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)

Pertussis toxin (PT), an oligomeric exotoxin of Bordetella pertussis containing five dissimilar subunits, is considered to be an essential immunogen in acellular and component pertussis vaccines against whooping cough. A rapid single-step procedure for isolating PT subunits was developed using reverse-phase high-performance liquid chromatography. Recoveries of individual subunits were 75% (S1), 70% (S2), greater than 90% (S3), greater than 90% (S4), and 50% (S5), as judged by SDS-PAGE and amino acid analysis. Lyophilized subunits were solubilized in urea followed by step-wise dialysis to remove the urea. All subunits were inactive in histamine sensitization, lymphocytosis, and hemagglutination assays. However, purified S1 retained residual NAD-glycohydrolase and ADP-ribosyltransferase activity. A partially active holotoxin could be generated by mixing the five individual subunits. All subunits were immunogenic in rabbits and mice. Monospecific antisera raised in both animal species were able to neutralize the PT-mediated clustering of Chinese hamster ovary cells, but active immunization of mice with single subunits failed to protect them in the intracerebral challenge assay. These subunit preparations therefore retained neutralizing determinants, but did not contain protective epitopes.
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PMID:Purification and immunological characterization of HPLC-purified pertussis toxin subunits. 165 40

The cholinergic agonist carbachol produces a concentration-dependent (half-maximum inhibitory concentration = 0.9 microM) decrease in the Na(+)-K(+)-adenosine triphosphatase (ATPase) activity of rabbit cardiac sarcolemma that occurred only in the presence of guanosine 5'-[gamma-thio]triphosphate (0.1 microM GTP gamma S) and reached 40% inhibition. The inhibition is blocked by the muscarinic receptor antagonist atropine (10 microM) and is abolished in sarcolemma treated with pertussis toxin (20 micrograms/ml) in the presence of 100 microM NAD. GTP gamma S alone reduces Na(+)-K(+)-ATPase activity by 45% (half-maximum inhibitory = 1 microM). The apparent affinity of the enzyme for GTP gamma S is increased approximately 10-fold in the presence of 1 microM carbachol. In sarcolemma solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS, 10 mM), the GTP gamma S-dependent inhibition of the Na(+)-K(+)-ATPase is also observed. Gel filtration of a CHAPS extract of sarcolemma on a Sepharose CL-6B column resulted in a separation of Na(+)-K(+)-ATPase and pertussis toxin-sensitive Gi activities. Na(+)-K(+)-ATPase activity that was separated on the column lost its sensitivity to the inhibitory action of guanine nucleotides. Inhibitory effects (20-30%) of guanosine 5'-triphosphate analogues [Gpp(NH)p, GTP gamma S, or Gpp(CH2)p] at micromolar concentrations were restored when the Na(+)-K(+)-ATPase activity was recombined with fractions that contained the pertussis toxin-sensitive Gi protein(s). Similar concentrations of guanosine 5'-triphosphate, guanosine 5'-diphosphate, guanosine-5'-[beta-thio]diphosphate, or App(NH)p were unable to induce the Gi protein-mediated attenuation of Na(+)-K(+)-ATPase activity in the reconstitution system.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Na(+)-K(+)-ATPase-G protein coupling in myocardial sarcolemma: separation and reconstitution. 165 96

The possible involvement of a GTP-binding protein in the regulation of Ca2+ channels by angiotensin II (Ang II) in vascular muscle cells was investigated by the whole-cell voltage-clamp method. Single cells were freshly isolated from guinea pig portal vein. The pipette solution contained high Cs+ to inhibit K+ currents and thereby isolate the Ca2+ channel current. Ba2+ (2 mM) was in the bath solution as a charge carrier for the Ca2+ channel. Application of Ang II (0.1-100 nM) produced an increase in peak amplitude of the Ba2+ current, with a shift of the current-voltage curve in the negative direction. These effects were inhibited by pretreatment with an antagonist of the Ang II receptor, [Sar1,Ile8]-Ang II. Presence of 0.1 mM GTP in the pipette solution stabilized the Ang II action, but 0.3-1.0 mM GDP-beta-S and 1.0 mM GTP-gamma-S inhibited it. GTP-gamma-S alone produced a slowly progressing increase in the basal (unstimulated) current amplitude. Preincubation of muscle tissues with pertussis toxin (1 micrograms/ml, for up to 6 hours at 36 degrees C) or intracellular application of preactivated pertussis toxin (1 micrograms/ml) plus NAD (1 mM) did not inhibit the Ang II action. Cholera toxin (10 micrograms/ml) also had no effect on the Ang II action. These results suggest that the Ang II stimulation of Ca2+ channels in smooth muscle of guinea pig portal vein may be mediated by a G protein that is insensitive to both pertussis toxin and cholera toxin.
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PMID:Involvement of a GTP-binding protein in stimulating action of angiotensin II on calcium channels in vascular smooth muscle cells. 166 Mar 61

We used standard microelectrode techniques to study alpha-1 adrenergic modulation of repolarization in canine Purkinje fibers. Our objectives were to subtype this alpha-1 receptor response pharmacologically, to determine whether alpha-1 adrenergic modulation of repolarization is dependent on the function of a pertussis toxin-sensitive G protein and to identify developmental changes in this alpha-1 response. Phenylephrine (Phe) induced a dose-dependent increase in transmembrane action potential duration at 50% (APD50) and 90% (APD90) repolarization. For the adult fibers, control APD50 and APD90 were 310 +/- 5 and 407 +/- 5 msec; after superfusion with Phe, 1 x 10(-6) M, the values were 350 +/- 6 and 468 +/- 8 msec, respectively (P less than .05). In 2- to 3-week-old dogs, control APD50 and APD90 were 170 +/- 14 and 255 +/- 10 msec; after superfusion with Phe, the values were 228 +/- 10 and 305 +/- 16 msec, respectively (P less than .05). Propranolol, 2 x 10(-7) M, did not affect the response to Phe. The alpha-1 blocker prazosin, 1 x 10(-7) M, and the alpha-1 receptor subtype selective antagonist, WB 4101, 1 x 10(-7) M, suppressed the response to Phe, but no effect on the response to Phe was seen with the subtype selective antagonist, chloroethylclonidine. In vivo pretreatment of dogs with pertussis toxin, 30 micrograms/kg i.v., decreased markedly the amount of G protein substrate available for subsequent in vitro ADP-ribosylation by pertussis toxin and [32P]NAD (from 7039 +/- 713 to 537 +/- 50 fmol/mg of protein in adult fibers and from 1134 to 62 fmol/mg of proteins in pooled young fibers). Pertussis toxin pretreatment increased the Phe-induced prolongation of APD50 and APD90 in the young and adult fibers.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A WB 4101-sensitive alpha-1 adrenergic receptor subtype modulates repolarization in canine Purkinje fibers. 167 17

The binding of ATP to pertussis toxin and its components, the A subunit and B oligomer, was investigated. Whereas, radiolabeled ATP bound to the B oligomer and pertussis toxin, no binding to the A subunit was observed. The binding of [3H]ATP to pertussis toxin and the B oligomer was inhibited by nucleotides. The relative effectiveness of the nucleotides was shown to be ATP greater than ATP greater than GTP greater than CTP greater than TTP for pertussis toxin and ATP greater than GTP greater than TTP greater than CTP for the B oligomer. Phosphate ions inhibited the binding of [3H]ATP to pertussis toxin in a competitive manner; however, the presence of phosphate ions was essential for binding of ATP to the B oligomer. The toxin substrate, NAD, did not affect the binding of [3H]ATP to pertussis toxin, although the glycoprotein fetuin significantly decreased binding. These results suggest that the binding site for ATP is located on the B oligomer and is distinct from the enzymatically active site but may be located near the eukaryotic receptor binding site.
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PMID:Binding of ATP by pertussis toxin and isolated toxin subunits. 169 50

The kinetic constants for the ADP-ribosylation of transducin were determined for the recombinant S1 subunit of pertussis toxin (rS1, composed of 235 amino acids) and two genetically derived deletion peptides, C180 and C195, which are composed of the 180 and 195 amino-terminal residues of the S1 subunit, respectively. Titration of NAD in the presence of a constant concentration of transducin (0.5 microM) showed that the KmappNAD in the ADP-ribosylation of transducin were similar, approximately 20 microM, for rS1, C195, and C180. In contrast, titration of transducin in the presence of a constant concentration of NAD (25 nM) showed that rS1 possessed a lower Kmapp(transducin) and greater kcat than either C195 or C180. Previous studies (Cortina, G., and Barbieri, J.T. (1991) J. Biol. Chem. 266, 3022-3030) showed that the 16 carboxyl terminal residues of the S1 subunit did not function in the ADP-ribosylation of transducin. It thus appears that residues between 195 and 219 of the S1 subunit are required for high affinity transducin binding and may be involved in the transfer of ADP-ribose to transducin. To localize the defect in the recognition of transducin by C180, rS1 and C180 were assayed for the ability to ADP-ribosylate either transducin or the purified alpha subunit of transducin (T alpha). Upon saturation of the target protein, rS1 ADP-ribosylated equivalent moles of transducin or T alpha, with the linear velocity of rS1-mediated ADP-ribosylation of transducin approximately 16-fold more rapid than the rate of ADP-ribosylation of T alpha. In contrast, the initial linear velocity of C180-mediated ADP-ribosylation of transducin was only 1.7-fold more rapid than the rate of ADP-ribosylation of T alpha. These data indicate that the amino-terminal 180 amino acids of S1 confer the specificity for ADP-ribosylation primarily through the interaction with T alpha, while residues between 195 and 219 of S1 confer high affinity binding to transducin primarily through the interaction, either directly or indirectly, with T beta gamma.
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PMID:The carboxyl terminus of the S1 subunit of pertussis toxin confers high affinity binding to transducin. 174 55

A synthetic peptide corresponding to amino acids 6-17 of the A subunit of pertussis toxin was synthesised and used for the immunization of Balb/c mice and the subsequent production of monoclonal antibodies (MAbs). This peptide contains a region of eight amino acids which is homologous to a region in the cholera toxin A subunit. The properties of two of the resultant MAbs are described. Both of the antibodies (CP7-3003F7, an IgG3 and CP7-3004G6X1, an IgG1) react in an ELISA with the peptide and with intact pertussis toxin, pertussis toxin A subunit and cholera toxin A subunit, but do not react significantly with pertussis toxin B subunit, intact cholera toxin, or cholera toxin B subunit. Competition ELISA assays in which the peptide, the intact toxins and the toxin subunits were compared with respect to their ability to inhibit the binding of the MAbs to peptide-coated ELISA plates demonstrated that only pertussis toxin A subunit was as active, on a molar basis, as the peptide. Western blot analyses of the holotoxins confirmed that both MAbs were reactive only with the toxin A subunits. The MAbs were unable to neutralize the activity of cholera toxin or pertussis toxin in a Chinese hamster ovary (CHO) cell assay. Both were also unable to neutralize either the ADP-ribosylation activity or the NAD-glycohydrolase activity of the pertussis toxin A subunit. The significance of these results with respect to the role of this conserved site in the activity of these two toxins is discussed.
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PMID:Monoclonal antibodies against the enzymatic subunit of both pertussis and cholera toxins. 177 7

A DNA encoding the human alpha 2-C10 adrenergic receptor was transfected into Rat 1 fibroblasts and clones selected on the basis of resistance to G418 sulfate. Two clones, one of which (1C) expressed some 3.5 pmol/mg membrane protein of the receptor as assessed by the specific binding of [3H]yohimbine and one (4D) which did not express detectable amounts of the receptor were selected for further study. When cholera toxin-catalyzed ADP-ribosylation was performed with [32P]NAD on membranes of these cells in the absence of added guanine nucleotides, radioactivity was incorporated into a polypeptide(s) of 40 kDa in addition to the 45- and 42-kDa forms of Gs alpha. Addition of the selective alpha 2 receptor agonist U.K.14304 enhanced markedly, in a dose-dependent manner, the cholera toxin-catalyzed [32P]ADP-ribosylation of the 40-kDa polypeptide(s), but not the 45- or 42-kDa polypeptides, in membranes of the 1C cells. Dose response curves for U.K.14304 enhancement of cholera toxin-labeling of the 40-kDa polypeptide(s) and stimulation of high affinity GTPase activity were identical. By contrast, U.K.14304 was ineffective in either assay in membranes from the 4D cells, demonstrating this effect to be dependent upon receptor activation. Furthermore, the alpha 2 receptor antagonist yohimbine blocked all effects of U.K.14304. The agonist promotion of cholera toxin-catalyzed ADP-ribosylation of Gi was completely blocked by guanine nucleotides. Whether GDP or GDP + fluoroaluminate (as a mimic of GTP) was used, blockade of the agonist effect was complete and indeed both conditions prevented agonist-independent labeling by cholera toxin of the 40-kDa polypeptide(s). Mg2+ produced an agonist-independent cholera toxin-catalyzed [32P]ADP-ribosylation of the 40-kDa polypeptide(s) but even in the presence of [Mg2+], agonist-stimulation of cholera toxin-labeling of the 40-kDa polypeptide(s) was observed and was additive with the effect of [Mg2+]. Agonist stimulation of cholera toxin-catalyzed ADP-ribosylation of Gi was completely attenuated by pretreatment of the cells with pertussis toxin, which prevents contact between receptors and G-proteins which are substrates for this toxin. By contrast, pretreatment of the cells with concentrations of cholera toxin able to "down-regulate" essentially all of the membrane-associated Gs alpha did not prevent agonist stimulation of cholera toxin-catalyzed ADP-ribosylation of Gi.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Agonist-dependent, cholera toxin-catalyzed ADP-ribosylation of pertussis toxin-sensitive G-proteins following transfection of the human alpha 2-C10 adrenergic receptor into rat 1 fibroblasts. Evidence for the direct interaction of a single receptor with two pertussis toxin-sensitive G-proteins, Gi2 and Gi3. 184 55

Gonad-stimulating substance (GSS) secreted from radial nerves induces meiotic maturation of starfish oocytes by stimulating production of 1-methyladenine (1-MeAde) in ovarian follicle cells. We have previously shown that cAMP mediates the action of GSS on 1-MeAde synthesis by starfish ovarian follicle cells. The present study examines the possible involvement of guanine nucleotide-binding regulatory proteins (G-proteins) and adenylate cyclase in the action of GSS on 1-MeAde production by starfish (Asterina pectinifera) follicle cells. GSS slightly stimulated adenylate cyclase activity in crude membrane preparations of follicle cells. GTP markedly enhanced this action of GSS in a dose-dependent manner. Nonhydrolyzable GTP analogs such as guanosine 5'-O-(3-thiotriphosphate) and 5'-guanylylimidodiphosphate, NaF, and forskolin also stimulated adenylate cyclase activity. In addition, chorela toxin (CT) stimulated adenylate cyclase activity in membrane preparations in the presence of NAD and GTP. Unlike adenylate cyclase, phosphodiesterase activity was not influenced by GSS. When crude membranes of follicle cells were incubated with [alpha-32P]NAD in the presence of CT and pertussis toxin, 45-kDa and 41-kDa proteins were ADP-ribosylated, respectively, suggesting the presence of two types (stimulatory and inhibitory) of G-proteins. It is concluded that G-proteins and adenylate cyclase play an important role in the action of GSS on 1-MeAde production by starfish ovarian follicle cells.
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PMID:Involvement of G-proteins and adenylate cyclase in the action of gonad-stimulating substance on starfish ovarian follicle cells. 184 1

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


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