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)

The structural organization of the low molecular mass form (43 kDa) of Bordetella pertussis adenylate cyclase was dissected taking advantage of the known sequence of the bacterial cya gene (Glaser, P., Ladant, D., Sezer, O., Pichot, F., Ullmann, A., and Danchin, A. (1988) Mol. Microbiol. 2, 19-30) and its low content of Trp and Met residues. Cleavage of the 43-kDa protein and of its complementary tryptic fragments (T25 and T18 peptides) with N-chlorosuccinimide and cyanogen bromide followed by sodium dodecyl sulfate-polyacrylamide gel analysis of digestion products allowed the following conclusions: (i) the catalytically active 43-kDa form of B. pertussis adenylate cyclase is within the first 400 residues of the protein encoded by the cya gene. T25 occupies the N-terminal domain of the protein (residues 1-235/237). Isolated T25 fragment exhibits a low but measurable enzymatic activity which indicates that it harbors the catalytic site; (ii) T18 which is the main calmodulin-binding domain, occupies the C-terminal segment of protein (residues 236/238-399) and is devoid of catalytic properties; (iii) the two complementary peptides T25 and T18 reassociated only in the presence of calmodulin, leading to significant recovery of the original activity. These results demonstrate that both fragments of the 43-kDa form of adenylate cyclase are essential for a high level of enzymatic activity.
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PMID:Characterization of the calmodulin-binding and of the catalytic domains of Bordetella pertussis adenylate cyclase. 253 1

Bordetella pertussis produces a calmodulin-sensitive adenylate cyclase that is associated with the whole bacteria and released into its culture media. Preparations of this enzyme invade animal cells, causing elevations in intracellular cAMP levels. Cell-associated adenylate cyclase accounted for 28% of the total adenylate cyclase activity while 72% was released into the culture supernatant. Over 90% of the cell-associated adenylate cyclase activity was sensitive to trypsin treatment of whole cells, indicating that the catalytic domain of the enzyme is localized on the outer surface of the bacterial cells. Enzyme activity was released from whole cells by treatment with SDS. This activity was resolved as a large form (Mr 215,000) by SDS-polyacrylamide gel electrophoresis. In contrast, the culture supernatant contained only the 45,000-dalton catalytic subunit. Enzyme activity released from spheroplasts by sonication was resolved into a large form (Mr 215,000) and a small form (Mr 45,000). The appearance of the small form with spheroplast formation was probably the result of proteolytic degradation. Antibodies generated against the catalytic subunit purified from culture supernatants cross-reacted with and immunoprecipitated both the large and small forms of adenylate cyclase isolated from bacterial cells. Furthermore, incubation of the cell-associated enzyme with a crude bacterial extract resulted in a time-dependent disappearance of the 215,000-dalton form and a concomitant increase in the amount of the smaller 45,000-dalton form. There was also a parallel increase in the ability of the cell-associated preparation to elevate intracellular cAMP levels in N1E-115 mouse neuroblastoma cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of the bacterial cell associated calmodulin-sensitive adenylate cyclase from Bordetella pertussis. 254 Jul 97

In order to identify molecular features of the calmodulin (CaM) activated adenylate cyclase of Bordetella pertussis, a truncated cya gene was fused after the 459th codon in frame with the alpha-lacZ' gene fragment and expressed in Escherichia coli. The recombinant, 604 residue long protein was purified to homogeneity by ion-exchange and affinity chromatography. The kinetic parameters of the recombinant protein are very similar to that of adenylate cyclase purified from B.pertussis culture supernatants, i.e. a specific activity greater than 2000 mumol/min mg of protein at 30 degrees C and pH 8, a KmATP of 0.6 mM and a Kd for its activator, CaM, of 0.2 nM. Proteolysis with trypsin in the presence of CaM converted the recombinant protein to a 43 kd protein with no loss of activity; the latter corresponds to the secreted form of B.pertussis adenylate cyclase. Site-directed mutagenesis of residue Trp-242 in the recombinant protein yielded mutants expressing full catalytic activity but having altered affinity for CaM. Thus, substitution of an aspartic acid residue for Trp-242 reduced the affinity of adenylate cyclase for CaM greater than 1000-fold. Substitution of a Gln residue for Lys-58 or Lys-65 yielded mutants with a drastically reduced catalytic activity (approximately 0.1% of that of wild-type protein) but with little alteration of CaM-binding. These results substantiated, at the molecular level, our previous genetic and biochemical studies according to which the N-terminal tryptic fragment of secreted B.pertussis adenylate cyclase (residues 1-235/237) harbours the catalytic site, whereas the C-terminal tryptic fragment (residues 235/237-399) corresponds to the main CaM-binding domain of the enzyme.
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PMID:Identification of residues essential for catalysis and binding of calmodulin in Bordetella pertussis adenylate cyclase by site-directed mutagenesis. 254 30

Bordetella pertussis organisms secrete adenylate cyclase, at least one form of which can invade host cells and appears to be a virulence factor. Treatment of urea extracts containing invasive cyclase of B. pertussis with trypsin, chymotrypsin, or subtilisin abolishes the ability to increase intracellular cyclic AMP levels in CHO cells (invasiveness) at concentrations that have minimal or no effects on adenylate cyclase activity. Higher protease concentrations can inhibit catalytic activity, and 1 microM calmodulin protects this catalytic activity, but not invasiveness, against proteolytic inhibition. Rabbit immunoglobulin G (IgG) fractions from antisera prepared against urea extracts inhibited invasiveness at 10-fold-lower concentrations than inhibited catalytic activity. One IgG from a rabbit immunized against a partially purified, noninvasive form of the B. pertussis adenylate cyclase inhibited catalytic activity but was ineffective against invasiveness. We conclude that these two properties of the adenylate cyclase are independent functions that reside on different domains of the same protein or on different proteins.
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PMID:Dissociation of catalytic and invasive activities of Bordetella pertussis adenylate cyclase. 254 62

A 2.7-kb cya A gene fragment encoding the amino-terminal end of the calmodulin-sensitive adenylate cyclase from Bordetella pertussis has been placed under the control of the lac promoter for expression in Escherichia coli. Following induction with isopropyl beta-D-thiogalactoside, calmodulin-sensitive adenylate cyclase activity was detected in a cell extract from E. coli. The expression vector directed the synthesis of a 90-kDa polypeptide that was recognized by rabbit polyclonal antibodies raised against the catalytic subunit of B. pertussis adenylate cyclase. Inspection of the deduced amino acid sequence of the cya A gene product revealed a sequence with homology to consensus sequences for an ATP-binding domain found in many ATP-binding proteins. On the basis of the analysis of nucleotide binding proteins, a conserved lysine residue has been implicated in the binding of ATP. A putative ATP-binding domain in the B. pertussis adenylate cyclase possesses an analogous lysine residue at position 58. To test whether lysine 58 of the B. pertussis adenylate cyclase is a crucial residue for enzyme activity, it was replaced with methionine by oligonucleotide-directed mutagenesis. E. coli cells were transformed with the mutant cya A gene, and the expressed gene product was characterized. The mutant protein exhibited neither basal nor calmodulin-stimulated enzyme activity, indicating that lysine 58 plays a critical role in enzyme catalysis.
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PMID:Site-directed mutagenesis of lysine 58 in a putative ATP-binding domain of the calmodulin-sensitive adenylate cyclase from Bordetella pertussis abolishes catalytic activity. 254 36

The adenylate cyclase activity of Bordetella pertussis in clinical isolates was measured in calmodulin-supplemented Stainer-Scholte broth by the rate of conversion of ATP to cyclic AMP. Analysis of 250 stock strains of Bordetella pertussis showed that measurable adenylate cyclase activity was produced by all strains. In clinical tests Bordetella pertussis was isolated from 135 (22%) of 605 swab samples. Increased adenylate cyclase activity was detected in 124 (92%) Stainer-Scholte broth cultures of these samples. A total of 475 swabs contained other bacteria or had no growth; only one of the Stainer-Scholte broth cultures of these swab samples contained measurable adenylate cyclase activity. The results indicate that testing for adenylate cyclase activity provides a specific and sensitive means for detecting Bordetella pertussis in clinical specimens.
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PMID:Detection of Bordetella pertussis by determination of adenylate cyclase activity. 255 Feb 32

The intracellular messengers that seem to be involved in renin secretion (RS) from juxtaglomerular cells (JG) are calcium (Ca), cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Unlike the majority of secretory systems, an increase in intracellular Ca concentration and calmodulin and protein kinase C activation inhibit RS. The intracellular Ca concentration in JG cells can be modified if: 1) the normal mechanisms of Ca extrusion of these cells is altered; 2) the calcium output is blocked by lanthanum; 3) the function of the voltage-sensitive Ca-channels is modified; 4) uptake or liberation of Ca from endoplasmic reticulum is modified; 5) plasmatic membrane is bypassed with calcium ionophores such as A 23187. 6) JG cells are stimulated by hormones that increase Ca and activate protein kinase C such as angiotensin II, vasopressin or alpha-1 adrenergic agonists; 7) extracellular Ca concentration increases or decreases. RS is stimulated by dibutyryl cAMP, cAMP phosphodiesterase inhibitors and by hormones and agents that activate adenylate cyclase (beta adrenergic agonists, bradykinin, histamine, forskolin and ethylcarboxamide adenosine). On the contrary, RS is inhibited by hormones and agents that inhibit adenylate cyclase such as: alpha-2 adrenergic agonists, neuropeptide Y, angiotensin II and cyclohexyladenosine. Pertussis toxin increases basal RS, blocks the inhibition by agents and hormones which inhibit adenylate cyclase and potentiate the stimulation produced by beta-adrenergic agonists. In JG cells, atrial natriuretic peptide inhibits RS, increases cGMP and decreases cAMP. The increase in cGMP correlates well with the inhibition of RS.
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PMID:[Intracellular messengers in the regulation of renin secretion]. 255 Oct 26

Cloning of higher eukaryotic genes has seldom been performed by complementation of a defective prokaryotic function. This is especially true in the case of functions that are normally absent from the prokaryotic host. We demonstrate here that it is possible to identify by complementation the cDNA from mouse brain, which encodes calmodulin (CaM) synthesis, in spite of the fact that the recipient strain, Escherichia coli, does not normally harbour a CaM function. A three-component cloning procedure in which a gene product requiring CaM for activity, adenylate cyclase from the pathogen Bordetella pertussis, was used to screen a cDNA library for cAMP synthesis in E. coli. The nucleotide sequence of the corresponding cDNA is also reported.
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PMID:Cloning and expression of mouse-brain calmodulin as an activator of Bordetella pertussis adenylate cyclase in Escherichia coli. 255 80

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


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