UMLS:C0043167 - FACTA Search

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Query: UMLS:C0043167 (pertussis)
19,595 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The exotoxin pertussis toxin (PT) produced by virulent Bordetella pertussis bacteria is regarded as the main virulence factor of the organism and held responsible for most of its pathological effects. Identification of functional sites on PT would greatly facilitate site-specific detoxification and thus also the development of a new vaccine. For the investigation of structure-function aspects of PT we have prepared and characterized eleven monoclonal antibodies (mAbs) (UB-A1, UB-A2, UB-A10, UB-B7, UB-B12, UB-D4, UB-D7, UB-D10, UB-F7, UB-G1, and UB-G12) directed at the native toxin. Only UB-B12 and UB-D10 recognized PT in Western blotting indicating that most of the mAbs were directed against conformational epitopes. The mAbs were assayed for their ability to interfere with the binding of PT in model receptor systems like a solid phase binding assay using fetuin as receptor moiety, hemagglutination of chymotrypsin-sensitized goose erythrocytes, and the PT-mediated induction of the clustered growth pattern (CGP) of Chinese hamster ovary (CHO) cells. Five of the eleven mAbs (UB-A1, UB-A2, UB-B7, UB-B12, and UB-D7) interfered with the binding of PT to fetuin on solid phase and with PT-mediated hemagglutination. UB-A2, UB-B7, and UB-B12 also inhibited the induction of the clustered growth pattern of CHO-cells. This indicates that the determinants recognized by these mAbs are associated with the formation of the carbohydrate recognition sites of PT. Thus, the monoclonal antibodies described in this study will be valuable tools in the further analysis of the structure-function relationship of pertussis toxin with respect to receptor recognition and binding.
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PMID:Characterization of monoclonal antibodies directed against domains of pertussis toxin involved in receptor recognition. 172 5

Trypsin digestion of pertussis toxin (PT) preferentially cleaved the S1 subunit at Arg-218 without detectable degradation of the B oligomer. The fragment produced, termed the tryptic S1 fragment, appears to remain associated with the B oligomer. Chymotrypsin digestion of PT also preferentially cleaved the S1 subunit without detectable degradation of the B oligomer. The chymotryptic S1 fragment possessed a slightly lower apparent molecular weight than the tryptic S1 fragment and was more accessible to the respective protease. Trypsin- and chymotrypsin-treated PT and PT required the presence of dithiothreitol and ATP for optimal enzymatic activity. Trypsin-treated PT showed approximately a 2-4-fold higher level of expression of ADP-ribosyltransferase and NAD-glycohydrolase activities than PT. Chymotrypsin-treated PT also exhibited approximately a 2-fold greater level of ADP-ribosyltransferase activity than PT. The observed increase in activity of protease-treated PT was due primarily to a shorter time for activation in PT mediated ADP-ribosylation of transducin. In addition, trypsin-digested PT possessed the same cytotoxic potential for Chinese hamster ovary cell clustering as PT. One possible role for the generation of a proteolytic fragment of the S1 subunit of PT would be to produce a catalytic fragment with increased efficiency for ADP-ribosylation of G proteins in vivo.
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PMID:Protease treatment of pertussis toxin identifies the preferential cleavage of the S1 subunit. 185 Jul 38

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

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

Platelet responses to agonists are believed to be mediated by at least two pertussis toxin-sensitive guanine nucleotide-binding (G) proteins: Gi which inhibits adenylyl cyclase and Gp, which stimulates phospholipase C. The present studies compare the properties of Gi and Gp and examine their interactions with the receptors for various platelet agonists. In permeabilized platelets and platelet membranes, pertussis toxin [32P]ADP-ribosylated a protein(s) (alpha 41) which migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis fractionally below rabbit and bovine alpha i (Mr = 41,000). Prior exposure of the platelets to an agonist inhibited the [32P]ADP-ribosylation of alpha 41 to an extent which correlated with the pattern of responses to that agonist. Thrombin, which elicited responses that were mediated by both Gi and Gp, decreased radiolabeling by greater than 90%. Epinephrine, which was functionally coupled only to Gi, decreased radiolabeling by 50%, as did vasopressin and platelet-activating factor (PAF), which were coupled only to Gp. U46619, a thromboxane analog which neither inhibited cAMP formation nor caused pertussis toxin-sensitive phosphoinositide hydrolysis, had no effect on 32P-ADP-ribosylation. These results suggest that either G alpha 41 regulates more than one enzyme or that alpha subunits from more than one G protein comigrate within alpha 41. Two-dimensional electrophoresis was used to test the latter possibility. Upon isoelectric focusing, alpha 41 resolved into two distinct subspecies. However, these appear to be minor variants rather than functionally distinct alpha subunits since: 1) both proteins produced the same proteolytic fragments after digestion with chymotrypsin or Staphylococcus aureus V8 protease and 2) preincubation of the platelets with agonists, including those which appear to interact in intact platelets solely with Gp (PAF and vasopressin) or solely with Gi (epinephrine), inhibited the [32P]ADP-ribosylation of both proteins to the same extent. The pattern of functional responses produced by some of the agonists was found to depend upon the conditions used for the assay. Although unable to inhibit cAMP formation in intact platelets, both PAF and vasopressin caused pertussis toxin-sensitive inhibition of adenylyl cyclase in isolated membranes. Collectively, these observations suggest that 1) in platelets a single pertussis toxin-sensitive, alpha 41-containing G protein may be involved in the regulation of both adenylyl cyclase and phospholipase C and 2) additional constraints which are altered during membrane isolation may help to determine which enzyme is coupled to which agonist.
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PMID:Interactions in platelets between G proteins and the agonists that stimulate phospholipase C and inhibit adenylyl cyclase. 283 6

Stimulation of normal rat splenic T cells with pertussigen (lymphocytosis-promoting factor, LPF, from Bordetella pertussis) resulted in the release of a soluble factor that enhanced the glycosylation of IgE-binding factors during their biosynthesis. The soluble factor was detected by the ability of a culture filtrate of LPF-stimulated spleen cells to switch a T cell hybridoma, 23A4, from the formation of unglycosylated IgE-binding factor to the formation of glycosylated IgE-binding factor. The glycosylation-enhancing factor (GEF) had affinity for D-galactose, and the binding of the factor to hybridoma cells via a cell surface galactose was essential for modulation of IgE-binding factors. The GEF was inactivated by irreversible inhibitors of serine proteases such as phenylmethylsulfonyl fluoride, diisopropylfluorophosphate, and p-nitrophenyl ethylpentylphosphonate but was not affected by nonphosphorylating analogues of the organophosphorus compounds. Benzamidine, a competitive and reversible inhibitor of trypsin, also inhibited the glycosylation of IgE-binding factors by GEF. The factor could be purified by absorption to p-aminobenzamidine agarose followed by elution with benzamidine. The capacity of GEF to enhance the glycosylation of IgE-binding factors was inhibited by synthetic substrates of trypsin but not by substrates of chymotrypsin, indicating that GEF is a trypsin-like enzyme. Indeed, trypsin, plasmin, and kallikrein enhanced the glycosylation of IgE-binding factors during their biosynthesis. An inhibitor of trypsin-like enzyme(s), N-alpha-p-tosyl-L-lysine chloromethylketone (TLCK), inhibited trypsin and plasmin but not kallikrein, and TLCK failed to inhibit the GEF-mediated enhancement of glycosylation. It was also found that bradykinin, the biologically active product of cleavage of kininogen by kallikrein, enhanced the glycosylation of IgE-binding factors. The results indicate that GEF is a kallikrein-like enzyme.
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PMID:Modulation of the biologic activities of IgE-binding factor. IV. Identification of glycosylation-enhancing factor as a kallikrein-like enzyme. 655 15

Sulfatides have been established recently as ligands for L-selectin, and we investigated whether they trigger transmembrane signals through ligation of L-selectin. We found that sulfatides trigger the increase of cytosolic free calcium in neutrophils and that this effect was strictly dependent on sulfation of the galactose ring, as non-sulfated galactocerebrosides were not stimulatory. Chymotrypsin and phorbol 12-myristate 13-acetate treatment of neutrophils caused shedding of L-selectin, but not of class I major histocompatibility complex antigens or beta 2 integrins, and blunted the capability of neutrophils to respond to sulfatides with an increase of cytosolic free calcium. Four different anti-L-selectin antibodies (DREG-200, LAM1/3, LAM1/6, and LAM1/10), but not four control antibodies directed against different surface molecules of neutrophils, also triggered an increase of cytosolic free calcium. The anti-L-selectin antibodies were stimulatory both if used in a soluble form, after cross-linking with anti-mouse F(ab')2 fragments, and immobilized to protein A of Staphylococcus aureus through the Fc fragment. With immobilized antibodies, an increase of cytosolic free calcium was found also by plating neutrophils on antibodies bound to protein A-coated coverslips and monitoring the increase of cytosolic free calcium by fluorescence microscopy. Both sulfatides and anti-L-selectin antibody effects were not inhibited by pertussis toxin, thus indicating that a pertussis toxin-sensitive GTP-binding protein was not involved in signal transduction. Sulfatides also triggered an increase of tumor necrosis factor-alpha and interleukin-8 mRNAs in neutrophils. Also to act as stimuli of cytokine mRNA expression, sulfatides required sulfation of the galactose ring, as non-sulfated galactocerebrosides were not stimulatory, and depended on expression of L-selectin, as shedding of this molecules induced by chymotrypsin blunted their effects. These findings suggest that L-selectin can transduce signals activating selective cell function.
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PMID:Sulfatides trigger increase of cytosolic free calcium and enhanced expression of tumor necrosis factor-alpha and interleukin-8 mRNA in human neutrophils. Evidence for a role of L-selectin as a signaling molecule. 750 38

The cyclic GMP mediated non-adrenergic non-cholinergic (NANC) relaxation in field stimulated bovine mesenteric artery and its modulation by various factors was studied. Electrical field stimulation of precontracted (Phe 2.5 microM or histamine 5 microM) bovine mesenteric arteries resulted in relaxations varying between 10-70% in different preparations. Tetrodotoxin (3 microM) completely blocked the inhibitory NANC response. Preincubation with high concentrations (100 microM-1 mM) of NG-nitro-L-arginine for 15 min. significantly reduced the relaxation induced by electrical field stimulation. Blockade of cyclooxygenases and prostaglandin synthesis by indomethacin had no effect on the relaxatory response to electrical field stimulation. Neither the alpha 2-adrenoceptor selective antagonist yohimbine (1 microM) nor the alpha 2-adrenoceptor selective agonist UK 14,304 (1 microM) had any significant effect on the electrical field stimulation-induced relaxation. Pertussis toxin (100 ng/ml) was without effect on relaxations elicited by electrical field stimulation. GTP in the concentration range 10 microM-1 mM slightly potentiated the relaxant response. N-carboxymethyl-Phe-Leu (an inhibitor of enkephalinase) or aprotinin (an inhibitor of several proteases) had no significant effect on the electrical field stimulation response. Addition of trypsin (100 U/ml) in combination with chymotrypsin (20 U/ml) significantly reduced the electrical field stimulation-induced relaxation. In the present study we have found indications for the involvement of nitric oxide and possibly also peptides in mediating the inhibitory NANC response (relaxation) in bovine mesenteric arteries.
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PMID:Involvement of nitric oxide and peptides in the inhibitory non-adrenergic, non-cholinergic (NANC) response in bovine mesenteric artery. 810 66

The limited proteolytic pattern of transducin, Gt, and its purified subunits with chymotrypsin were analyzed and the cleavage sites on the alpha t subunit were identified. The alpha t subunit in the GTP gamma S bound form was cleaved into a major 38 kD fragment, whereas alpha t-GDP was progressively digested into 38, 23, 21, and 15 kD fragments. The beta gamma t subunit was not very sensitive to proteolytic digestion with chymotrypsin. The gamma t subunit was not cleaved and only a small portion of beta t was digested into several fragments. In order to determine which proteolytic fragment of alpha t still contained the carboxyl terminal region, chymotrypsinization was carried out using Gt previously 32P-labeled at Cys347 by pertussis toxin-catalyzed ADP-ribosylation. The 32P-label was mainly associated with the alpha t subunit and a 15 kD fragment. The 23 and 21 kD fragments were not 32P-labeled. Analysis of amino terminal sequences of 38, 21, and 15 kD proteolytic bands allowed the identification of the major cleavage sites. Chymotrypsin had two cleavage sites in the amino terminal region of alpha t, at Leu15 and Leu19. Chymotrypsin removed 15-19 amino acid residues from the amino terminus of alpha t, generating two peptides (38 kD) which comigrates in gel electrophoresis. Chymotrypsin also cleaved at Trp207 in a conformation-dependent manner. Trp207 of alpha t-GTP gamma S was resistant to proteolysis but alpha t-GDP and the 38 kD fragments of alpha t-GDP produced the 23 and 21 kD fragments, respectively, and a 15 kD fragment containing the carboxyl terminus.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Tryptophan207 is involved in the GTP-dependent conformational switch in the alpha subunit of the G protein transducin: chymotryptic digestion patterns of the GTP gamma S and GDP-bound forms. 848 7

Pertussis toxin (PT)-catalyzed ADP-ribosylation of transducin (Gt) is stimulated by ATP. In the absence of ATP, PT exhibited an approximately 20-fold lower linear velocity than the recombinant S1 subunit (rS1) in catalyzing the ADP-ribosylation of Gt. In the presence of 0.1 mM ATP, the linear velocities of rS1 and PT were essentially identical. ATP increased the kcat of PT-catalyzed ADP-ribosylation of Gt without altering the Kmapp for either Gt or NAD. Further, in the presence of ATP, PT exhibited similar kinetic constants under conditions of variable Gt and variable NAD as rS1 in catalyzing the ADP-ribosylation of Gt. The S1 subunit of PT was cleaved by chymotrypsin to a single immunoreactive peptide in the absence of ATP, while three immunoreactive peptides were generated in the presence of ATP. The S1 subunit of PT was not cleaved by trypsin in the absence of ATP, at the concentrations of trypsin used, while two immunoreactive peptides were produced in the presence of ATP. The immunoreactive peptides produced either by chymotrypsin or trypsin cleavage of the S1 subunit of PT in the presence of ATP were indistinguishable from those produced by cleavage of rS1 with the same protease. Chymotryptic and tryptic cleavage of rS1 was not altered by ATP. When PT was incubated with ATP prior to Bio-Gel P-100 gel filtration, approximately 8% of the S1 subunit dissociated from the B oligomer, as determined by ADP-ribosyltransferase assays of the column eluant. This increased to 20% when ATP was included in the column buffer. The presence of dithiothreitol and NAD in addition to ATP did not affect the amount of dissociated S1 subunit. Our data further indicated that activation of PT by ATP was a reversible process. Together, these data showed that ATP quantitatively converted the S1 subunit of PT to a form which was kinetically and conformationally identical with rS1, while only a fraction of the S1 subunit was dissociated from the B oligomer. These results indicate that both S1 subunit which is bound to the B oligomer as well as dissociated S1 subunit are capable of catalyzing the ADP-ribosylation of Gt.
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PMID:Molecular characterization of the in vitro activation of pertussis toxin by ATP. 850 98

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