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)

Transducin, the signal coupling protein of retinal rod photoreceptor cells, is one of a family of G proteins that can be inactivated by pertussis toxin. We have investigated the nature of this inactivation in order to determine (1) whether it requires the toxin-catalyzed transfer of ADP-ribose from NAD+ to cysteine-347 of the alpha subunit and (2) whether it involves locking the alpha subunit in the inactive conformation characteristic of its GDP-bound state, or is limited to disruption of binding to photoexcited rhodopsin (R*). Our results indicate that all observed effects of pertussis toxin treatment, including a shift in the electrophoretic mobility of transducin's alpha subunit and functional inactivation, require NAD+ and that the appearance of the shift parallels incorporation of ADP-ribose. We have also found that, apart from interactions with photoexcited rhodopsin, the functional properties of ADP-ribosylated transducin are essentially the same as those of unmodified transducin. Normal spontaneous nucleotide exchange kinetics and the ability to activate cGMP phosphodiesterase are preserved following quantitative ADP-ribosylation, as are the abilities to hydrolyze GTP, to bind to a dye affinity column, and to display enhanced fluorescence upon addition of Al3+ and F-. Thus, ADP-ribosylation merely blocks catalysis of transducin nucleotide exchange by R* and does not lock transducin in an inactive state.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Nucleotide exchange and cGMP phosphodiesterase activation by pertussis toxin inactivated transducin. 166 Nov 43

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

Bovine brain microtubule protein, containing both tubulin and microtubule-associated proteins, undergoes ADP-ribosylation in the presence of [14C]NAD+ and a turkey erythrocyte mono-ADP-ribosyltransferase in vitro. The modification reaction could be demonstrated in crude brain tissue extracts where selective ADP-ribosylation of both the alpha and beta chains of tubulin and of the high molecular weight microtubule-associated protein MAP-2 occurred. In experiments with purified microtubule protein, tubulin dimer, the high molecular weight microtubule-associated protein MAP-2, and another high molecular weight mirotubule-associated protein which may be a MAP-1 species were heavily labeled. Tubulin and MAP-2 incorporated [14C]ADP-ribose to an average extent of approximately 2.4 and 30 mol of ADP-ribose/mol of protein, respectively. Assembly of microtubule protein into microtubules in vitro was inhibited by ADP-ribosylation, and incubation of assembled steady-state microtubules with ADP-ribosyltransferase and NAD+ resulted in rapid depolymerization of the microtubules. Thus, the eukaryotic enzyme can ADP-ribosylate tubulin and microtubule-associated proteins to much greater extents than previously observed with cholera and pertussis toxins, and the modification can significantly modulate microtubule assembly and disassembly.
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PMID:Microtubule protein ADP-ribosylation in vitro leads to assembly inhibition and rapid depolymerization. 173 82

Systemic infusion of angiotensin II, a potent agonist, using doses that are initially subpressor, eventually produces sustained blood pressure elevation and reductions in glomerular capillary ultrafiltration coefficient characterized by enhanced signal transduction to angiotensin II and other agonists. In this setting, there is a significant increased affinity of angiotensin II binding to smooth muscle and glomerular mesangial receptors and enhanced sensitivity and magnitude of angiotensin II-induced decrements in cyclic AMP. Since G proteins are important modulators of binding and signal transduction, the present studies were designed to test the hypothesis that differences in the relative amounts of G proteins may be present and have accounted for differences observed. G proteins were identified and quantitated by isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, radiolabeling in the presence of activated toxins with [gamma-32P]NAD+, immunoprecipitation, and immunoblotting. A 168% and 465% increase in pertussis toxin-catalyzed ADP ribosylation of alpha 40-41 was found in angiotensin II-treated groups over control groups for glomerular and mesenteric membranes, respectively. Immunoblotting revealed a 250% and 35% increase in the levels of the Gi isoforms alpha i-2 and alpha i-3, respectively, and a decrease of 53% in alpha i-1 from the angiotensin II-treated group. No differences were observed in cholera toxin labeling or immunoblotting of Gs. These results demonstrate multiple mechanisms whereby angiotensin-induced signal transduction can be modulated involving both the receptors and G proteins. These observed differences in G proteins in systemic and renal vasculature accompanying angiotensin II infusion suggest the possibility of a regulatory role in the pathophysiology of angiotensin II-induced hypertension and renal disease.
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PMID:Angiotensin II-induced changes in guanine nucleotide binding and regulatory proteins. 173 48

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

Pertussis toxin (PTX) catalyzes the ADP-ribosylation of the alpha-subunit of GTP-binding proteins (G-proteins) in the presence of NAD+. Pertussis toxin also decreases the electrophoretic mobility of the alpha-subunit on urea SDS PAGE. This effect of PTX has been suggested to be a property of the toxin different from its ability to catalyze ADP-ribosylation. However, the present report provides evidence to the contrary; ie, this mobility shift required the ADP-ribosylation of alpha-subunits. This conclusion was based on: (1) in the presence of increasing concentrations of NAD+ (0.026-1.3 microM), there was a linear increase in the formation of the slower migrating alpha-subunit as measured by immunoblotting with selective antisera, (2) addition of NADase to the incubation mixture completely eliminated the formation of this protein, and (3) increasing concentrations of nicotinamide (50-250 mM), which inhibits ADP-ribosylation, decreased the amount of the slower migrating alpha-subunit. Thus, in addition to PTX, NAD+ was required for the mobility shift and the slower migrating alpha-subunit is likely the ADP-ribosylated form.
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PMID:Requirement of ADP-ribosylation for the pertussis toxin-induced alteration in electrophoretic mobility of G-proteins. 183 88

Certain microbial toxins are ADP-ribosyltransferases, acting on specific substrate proteins. Although these toxins have been of great utility in studies of cellular regulatory processes, a simple procedure to directly study toxin-catalyzed ADP-ribosylation in intact cells has not been described. Our approach was to use [2-3H]adenine to metabolically label the cellular NAD+ pool. Labeled proteins were then denatured with SDS, resolved by PAGE, and detected by flurography. In this manner, we show that pertussis toxin, after a dose-dependent lag period, [3H]-labeled a 40-kD protein intact cells. Furthermore, incubation of the gel with trichloroacetic acid at 95 degrees C before fluorography caused the release of label from bands other than the pertussis toxin substrate, thus, allowing its selective visualization. The modification of the 40-kD protein was ascribed to ADP-ribosylation of a cysteine residue on the basis of inhibition of labeling by nicotinamide and the release of [3H]ADP-ribose from the labeled protein by mercuric acetate. Cholera toxin catalyzed the [3H]-labeling of a 46-kD protein in the [2-3H]adenine-labeled cells. Pretreatment of the cells with pertussis toxin before the labeling of NAD+ with [2-3H]adenine blocked [2-3H]ADP-ribosylation catalyzed by pertussis toxin, but not that by cholera toxin. Thus, labeling with [2-3H]adenine permits the study of toxin-catalyzed ADP-ribosylation in intact cells. Pasteurella multocida toxin has recently been described as a novel and potent mitogen for Swiss 3T3 cell and acts to stimulate the phospholipase C-mediated hydrolysis of polyphosphoinositides. The basis of the action of the toxin is not known. Using the methodology described here, P. multocida toxin was not found to act by ADP-ribosylation.
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PMID:A novel approach to detect toxin-catalyzed ADP-ribosylation in intact cells: its use to study the action of Pasteurella multocida toxin. 183 59

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

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