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)

Adenylate cyclase (AC) toxin from Bordetella pertussis enters cells to cause supraphysiologic increases in cAMP. AC toxin is also hemolytic. Substitution of Lys-58 with a methionine residue by site-directed mutagenesis of the structural gene for AC toxin, cyaA, and introduction of this mutation onto the B. pertussis chromosome results in an organism that synthesizes an enzyme-deficient AC toxin molecule. This mutant toxin molecule exhibits 1000-fold reduction in enzymatic activity relative to wild-type and has no toxin activity in J774 cells. The enzyme-deficient toxin molecule is not, however, impaired in its ability to lyse sheep red blood cells. In order to ascertain the importance of these two separate activities of AC toxin in vivo the enzyme-deficient organisms were used to infect infant mice. The hemolytic, enzyme-deficient mutant organisms are reduced in virulence relative to wild-type organisms after intranasal challenge indicating that, although the enzymatic activity of AC toxin does not contribute to hemolysis, it is this property of the toxin which is important for virulence of B. pertussis.
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PMID:Enzymatic activity of adenylate cyclase toxin from Bordetella pertussis is not required for hemolysis. 131 23

The baculovirus-based expression system was adapted to express alpha subunits of the complete (alpha i3) and an amino-terminally truncated (alpha i3') form of Gi3 and of two complete forms of Gs (alpha s-L and alpha s-S). Subunits encoded in full length cDNAs were obtained with yields of 40-60 mg of recombinant protein/liter of cells, of which alpha i3 was between 30 and 50% soluble, but alpha s subunits were only 5-10% soluble. Only the complete alpha i3 was myristoylated. alpha i3 was purified in four steps. The purified protein bound 0.8-0.9 mol of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) per mol of protein and had one predominant contaminant which was identified as a truncated form that begins with methionine 18 instead of methionine 1. Both the full length alpha i3 and the truncated alpha i3' formed trimers with human erythrocyte beta gamma as seen by their migration in sucrose density gradients and by an increased rate of ADP ribosylation by pertussis toxin, but compared to alpha i3, alpha i3' interacted with beta gamma with a reduced affinity and dissociated upon warming. At 32 degrees C, only full length alpha i3 was ADP-ribosylated; at 4 degrees C, alpha i3 and alpha i3' were both ADP-ribosylated, with the truncated form requiring approximately 200-fold higher concentrations of beta gamma. A genetically engineered alpha i3' (alpha i3[18-354]) was also expressed in Sf9 cells. Yields, assessed as saturable GTP gamma S binding sites, were 3-5 mg per liter. Scatchard analysis showed that truncation of the amino terminus interferes with the ability of Mg2+ to promote high affinity binding of GTP gamma S. We conclude that the G protein alpha subunit amino terminus is not essential for interaction with beta gamma dimers, but rather is important in determining the affinity of the alpha subunit for both the beta gamma dimers and guanine nucleotide.
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PMID:A truncated recombinant alpha subunit of Gi3 with a reduced affinity for beta gamma dimers and altered guanosine 5'-3-O-(thio)triphosphate binding. 133 51

A somatostatin (SRIF) receptor and its associated Gi regulatory proteins was purified from GH4C1 rat pituitary cells by: 1) saturation of the membrane-bound receptor with biotinyl-NH-[Leu8,D-Trp22,Tyr25] SRIF28 (bio-S28); 2) solubilization of receptor-ligand (R.L) complex with deoxycholate-lysophosphatidylcholine (D.L); 3) adsorption of solubilized receptor-ligand complex to immobilized streptavidin; and 4) elution of receptor and G-protein by GTP. The receptor, a glycoprotein with an average M(r) of 85,000, was then purified to substantial homogeneity on immobilized wheat germ agglutinin. The 85-kDa glycoprotein was identified as a SRIF receptor by several criteria. (a) It had the same size as the chemically cross-linked R.[125I]L complex. (b) Yield of the purified protein increased and plateaued in the same range of bio-S28 concentrations where specific high affinity binding reached saturation. (c) It was copurified with appropriate G-protein subunits. The 85-kDa receptor and two other proteins with M(r) values of 35,000 and 40,000, the sizes of G beta and G alpha, did not appear in eluates from control streptavidin columns done with SRIF receptors loaded with nonbiotinylated S14. The 40-kDa protein was identified as a Gi alpha by ADP-ribosylation from [32P]NAD catalyzed by pertussis toxin. (d) Both the chemically cross-linked R.[125I]L complex and SRIF receptor purified from [35S]methionine-labeled GH4C1 cells were reduced in size to about 38 kDa by endoglycosidase F. (e) Amino acid sequence from the purified receptor was nearly identical with that of a recently cloned SRIF receptor subtype.
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PMID:Purification of a pituitary receptor for somatostatin. The utility of biotinylated somatostatin analogs. 135 97

The regulated secretory pathway comprises accelerated discharge of proteins in response to hormonal stimuli, their presence in secretory granules (SG), and a long intracellular residence time. Dexamethasone induction of AR42J results in an increase in granule content and responsiveness to cholecystokinin (CCK). We studied the effects of conditions implicated in sorting of secretory proteins into the regulated pathway using [35S]methionine pulse-chase protocols that examine transport of secretory proteins from the rough endoplasmic reticulum (RER)----SG and specifically from the Golgi complex (GC)----SG. The latter uses a chase at 20 degrees C to allow accumulation of labeled proteins in the trans-Golgi, followed by a shift to 37 degrees C that initiates their transport to SG under test conditions. Quantitation of CCK-8-stimulated discharge of prestored amylase and of newly synthesized labeled proteins that have entered SG during the chase enables us to examine the effect of perturbants over selected parts of the pathway. The effects of acidic intracellular compartments, the cytoskeleton, protein synthesis, ATP, and temperature on pre- and post-Golgi entry of proteins into the regulated pathway were studied. NH4Cl, monensin, Na azide, incubation at 20 degrees C, and pertussis toxin retarded RER----SG transport without affecting amylase discharge. Only incubation with 20 mM NH4Cl or 1 microM monensin inhibited transfer of newly synthesized proteins from the late GC----SG. RER----Golgi or intra-Golgi transport thus appears to require ATP and possibly guanosine 5'-triphosphate (GTP)-binding proteins. Acidic compartments appear to be essential for sorting of secretory proteins from the GC----SG.
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PMID:Perturbation of regulated secretion in the pancreatic acinar cell line, AR42J. 137 45

Calcium currents can be modulated by receptor activation of the GTP-binding protein G(o). We have determined whether the two forms of G(o), Go1 and Go2, differentially regulate calcium current magnitude. Using identified neurons of the pond snail Helisoma, we demonstrate that a high-voltage-activated (HVA) calcium current is reduced by addition of the neuropeptide Phe-Met-Arg-Phe-amide (FMRFamide) and that this inhibition is mediated by a pertussis toxin (PTX)-sensitive G protein pathway. Using this calcium current as an assay for G protein activity, we microinjected GTP gamma S-activated alpha-subunits of G proteins into neuronal somata. We demonstrate that the calcium current is differentially regulated by the two forms of alpha o. Microinjection of alpha o2*, but not alpha o1*, reduces calcium current magnitude.
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PMID:Microinjection of the alpha-subunit of the G protein Go2, but not Go1, reduces a voltage-sensitive calcium current. 141 84

1. The modulation of the voltage-activated Ca2+ current by the neuropeptide Phe-Met-Arg-Phe-NH2 (FMRFa) was investigated in dissociated central neurons from Helix aspersa using whole-cell voltage-clamp recording techniques. External Ba2+ was always used as the charge carrier in this study, and the intracellular Ca2+ concentration was buffered to 20 nM with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). 2. Run-down of the Ca2+ currents was not a problem as long as the neurons were dialyzed with a patch electrode filling solution containing ATP (1 or 2 mM). In ATP-dialyzed neurons, the rate of inactivation of the calcium current increased with time without any significant change in the rate of activation. However, when neurons were dialyzed with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S; 100 microM; with ATP), the rate of inactivation decreased with time. There was no effect of GTP gamma S on the rate of activation of the Ca2+ current. This suggests that guanosine 5'-triphosphate (GTP)-binding proteins (G proteins) are able to modulate the rate of inactivation of the Ca2+ current in Helix neurons. 3. FMRFa both decreased and enhanced the amplitude of the Ca2+ current in these neurons. This inhibition was observed in most neurons, while the enhancement was observed in 20% of the neurons. Although the enhancement usually was preceded by the inhibitory response, sometimes the enhancement was observed separately. 4. The FMRFa-induced inhibition of the Ca2+ current usually consisted of a decrease in both the amplitude and the rate of inactivation of this current, effects that were reduced as the membrane potential was stepped to more depolarized potentials. A pertussis toxin (PTX)-sensitive G protein mediated this response, whereas no evidence was found to suggest the involvement of any known intracellular messenger. Therefore this inhibition may have resulted from a direct coupling between the FMRFa receptor and the Ca2+ channels via a PTX-sensitive G protein. 5. Arachidonic acid (100 microM) irreversibly reduced the amplitude of the Ca2+ current, but it did not alter the relative inhibition of this current by FMRFa. 6. The FMRFa-induced enhancement of the Ca2+ current was difficult to study because it was observed infrequently, and was rarely observed independently of the FMRFa-induced inhibitory response. In addition, the ability of FMRFa to enhance this current usually disappeared with time.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The neuropeptide FMRFa both inhibits and enhances the Ca2+ current in dissociated Helix neurons via independent mechanisms. 165

Human polymorphonuclear neutrophils (PMN) respond to ATP with an elevation in intracellular calcium and a marked enhancement of O2-production in response to stimulation by the chemotactic peptide N'-formyl-Met-Leu-Phe (FMLP). These pertussis toxin-sensitive pathways appear to be mediated by a nucleotide receptor(s) on the surface of human PMN. In the current study, we have examined the binding to intact human PMN of the ATP analog, adenosine 5'-O-(3-thio[35S] triphosphate) [( 35S]ATP gamma S). On the basis of Scatchard analysis, the binding of [35S]ATP gamma S involves at least two sites, one of high and one of low affinity. In the presence of sodium thiophosphate, a compound which did not affect intracellular increases in calcium induced by ATP or N'-formyl-Met-Leu-Phe, a significant fraction of the [35S]ATP gamma S binding was eliminated. This reduction involved both high and low affinity binding of [35S]ATP gamma S and was related to a reduction in numbers of binding sites. The Kd values for the high affinity binding site were unaffected by the presence of sodium thiophosphate, although the low affinity Kd values were numerically increased by 2-fold. In the presence of thiophosphate, [35S]ATP gamma S binding was specific, saturable, and reversible, and was related to a single class of high affinity (Kd = 36 +/- 19 nM) binding sites (184 +/- 144 sites/cell), together with a second class of low affinity (Kd = 1110 +/- 503 nM) binding sites (13,562 +/- 6,851 sites/cells). Competitive binding experiments, based on the ability of nucleotides and ATP analogs to block [35S]ATP gamma S binding to PMN, revealed a rank order of ATP gamma S greater than ATP greater than 2-MeS-ATP = 8-Bromo ATP greater than ADP = ITP greater than AMP-PCP = GTP much greater than CTP. A comparison between the ability of nucleotides to compete with [35S]ATP gamma S binding and their ability to induce a biologic response (elevation of intracellular calcium) revealed a close correlation (r2 = 0.83). These findings support the possibility of a common nucleotide PMN receptor functionally linked to a cellular response which involves increases in intracellular calcium.
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PMID:Adenosine-5'-O-(3-thiotriphosphate) binding to human neutrophils. Evidence for a common nucleotide receptor. 165 77

We have previously shown that bradykinin-induced production of second messengers such as inositol trisphosphate and diacylglycerol in neurotumor cells is inhibited by raising cellular cyclic AMP levels, which in turn inhibit phospholipase C. A monoclonal antibody to phospholipase C-II immunoprecipitated the 140-kDa form of phospholipase C-II from [35S]methionine/[3H]eucine-labeled cells, but not [32P]orthophosphate-labeled phospholipase C-II, following treatment with either forskolin or dibutyryl cyclic AMP. This suggested that phospholipase C is not the target for cyclic AMP-dependent protein kinase-mediated phosphorylation. In vitro studies confirmed that phospholipase C activity was inhibited by raising cellular cAMP levels, and partial sensitivity to Bordetella pertussis toxin suggested the involvement of a GTP-binding protein which could be the target for protein kinase A. The involvement of a GTP-binding protein in coupling the bradykinin receptor to phospholipase C was further suggested by the ability of both guanosine 5'-O-(thio-triphosphate) and fluoride (NaF) to release inositol phosphates from NCB-20 cell membranes previously labeled with [3H]inositol. Both effects were blocked by pretreatment of the cells with protein kinase A activators, further suggesting a GTP-binding protein as the target for protein kinase A-mediated phosphorylation. When whole NCB-20 cell extracts were blotted onto nitrocellulose and incubated with [alpha- 32P]GTP, a major 24-kDa band plus minor bands at 22 and 20 kDa were revealed by autoradiography. A pH 3.0/6.0 soluble (basic protein) NCB-20 cell extract revealed the major 24-kDa band plus the 20-kDa band, and similar basic proteins were shown to be heavily phosphorylated following [32P]orthophosphate labeling and pretreatment with forskolin. The size and ability to bind GTP on Western blots are characteristic of the ras, rho, smg, etc. family of GTP-binding proteins recently suggested to be the much sought after GPLC (Lapetina, E.G., Lacal, J. C., Reep, B. R., and Molina y Vedia, L. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 3131-3134; Wang, P., Nishihata, J., Takabori, E., Yamamoto, K., Toyoshima, S., and Osawa, T. (1989) J. Biochem. (Tokyo) 105, 461-466; Nagata, K.-I., Nagao, S., and Nozawa, Y. (1989) Biochem. Biophys. Res. Commun. 160, 235-242). We propose that GPLC is uniquely sensitive to protein kinase A-mediated phosphorylation and that phosphorylation inhibits stimulus-secretion coupling in these cells.
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PMID:Phospholipase C activity in NCB-20 cells is inhibited by protein kinase A-mediated phosphorylation of low molecular mass GTP-binding proteins. 169 Nov 76

The alpha-subunit of Gi-2, in addition to that of Gs (GTP-binding proteins involved in adenylate cyclase inhibition and stimulation, respectively) was ADP-ribosylated by cholera toxin in HL-60 cell membranes when a chemotactic receptor was stimulated by formyl-Met-Leu-Phe (fMLP), and the sites modified by cholera and pertussis toxins on the alpha-subunit of Gi-2 were different (Iiri, T., Tohkin, M., Morishima, N., Ohoka, Y., Ui, M., and Katada, T. (1989) J. Biol. Chem. 264, 21394-21400). In order to investigate how the functions of Gi-2 were modified by cholera toxin, the ADP-ribosylated and unmodified proteins were purified from HL-60 cell membranes that had been incubated in the presence and absence of cholera toxin, respectively. The modified Gi-2 displayed unique properties as follows. 1) The ADP-ribosylated alpha-subunit had a more acidic pI than the unmodified one, leading to a partial resolution of the modified Gir2 trimer from the unmodified protein by an anion column chromatography. 2) When the purified proteins were incubated with [gamma-32P]GTP, the radioactivity was more greatly retained in the modified Gi-2 than in the unmodified protein. 3) The actual catalytic rate (kcat) of GTP hydrolysis was, indeed, markedly inhibited by cholera toxin-induced modification. 4) There was an increase in the apparent affinity of Gi-2 for GDP by cholera toxin-induced modification. 5) The modified Gi-2 exhibited a low substrate activity for pertussis toxin-catalyzed ADP-ribosylation. 6) A high-affinity fMLP binding to HL-60 cell membranes was more effectively reconstituted with the ADP-ribosylated Gi-2 than with the unmodified protein. These results suggested that the agonist-fMLP receptor complex was effectively coupled with the ADP-ribosylated Gi-2, resulting in the GTP-bound form, and that the hydrolysis of GTP on the modified alpha-subunit was selectively attenuated. Thus, cholera toxin ADP-ribosylated Gi-2 appeared to be not only a less sensitive pertussis toxin substrate but also an efficient signal transducer between receptors and effectors.
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PMID:Modification of the function of pertussis toxin substrate GTP-binding protein by cholera toxin-catalyzed ADP-ribosylation. 173 Jun 31

Since human polymorphonuclear neutrophils (PMN) exposed to ATP or its poorly hydrolyzable analogue, adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S), respond with increases in intracellular calcium and enhanced O2- responses to the chemotactic peptide N'-formyl-Met-Leu-Phe (fMLP), we systematically evaluated responses of PMN to various nucleotides. The P2X and P2Y receptor agonists, 2-methylthioadenosine triphosphate and beta, gamma-methyleneadenosine triphosphate, failed to induce increases in intracellular calcium and did not desensitize PMN to increases in intracellular calcium induced by ATP gamma S. Since it has been suggested that P2Z receptor occupancy with the ATP4- species caused nonselective increases in cell permeability, the ability of ATP to induce increases in intracellular calcium was evaluated in the presence and absence of extracellular Ca2+ and Mg2+. In the presence of these cations, 5-fold greater concentrations of ATP were required. The effects of ATP4- were not associated with changes in cell membrane permeability. This suggests that ATP4- is the active species but that its effect on PMN is not linked to a nonselective increase in permeability of the cell membrane. With respect to responses of PMN to purine and pyrimidine nucleotides as defined by increases in intracellular calcium, the rank order of potency for the nucleotides was ATP = UTP greater than ATP gamma S greater than or equal to ITP greater than GTP greater than or equal to CTP. These responses were blocked by pretreatment of PMN with pertussis toxin. Prior exposure of PMN to ATP gamma S blocked cellular responses (calcium increases) to these nucleotides but not to fMLP. Likewise, exposure of PMN to any nucleotides blocked subsequent cellular responses to ATP gamma S but not to fMLP. These data support the concept that nucleotide responses of PMN utilize either a common receptor or a common signal transduction pathway involving a guanine nucleotide binding protein in events leading to elevations in intracellular calcium. Nucleotide interaction with PMN does not follow the established pattern of responses associated with P2X or P2Y purinergic receptor occupancy.
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PMID:Nucleotide responses of human neutrophils. 184 54


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