytic and the toxic functions. It penetrates the host cell directly from the plasma membrane and is concomitantly inactivated by a proteolytic degradation.
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PMID:Invasive adenylate cyclase toxin of Bordetella pertussis. 256 Feb 73

Mutants of Bordetella pertussis deficient in virulence-associated factors were identified by using the transposon Tn5 lac. Tn5 lac is a derivative of Tn5 which generates promoter fusions for beta-galactosidase. Tn5 lac insertions in the vir-regulated genes of B. pertussis were identified by selecting for kanamycin-resistant mutants that expressed beta-galactosidase when the vir-regulated genes were expressed but not when the vir-regulated genes were turned off. Fourteen different mutations in vir-regulated genes were identified. Two mutants were deficient in the production of the filamentous hemagglutinin, two mutants were deficient in the production of adenylate cyclase toxin and hemolysin, and one mutant was deficient in the production of dermonecrotic toxin. One insertion mapped adjacent to the pertussis toxin gene, but the mutant produced pertussis toxin. The phenotypes of the remaining eight mutants were not determined, but the mutants did not appear to be deficient in the production of the 69,000-dalton outer membrane protein (agglutinogen 3) or the capsule. Screening for mutations in either of the fimbrial genes proved to be problematic since the parental strain was found to switch from a fimbriated to a nonfimbriated state at a high frequency, which was suggestive of the metastable expression of pili in other bacteria. We used Southern blot analysis with a 30-mer specific for the fimbrial sequences. No bands with the predicted increase in size due to the 12 kilobases from Tn5 lac were observed, which suggests that none of these genes were mutated. Southern blot analysis also revealed that seven of the eight unidentified mutations mapped to different restriction fragments, which suggests that they could be deficient in as many as seven different genes.
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PMID:Use of the promoter fusion transposon Tn5 lac to identify mutations in Bordetella pertussis vir-regulated genes. 256 47

Different aspects of lethal infection of infant mice with Bordetella pertussis were examined. Mutants deficient in vir-regulated genes were tested for the ability to cause a lethal infection in the infant mouse model. Adenylate cyclase toxin-hemolysin and pertussis toxin were required to cause a lethal infection at low doses. Mixed infection caused by challenging the mice with an equal number of pertussis toxin and adenylate cyclase toxin-hemolysin mutants at a dose at which neither alone was lethal was also unable to cause a lethal infection. Production of the filamentous hemagglutinin and the dermonecrotic toxin was not required to cause a lethal infection. Nine other mutants in vir-regulated genes whose phenotypes have yet to be determined were also tested. Only two of these mutants were impaired in the ability to cause a lethal infection. Expression of fimbriae does not appear to affect the dose required to cause a lethal infection; however, fimbrial expression was correlated with the later stages of a nonlethal, persistent infection. Growth of the bacteria in MgSO4, a condition which reversibly suppresses expression of the genes required for virulence, did not alter the ability of the bacteria to cause a lethal infection. Auxotrophic mutants deficient in leucine biosynthesis were as virulent as the parental strain; however, mutants deficient in methionine biosynthesis were less virulent. A B. parapertussis strain was much less effective in promoting a lethal infection than any of the wild-type B. pertussis strains examined. A persistent infection in the lungs was observed for weeks after challenge for mice given a sublethal dose of B. pertussis, and transmission from infected infants to the mother was never observed.
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PMID:Lethal infection by Bordetella pertussis mutants in the infant mouse model. 257 61

Stimulation of normal rat splenic T cells with pertussigen (lymphocytosis-promoting factor from Bordetella pertussis) resulted in the release of a soluble factor that enhanced the assembly of N-linked oligosaccharides to IgE-binding factors during their biosynthesis. The glycosylation-enhancing factor (GEF) is a kallikrein-like enzyme and is purified by absorption to p-aminobenzamidine-Agarose followed by elution with benzamidine. Incubation of normal mouse mast cells with affinity-purified GEF or bradykinin, a product of cleavage of kininogen by kallikrein, resulted in the release of histamine and arachidonate from the cells. Passive sensitization of mast cells with mouse IgE antibody, followed by pretreatment of the cells with a suboptimal concentration of GEF, resulted in an enhancement of antigen-induced histamine release. It was found that GEF and bradykinin induced the same biochemical events in mast cells as those induced by bridging of IgE receptors. Both GEF and bradykinin induced phospholipid methylation and an increase in intracellular cyclic AMP (cAMP). Incorporation of 3H-methyl groups into phospholipids and intracellular cAMP levels both reached a maximum 30 sec after challenge with GEF or bradykinin, and then declined to base-line levels within 2 to 3 min. These biochemical events were followed by 45Ca influx and histamine release; 45Ca uptake reached a plateau value at 2 min, and histamine release reached a maximum at 5 to 8 min. The initial rise in cAMP induced by GEF (or bradykinin) was not inhibited by indomethacin, indicating that the activation of adenylate cyclase is not the result of prostaglandin synthesis. In both IgE-mediated and GEF-induced histamine release, inhibitors of methyltransferases, such as 3-deaza adenosine and L-homocysteine thiolactone, inhibited not only phospholipid methylation but also the cAMP rise and subsequent Ca2+ uptake and histamine release. The results indicate that GEF induces activation of methyltransferases and that phospholipid methylation is involved in the cAMP rise, Ca2+ uptake, and histamine release. The induction of the same biochemical events in the same sequence by bridging of IgE receptors and by GEF (bradykinin) supports the hypothesis that receptor bridging induces the activation of serine protease(s) and cleavage products of this enzyme in turn activate methyltransferases in mast cells.
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PMID:Release of histamine and arachidonate from mouse mast cells induced by glycosylation-enhancing factor and bradykinin. 257 23

Bordetella pertussis and Bordetella parapertussis are both causative agents of whooping cough outbreaks. Although not expressing the pertussis toxin, B. parapertussis induces, in a murine model, an acute hemorrhagic edematous alveolitis, similar to that observed with B. pertussis. These data suggest that the pertussis toxin may only play an accessory role in the acute pulmonary syndrome observed during Bordetella infection. Both with B. pertussis and B. parapertussis, the ability to induce lethal pulmonary lesions is associated with enhanced in vitro adenylate cyclase expression and activity. We also demonstrate that passive immunization with specific anti-B. pertussis adenylate cyclase antibodies or active immunization with purified B. pertussis secreted adenylate cyclase protect mice against a lethal respiratory challenge with B. pertussis or B. parapertussis. Our results suggest that adenylate cyclase might be the primary cytotoxin responsible for mouse pulmonary lesions during respiratory tract infection with B. pertussis or with the related species B. parapertussis and is a protective antigen of B. pertussis.
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PMID:Bordetella adenylate cyclase is a virulence associated factor and an immunoprotective antigen. 262 29


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