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)

The pharmacological specificity of the mGluR1 alpha subtype of the metabotropic glutamate receptor (mGluR) was examined in a cloned baby hamster kidney cell line (BHK-ts13) measuring [3H]glutamate binding and inositol phosphate (PI) hydrolysis. PI-hydrolysis was maximally stimulated by quisqualate (1112 +/- 105% of basal), glutamate (1061 +/- 70% of basal), ibotenate (1097 +/- 115% of basal) and beta-N-methylamino-L-alanine (BMAA) (1010 +/- 104% of basal). In contrast, the maximal stimulation of PI-hydrolysis by (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylic acid (t-ACPD) was only 673 +/- 78% of the basal level. The relative order of potency was quisqualate > glutamate > ibotenate > t-ACPD > BMAA. Agonist-stimulated PI-hydrolysis was attenuated (25 +/- 4% inhibition) by L-2-amino-3-phosphonopropionic acid and partially blocked (44 +/- 7%) by pertussis toxin treatment. Saturation binding studies with [3H]glutamate on membranes prepared from BHK-ts13 cells expressing the mGluR1 alpha subtype showed that glutamate binds to a single affinity state of this receptor with a limited capacity (Kd = 296 nM, Bmax = 0.8 pmol/mg protein). In competition experiments, [3H]glutamate was displaced by quisqualate, glutamate, ibotenate, t-ACPD and BMAA with a rank order of potency similar to that found for stimulation of PI-hydrolysis.
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PMID:A pharmacological characterization of the mGluR1 alpha subtype of the metabotropic glutamate receptor expressed in a cloned baby hamster kidney cell line. 769 Jun 72

Pseudomonas aeruginosa exotoxin A (ETA) is a member of the family of bacterial ADP-ribosylating toxins which use NAD+ as the ADP-ribose donor. By analogy to diphtheria and pertussis toxins, the His440 residue of ETA has been proposed to be one of the critical residues within the active site of the toxin. In this study the role of the His440 residue was explored through site-directed mutagenesis which resulted in the production of ETA proteins containing Ala, Asn, and Phe substitutions at the 440 position. The His440-substituted ETA proteins were purified and analyzed. All substitutions at the 440 site displayed severely reduced ADP-ribosylation activity (> 1000-fold). However, NAD glycohydrolase activity remained intact and in the case of ETAH440N actually increased 10-fold. NAD+ binding is not affected by substitutions at the 440 site as indicated by similar Km values for the ETA variants tested. Conformational integrity of the mutant toxins appears to be largely unaffected as assessed by analysis with a conformation-sensitive monoclonal antibody as well as sensitivity to proteinase digestion. In view of the location of His440 residue within or close to the proposed NAD(+)-binding site, these results suggest that His440 may be a catalytic residue involved in the transfer of the ADP-ribose moiety to the EF-2 substrate.
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PMID:Active site mutations of Pseudomonas aeruginosa exotoxin A. Analysis of the His440 residue. 782 95

Previous results showed that within 30 s after glutamate stimulation of cultured rat hippocampal pyramidal neurons there occurred an elevation of Ca2+ and diacylglycerol, and the phosphorylation of three acidic protein kinase C substrates, i.e., an 87-kDa protein known as myristoylated alanine-rich C kinase substrate and a 120- and a 48-kDa protein. In addition, it was suggested that a metabotropic-type glutamate receptor might be responsible for the phosphorylation observed. This work examines the ability of metabotropic and inotropic glutamate receptor agonists to quickly activate phospholipases in 1.26 mM versus 50 nM extracellular Ca2+ by measuring the generation of inositol phosphates. NMDA, quisqualate, and trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid did not stimulate the generation of inositol phosphates in the presence of normal or low extracellular Ca2+ in pyramidal neurons. Kainate stimulated the production of inositol phosphates in the presence of 1.26 mM extracellular Ca2+ but not in 50 nM extracellular Ca2+. Other than glutamate, only ibotenate was able to stimulate the generation of inositol phosphatases in both normal and low extracellular Ca2+. The maximal response to ibotenate was approximately equal to that of glutamate, when pyramidal neurons were stimulated in 50 nM extracellular Ca2+. The generation of inositol phosphates by glutamate and ibotenate could be partially blocked (50-60% reduction) by pretreatment of neurons with pertussis toxin (250 ng/ml), suggesting that a GTP-binding protein might be involved. In addition, ibotenate stimulated the immediate phosphorylation of the same three protein kinase C substrates as glutamate. The NMDA receptor blocker MK-801 had no effect on this phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:An ibotenate-selective metabotropic glutamate receptor mediates protein phosphorylation in cultured hippocampal pyramidal neurons. 790 44

Previous studies have demonstrated that the alpha 2A-adrenergic receptor (alpha 2A AR) incorporates [3H]palmitate and that replacement of Cys442 by Ala or Ser eliminates detectable acylation without perturbing coupling to pertussis toxin-sensitive GTP-binding proteins (Kennedy, M. E., and Limbird, L. E. (1993) J. Biol. Chem. 268, 8003-8011) or, as shown here, without perturbing agonist-dependent receptor phosphorylation, in contrast to the consequences of eliminating beta 2-adrenergic receptor acylation. As a first step in revealing the functional role for this post-translational modification at the alpha 2A AR, we explored sequences in the alpha 2AAR which confer alpha 2AAR acylation and whether or not [3H]palmitoylation of the alpha 2AAR is dynamic. Deletion of the 7 terminal amino acids distal to Cys442 of the alpha 2AAR did not eliminate detectable [3H]palmitoylation of the alpha 2AAR, whereas truncation to Leu441 did, indicating both that Cys442 is the likely site for acylation and that sequences distal to Cys442 are not required for acylation at Cys442. Since mutation of sequences proximal to Cys442 altered overall receptor structure, based on markedly reduced detectable adrenergic receptor binding, proximal motifs required for palmitoylation of the alpha 2AAR could not be explored further. When the turnover of [35S]Met/Cys-labeled alpha 2AAR was compared with the turnover of the [3H]palmitate-labeled alpha 2AAR, it was of interest that agonist treatment accelerated the half-life of decay of the [3H]palmitate-labeled alpha 2AAR without detectable receptor down-regulation, providing evidence that the acylation of the alpha 2AAR may be a dynamic process.
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PMID:Palmitoylation of the alpha 2A-adrenergic receptor. Analysis of the sequence requirements for and the dynamic properties of alpha 2A-adrenergic receptor palmitoylation. 798 67

The 85-kDa cytoplasmic phospholipase A2 (cPLA2) is the major hormone and growth factor-regulated enzyme that catalyzes release of arachidonic acid in mammalian cells. Activation of cPLA2 requires elevation of intracellular Ca2+ and the phosphorylation of the cPLA2 enzyme by mitogen-activated protein (MAP) kinase. Down-regulation of protein kinase C by phorbol esters or pertussis toxin catalyzed ADP-ribosylation of Gi proteins inhibits thrombin and ATP receptor-stimulated MAP kinase and arachidonic acid release, indicating that functional protein kinase C and Gi proteins are required for G protein regulation of arachidonic acid release. A mutant G alpha i2 subunit having Gly203 mutated to Thr (alpha i2G203T) inhibited thrombin and ATP receptor stimulation of arachidonic acid release independent of adenylyl cyclase inhibition, Ca2+ mobilization, and MAP kinase activation. Overexpression of the wild-type alpha i2 polypeptide or the inactive mutant alpha i2G204A (Gly204 mutated to Ala) polypeptide had no effect on thrombin or ATP receptor stimulation of arachidonic acid release. The phenotype observed with expression of the mutant alpha i2G203T polypeptide defines a role for Gi2 in the control of cPLA2 activity and subsequent arachidonic acid release in addition to the regulation of intracellular Ca2+ levels and MAP kinase activity.
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PMID:Expression of a mutant Gi2 alpha subunit inhibits ATP and thrombin stimulation of cytoplasmic phospholipase A2-mediated arachidonic acid release independent of Ca2+ and mitogen-activated protein kinase regulation. 829 38

Molecular modeling and alignment of the primary amino acid sequence of the S1 subunit (S1) of pertussis toxin (PT) with other members of the family of ADP-ribosylating bacterial exotoxins predicted that tyrosine-98 (Y98) of S1 was a conserved residue among these exotoxins. To extend our understanding of the relationship between pertussis toxin and the other ADP-ribosylating exotoxins, we defined the function of Y98 of S1. Using site-directed mutagenesis, Y98 of S1 was substituted with alanine (Y98A), leucine (Y98L), histidine (Y98H), and phenylalanine (Y98F). Mutations were analyzed in the C180 peptide and C219 peptide, recombinant derivatives of S1 which contain the first 180 and 219 amino-terminal residues of S1, respectively. Periplasmic extracts containing the Y98n peptides expressed similar specific activities for the ADP-ribosylation of transducin (Gt) as the periplasmic extract containing wild-type peptides. Mutations at Y98 influenced the subcellular localization of the respective Y98n peptide. The majority of the wild-type Y98 and Y98F peptides localized to the periplasmic extract, while the majority of Y98A and Y98L peptides were associated with the insoluble bacterial outer membrane. Purified C180Y98A and C180Y98F and partially purified C180Y98H peptides possessed similar specific activities for the ADP-ribosylation of Gt as the wild-type C180 peptide. KmNAD and kcat for C180Y98A and C180Y98F in the NAD glycohydrolase reaction were similar to the wild-type C180 peptide. These data show that the R group of Y98 does not participate in the ADP-ribosylation of Gt, but appears to contribute to the proper folding of S1.
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PMID:Biochemical analysis of mutations at tyrosine-98 of the S1 subunit of pertussis toxin. 831 78

Novel protein kinases that may participate in the signal transduction pathways of neutrophils were sought by a procedure based on the ability of these enzymes to undergo renaturation and catalyze the phosphorylation of a peptide substrate fixed in a gel. We report that neutrophils contain four uncharacterized protein kinases with molecular masses of about 69, 63, 49, and 40 kDa, which are rapidly activated upon stimulation of these cells with the chemoattractant fMet-Leu-Phe. These kinases can catalyze the phosphorylation of a peptide that corresponds to residues 297-331 of the 47-kDa subunit of the NADPH oxidase system (p47-phox). A peptide that corresponds to residues 153-178 of the human myristolyated alanine-rich C kinase substrate (MARCKS) protein was also a substrate for the 69- and 63-kDa kinases. The time course for the activation of these enzymes was similar to the phosphorylation of p47-phox and MARCKS in intact neutrophils. In contrast, stimulation of these cells with 4 beta-phorbol 12-myristate 13-acetate, the calcium ionophore A23187, or the combination of these agonists did not activate these enzymes. Activation of the 63- and 40-kDa protein kinases was blocked by pertussis toxin, calyculin A, and staurosporine. Several other unidentified protein kinases were also active with these peptides but did not exhibit enhanced activity after cell stimulation with this method.
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PMID:Stimulation of neutrophils with a chemoattractant activates several novel protein kinases that can catalyze the phosphorylation of peptides derived from the 47-kDa protein component of the phagocyte oxidase and myristoylated alanine-rich C kinase substrate. 834 15

The alpha 2A-adrenergic receptor (alpha 2AAR) is coupled to a variety of effectors via pertussis toxin-sensitive GTP-binding proteins. Like most members of the G-protein-coupled receptor superfamily, the primary structure of the alpha 2AAR possesses a putative consensus sequence for palmitoylation in the COOH terminus at Cys-442. This study demonstrates that the alpha 2AAR incorporates [3H] palmitic acid in metabolic labeling studies and that mutation of Cys-442 to Ala or Ser eliminates detectable 3H-palmitoylation. However, mutation of Cys-442 does not alter adrenergic ligand specificity or allosteric modulation by amphipathic agents, such as amiloride analogs. Since reports in the literature suggest that a homologous mutation in the beta 2-adrenergic receptor attenuates coupling to Gs (O'Dowd, B. F., Hnatowich, M., Caron, M. G., Lefkowitz, R. J., and Bouvier, M. (1989) J. Biol. Chem. 264, 7564-7569) whereas chemical removal of palmitate from bovine rhodopsin enhances coupling to Gt (Morrison, D. F., O'Brien, P. J., and Pepperberg, D. R. (1991) J. Biol. Chem. 266, 20118-20123), we examined if mutation of Cys-442 and parallel loss of detectable palmitoylation alter alpha 2AAR coupling to G-proteins. Several independent cell lines of Madin-Darby canine kidney II cells expressing wild-type (Cys-442) or mutant (Ala-442 and Ser-442) alpha 2AARs were established. Metabolic labeling of Madin-Darby canine kidney cells expressing wild-type (Cys-442) or mutant (Ala-442) alpha 2AARs with [3H]palmitic acid indicated that only wild-type Cys-442-containing receptors incorporated [3H]palmitate, monitored following isolation of the alpha 2AAR detergent extracts using yohimbine-agarose chromatography. Receptor-G-protein coupling was assessed by evaluating sensitivity of receptor-agonist interactions to guanine nucleotides in competition for [3H]yohimbine antagonist binding, guanyl-5'-yl imidotrisphosphate sensitivity of pertussis toxin-sensitive p-[125I]iodoclonidine agonist binding, and agonist-stimulated guanosine 5'-O-(thiotriphosphate) binding. Using all three approaches, no detectable change in alpha 2AAR-G-protein coupling was apparent, in contrast to apparent opposite effects on the beta 2-adrenergic receptor-Gs and rhodopsin-Gt coupling reported previously by others. One interpretation is that this conserved cysteine may play differing roles at different receptor-G-protein interfaces. Alternatively, this shared structural motif may play a role in not yet investigated pathways, such as receptor expression, turnover, and localization.
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PMID:Mutations of the alpha 2A-adrenergic receptor that eliminate detectable palmitoylation do not perturb receptor-G-protein coupling. 838 31

The cDNA for the alpha i1 protein that had undergone site-directed mutagenesis to change glycine-2 to alanine was ligated into a baculovirus transfer vector. A recombinant virus was obtained by transfecting Sf9 cells with both the wild-type baculovirus DNA and the transfer vector and screening for recombinant plaques. Infection with the recombinant virus led to a high level of expression of the mutated alpha i1 protein in the soluble fraction of the cell. The protein was purified by ammonium sulfate precipitation, gel filtration, and immobilized dye chromatography. The typical yield was 7.5 mg from two 800-ml cultures. The protein showed immunoreactivity to three different alpha i-specific antibodies. It bound guanosine 5'-(gamma-thio)triphosphate (GTP gamma S) with a stoichiometry of 0.63 to 0.91 mol/mol and with a rate constant (kGTP gamma S) of binding of 0.126 min-1. When GTP gamma S bound, the protein was protected from complete tryptic cleavage. The recombinant protein was able to undergo pertussis toxin-catalyzed ADP ribosylation and bind beta gamma subunits but with a reduced affinity compared to that of alpha transducin. Thus using a recombinant baculovirus, a nonmyristylated G protein alpha subunit was abundantly expressed in Sf9 cells and milligram quantities of a functional protein were easily purified.
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PMID:Baculovirus expression and purification of a soluble, mutant G-protein alpha subunit. 842 10

Tracheal cytotoxin (TCT) is a disaccharide-tetrapeptide released by Bordetella pertussis, the causative agent of pertussis (whooping cough). We have previously determined the structure of TCT to be GlcNAc-1,6-anhydro-MurNAc-L-Ala-gamma-D-Glu-meso-A2pm-D-Ala, where MurNAc = N-acetylmuramic acid and A2pm = diaminopimelic acid. Purified TCT reproduces the respiratory cytopathology observed during pertussis, including ciliostasis and extrusion of ciliated cells. We have tested structural analogs of TCT for their ability to reproduce native TCT toxicity in explanted hamster tracheal tissue and hamster trachea epithelial (HTE) cell cultures. Other investigators have evaluated many of these analogs, which are muramyl or desmuramyl peptides, for muramyl peptide activities such as immunopotentiation, induction of slow-wave sleep, and pyrogenicity. Four desmuramyl peptides were produced in our laboratory from B. pertussis peptidoglycan or by chemical synthesis, including unusual peptides containing alpha-aminopimelic acid in place of A2pm. Based on the relative ability of compounds to inhibit DNA synthesis in HTE cells, truncated analogs lacking A2pm entirely or lacking only the side-chain amine or carboxyl group of A2pm were less active than TCT by a factor of at least 1000. All active analogs included a native or near-native peptide moiety, independent of the presence, absence, or substitution of the sugar moiety. We conclude that the structural requirements for TCT toxicity differ considerably from those for most other muramyl peptide activities, in that the disaccharide moiety is irrelevant for toxicity and both the free amino and carboxyl groups of the A2pm side chain are required for activity.
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PMID:Bordetella pertussis tracheal cytotoxin and other muramyl peptides: distinct structure-activity relationships for respiratory epithelial cytopathology. 846 Jan 47


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