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)

A competitive ELISA has been used to monitor the release of fimbriae from 1.2.3 serotype of B. pertussis after treatment by different methods and fimbriae have been purified from homogenates or KSCN extracts by chromatography. Fimbriae purified from different serotypes have been studied by inhibition of bacterial agglutination, immunodiffusion and SDS-polyacrylamide gel electrophoresis. Fimbriae purified from a 1.2 serotype have been labelled in immunoelectron microscopy with a IgM monoclonal antibody to agglutinogen 2 and evidence is presented that fimbriae purified from a 1.3 serotype also carry the agglutinogen 3 specificity. A difference in subunit molecular weight has been found between fimbriae purified from 1.2 and 1.3 serotypes.
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PMID:Release and purification of fimbriae from Bordetella pertussis. 287 1

One serotype antigen, agglutinogen 3, from Bordetella pertussis, (strain M2, serotype 1.3), has been purified. The purification procedure included acetone drying of cells harvested from shaking cultures. Agglutinogens were extracted in phosphate buffered saline. Crude extract was heat treated at 80 degrees C for 5 min and precipitated by ammonium sulphate between 25 and 60% saturation at 4 degrees C, providing 50% of the total activity and a five-fold purification. Further purification was attempted by gel filtration chromatography using a TSK-G3000 SW column. The ammonium sulphate precipitated fraction was also separated by anion exchange chromatography using a Mono Q HR 5/5 column. The purification work indicated that agglutinogen 3 is associated with several other substances and that this property can lead to purification difficulties. The isolation procedure was monitored by an agglutination-inhibition assay. The peak fraction from the ion exchange chromatography was purified up to 27-fold according to the specific activities (inhibition units per mg protein). The yield was only 1% due to severe loss of activity. In the gel filtration chromatography agglutinogen 3-activity eluted with a maximum activity corresponding to a molecular weight near 450,000. SDS-PAGE analysis indicated that agglutinogen 3 might have a subunit molecular weight of 20,800.
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PMID:Purification and preliminary characterization of agglutinogen 3 from Bordetella pertussis. 287 4

Bordetella pertussis, the causative organism of whooping cough, produces a calmodulin-sensitive adenylate cyclase. Confer & Eaton [(1982) Science 217, 948-950] have shown that an extract from B. pertussis increases intracellular cyclic AMP levels in neutrophils and suggested that this increase is caused by the bacterial adenylate cyclase which penetrates these cells. We demonstrate in the present study that adenylate cyclase activity in lysates from lymphocytes exposed to a partially purified preparation of the bacterial enzyme has properties completely different from those of the intrinsic membrane-bound enzyme. Adenylate cyclase activity in lysates from lymphocytes exposed to the invasive enzyme is insensitive to N-ethylmaleimide, readily inactivated by acetic anhydride and relatively stable to SDS. Similar properties are exhibited by the bacterial enzyme itself. By contrast, the intrinsic membrane-bound enzyme activated by forskolin and guanosine 5'-gamma-thiotriphosphate is sensitive to N-ethylmaleimide and SDS and relatively stable to acetic anhydride. This strongly supports the notion that B. pertussis adenylate cyclase penetrates cells. Using the partially purified preparation of the invasive enzyme, we have studied the kinetics of its penetration. The intracellular catalytic activity reaches a steady state within 20 min, irrespective of enzyme or cell concentration. Steady-state levels are maintained for at least 2 h provided that the invasive enzyme is present in the incubation medium. Upon its removal, a rapid decrease (t1/2 approximately equal to 15 min) in the intracellular cyclase level is observed. This decrease reflects intracellular inactivation of the bacterial enzyme and is not caused by the release of the enzyme to the cell medium.
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PMID:The invasive adenylate cyclase of Bordetella pertussis. Properties and penetration kinetics. 288 19

Bordetella pertussis was able to grow in vitro under conditions where the only iron present was bound to the iron-binding proteins ovotransferrin, transferrin or lactoferrin. Under these conditions the bacteria produced neither hydroxamate nor phenolate-catecholate siderophores to assist in the procurement of iron. Examination of B. pertussis outer-membrane preparations by SDS-PAGE and immunoblotting showed that the iron-binding protein ovotransferrin was bound directly to the bacterial surface. Assays of the binding of radiolabelled transferrin by the bacteria showed that the association was a specific process and that there was turnover of the bound proteins. Competitive binding assays indicated that lactoferrin could be bound in the same way. It is suggested that B. pertussis obtains iron directly from host iron-binding proteins during infection.
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PMID:Interaction of lactoferrin and transferrins with the outer membrane of Bordetella pertussis. 288 36

Agglutinogen 2 (AGG2) of Bordetella pertussis is a fimbrial antigen and therefore a potential adhesin and acellular vaccine component. AGG2 was found to dissociate only under harsh conditions into the subunits of mol. wt. 22500 seen in SDS-PAGE. Results from studies of agglutinogen 3 (AGG3) are presented which confirm previous findings from this Laboratory that AGG3 is also a fimbrial protein but with a subunit mol. wt. of 22000. The amino acid sequence of AGG2, deduced from the nucleotide sequence of the gene encoding it, was used as a basis for synthesis of three peptides. Coupled to Keyhole Limpet Haemocyanin (KLH), the peptides were immunogenic in mice, inducing antibodies which bound well to homologous peptide in ELISA but poorly to intact fimbriae. Monoclonal and polyclonal serotype-specific antibodies failed to react significantly with the peptides or their KLH-conjugates. These results indicate that the synthetic peptides do not represent the serotype 2 epitope. Mice immunized with purified AGG2 or AGG3 were found to be protected against respiratory infection with B. pertussis. Results presented here indicate that this protection is, to a large extent, serotype-specific and that immunization of mice with AGG2 or AGG3 can lead to a change in serotype of the infecting strain. These results are analogous to findings from epidemiological studies of the protection induced in children by whole cell vaccines. They reaffirm the importance of both AGG2 and AGG3 as components of whole cell and acellular vaccines.
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PMID:Agglutinogens and fimbriae of Bordetella pertussis. 290 20

Adenylate cyclase activity in renal papillary membranes was stimulated by both vasopressin and the adenosine agonist 5'-N-ethylcarboxamidoadenosine (NECA). The stimulations mediated by the two receptors were additive at all concentrations and interacted differently with other AC-stimulatory factors viz cholera toxin, pertussis toxin and fluoride ion. Treatment of papillary tubules with cholera toxin increased cyclase activity from 4.5 +/- 1.5 to 110 +/- 9.1 (SE, n = 5) pmol/min/mg protein. Maximally effective concentrations of vasopressin increased activity in control preparations to 10.3 +/- 2.8 (an increase of 5.8 +/- 1.3). In cholera toxin treated preparations, vasopressin increased activity to 138.9 +/- 14.5 (an increase of 28.9 +/- 5.4, n = 5; p less than .01). Pertussis toxin increased activity to 9.1 +/- 3.0. The response to vasopressin was enhanced such that the absolute maximum increase in activity was 12.6 +/- 3.9 (n = 5; p less than .01). Addition of the two toxins together produced a greater than additive stimulation to 145 +/- 36. Maximum increase in activity caused by vasopressin was further enhanced to 48 +/- 13 (n = 5; p less than .01). In contrast, cyclase stimulation by NECA was additive with stimulations by the two toxins, separately and in combination. The NECA stimulation however, was enhanced in the presence of fluoride ion while the vasopressin stimulation was additive at all concentrations. Papillary membranes contained two different cyclase-stimulatory coupling proteins with alpha-subunits of MW's 46,600 +/- 450 (SE, n = 6) and 41,500 +/- 480 (SE, n = 6) as identified on SDS-polyacrylamide gel electrophoresis following cholera toxin labeling. Taken together, these data suggest that two adenylate cyclase-stimulatory coupling mechanisms with different properties are operative in renal papillary membranes.
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PMID:Evidence for two different stimulatory adenylate cyclase coupling mechanisms in rat renal papilla. 294 38

Bovine peripheral neutrophils contain high levels of a 40-kDa pertussis toxin substrate, which was found highly enriched in a light membrane fraction upon subcellular fractionation of neutrophil homogenates. The 40-kDa pertussis toxin substrate, referred to as alpha n, was purified to near homogeneity from this fraction by sequential ion-exchange, gel-filtration and hydrophobic chromatography. Purified alpha n was shown to interact with beta gamma subunits, undergo ADP-ribosylation by pertussis toxin, and bind guanine nucleotides with high affinity. The mobility of purified alpha n on SDS/polyacrylamide gels was intermediate between those of the alpha subunits of Gi and Go, purified from bovine brain, and slightly lower than the mobility of the alpha subunit of transducin (Gt). Several polyclonal antisera against the alpha subunits of bovine Gt and Go did not react with alpha n on immunoblots. CW 6, a polyclonal antiserum reactive against the bovine alpha i, reacted only minimally with alpha n. These results suggest that the major pertussis toxin substrate of bovine neutrophils, designated Gn, is structurally different from previously identified pertussis toxin substrates and may represent a novel guanine-nucleotide-binding protein.
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PMID:Purification and immunochemical characterization of the major pertussis-toxin-sensitive guanine-nucleotide-binding protein of bovine-neutrophil membranes. 310 40

A novel G protein which appears to couple chemotactic peptide receptors to a polyphosphoinositide phospholipase C has been purified from rabbit neutrophils. Neutrophil membranes were solubilized with sodium cholate and fractionated by successive anion exchange, gel filtration and hydrophobic chromatography. Guanosine-5'-(3-O-thio)triphosphate binding activity was purified 170-fold from the soluble extract. The alpha-subunit of the purified G protein was identified by pertussis toxin-catalyzed ADP-ribosylation, and found to have an Mr of 40,000. The beta-subunit (Mr 36,000) comigrated on SDS-polyacrylamide gel electrophoresis with the beta-subunits of bovine brain Gi and Go. The neutrophil pertussis toxin substrate is highly unstable in cholate solution unless 30% ethylene glycol is added. Structural and functional analysis of this novel G protein will advance our understanding of the molecular mechanisms of coupling of receptors to phospholipase C.
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PMID:Identification and purification of a novel G protein from neutrophils. 311 83

A guanine-nucleotide-binding protein (G-protein) was purified from cholate extracts of bovine brain membranes by sequential DEAE-Sephacel, Ultrogel AcA-34, heptylamine-Sepharose and Sephadex G-150 chromatography. Guanosine 5'-[gamma-[35S]thio]triphosphate (GTP[35S])-binding activity copurified with a 25,000 Da peptide and a 35,000-36,000 Da protein doublet. Neither pertussis toxin nor cholera toxin catalysed the ADP-ribosylation of a protein associated with the GTP[35S]-binding activity. Photoaffinity labelling of the purified protein with 8-azido[gamma-32P]GTP indicated that the GTP-binding site resides on the 25,000 Da protein. The 35,000-36,000 Da protein doublet was electrophoretically indistinguishable from the beta-subunits of other GTP-binding proteins, and the 36,000 Da protein was recognized by antiserum to oligomeric Gt. The purified protein specifically bound 17.2 nmol of GTP[35S]/mg of protein. The Kd of the binding site for radioligand was approx. 15 nM. The brain GTP-binding protein co-migrated during SDS/polyacrylamide-gel electrophoresis with a GTP-binding protein, named Gp, purified from human placenta [Evans, Brown, Fraser & Northup (1986) J. Biol. Chem. 261, 7052-7059], and cross-reacted with antiserum raised against the placental protein, but not with antiserum raised to brain Go. SDS/polyacrylamide-gel electrophoresis of the brain and placental GTP-binding proteins in the presence of Staphylococcus aureus V8 protease yielded identical peptide maps.
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PMID:Identification and purification from bovine brain of a guanine-nucleotide-binding protein distinct from Gs, Gi and Go. 312 Jun 96

A soluble inositolphospholipid-specific phospholipase C (PI-phospholipase C) has been purified 5,800-fold from the cytosolic fraction of calf thymocytes. The purification was achieved by sequential column chromatographies on DEAE-Sepharose CL-6B, heparin-Sepharose CL-6B, Sephacryl S-300, Mono S, and Superose 12, followed by column chromatography on Sephadex G-100 in the presence of 1% sodium cholate. The enzyme thus purified was found to be homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the enzyme was estimated to be 68 kDa by SDS-PAGE. The enzyme is specific for inositol phospholipids. Phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate (PIP2) were hydrolyzed, but phosphatidylcholine and phosphatidylethanolamine were not affected by the enzyme. GTP gamma S-binding activity was detected in the enzyme fractions after all the purification steps, but not in the final enzyme preparation. The PI-phospholipase C and GTP gamma S-binding activities in the partially purified enzyme preparation could be separated by the column chromatography on Sephadex G-100 only in the presence of 1% sodium cholate. Thus, the soluble PI-phospholipase C has affinity to a GTP-binding protein. SDS-PAGE of the GTP-binding fractions eluted from the Sephadex G-100 column gave three visible bands of 54, 41, and 27 kDa polypeptide was specifically ADP-ribosylated by pertussis toxin. Furthermore, it was found that GTP and GTP gamma S (10 microM and 1 mM) could enhance the PIP2 hydrolysis activity of the partially purified enzyme in the presence of 3 mM EGTA, but the purified enzyme after separation from the GTP-binding activity was not affected by GTP and GTP gamma S. The soluble PI-phospholipase C of calf thymocytes may be not only physically but also functionally associated with a GTP-binding protein.
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PMID:Physical and functional association of cytosolic inositolphospholipid-specific phospholipase C of calf thymocytes with a GTP-binding protein. 312 64


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