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)

Phospholipid base exchange activity using choline as substrate was detected in plasma membranes (PM) and other subcellular fractions of rat liver, with microsomes (MS) showing the highest specific activity. In contrast, phospholipase D activity was only detected in PM. In PM, choline exchanged for phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS), whereas ethanolamine exchanged for PE and PS, and serine exchanged for PS. Ca2+ (10 microM or higher) stimulated choline incorporation into PC in MS and PM, whereas Mg2+ (10 microM or higher) stimulated it only in PM. Ethanolamine and serine incorporation into PM phospholipids was also stimulated by Ca2+, and inositol incorporation by Mn2+. Phospholipase D activity was substantial in the presence of EGTA and was slightly stimulated by Ca2+ concentrations less than 500 microM. It was undetectable without Mg2+. Low concentrations of oleate (1 mM or less) stimulated phospholipase D activity. These concentrations inhibited choline base exchange activity, whereas higher concentrations (3-8 mM) were stimulatory. Comparison of the subcellular distribution and Ca2+, Mg2+, and oleate effects on choline base exchange and phospholipase D activities supports the view that they are catalyzed by different enzymes. The incorporation of choline, but not ethanolamine or serine, into the phospholipids of PM, but not MS, was stimulated by micromolar concentrations of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) and other slowly hydrolyzable analogues of GTP. GDP, GMP, and other nucleoside triphosphates and their analogues were ineffective. GTP gamma S stimulation of base exchange activity was dependent upon Mg2+ and was inhibited by high concentrations of guanosine 5'-O-2-(thio)diphosphate. In the presence of low concentrations of GTP gamma S, ATP and its slowly hydrolyzable analogues stimulated base exchange activity. Dose-response curves for these nucleotides revealed a potency order consistent with mediation by purinergic receptors of the P2Y type. Base exchange activity stimulated by ATP plus GTP gamma S or GTP gamma S alone was not altered by treatment with pertussis or cholera toxins. These results suggest that the choline base exchange activity of liver PM is regulated by a pertussis toxin-insensitive G-protein linked to P2Y purinergic receptors.
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PMID:Phospholipid base exchange activity in rat liver plasma membranes. Evidence for regulation by G-protein and P2y-purinergic receptor. 131 19

Loss of sensitivity to thrombin following an initial response is characteristic of a number of cell types, including platelets. It has recently been proposed that thrombin receptors resemble other G protein-coupled receptors, but that activation involves a novel mechanism in which thrombin cleaves the receptor, exposing a new N terminus that serves as the ligand for the receptor. Based upon this model, we have examined the mechanism of thrombin receptor desensitization by comparing the effects of thrombin with those of a peptide corresponding to the N-terminal sequence of the receptor following proteolysis by thrombin: SFLLRNPNDKYEPF or TRP42/55. Like thrombin, TRP42/55 stimulated pertussis toxin-sensitive inositol 1,4,5-trisphosphate formation, raised cytosolic Ca2+, and inhibited cAMP formation in the megakaryoblastic HEL cell line. Exposure to either thrombin or TRP42/55 desensitized the cells to both, but not to a third agonist, neuropeptide Y. The rate of recovery after desensitization depended upon the order of agonist addition. Resensitization of the cell to thrombin following a brief exposure to thrombin required up to 24 h and could be inhibited with cycloheximide. Resensitization to TRP42/55 after exposure to thrombin, or to thrombin after exposure to TRP42/55, on the other hand, was detectable within 30 min and could be inhibited by serine/threonine phosphatase inhibitors, but not by cycloheximide. Loss of responsiveness to thrombin and TRP42/55 was also observed following addition of the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). However, while the protein kinase inhibitor staurosporine completely prevented the desensitization caused by TPA, it had only a limited effect on the desensitization caused by TRP42/55. These results demonstrate that the G protein-mediated effects of thrombin can be reproduced by a receptor-derived peptide and suggest that desensitization occurs by at least two mechanisms. The first, which is seen with thrombin, but not TRP42/55, involves proteolysis and requires protein synthesis for recovery. The second, which occurs with TRP42/55 and TPA, as well as with thrombin, involves phosphorylation, possibly of the receptor itself. Although protien kinase C is activated by thrombin and is presumably responsible for the desensitization caused by TPA, it does not appear to play a major role in receptor desensitization caused by thrombin and TRP42/55. This suggests that other kinases, such as those which inactivate adrenergic receptors and rhodopsin, are involved in the down-regulation of thrombin receptor function.
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PMID:Homologous desensitization of HEL cell thrombin receptors. Distinguishable roles for proteolysis and phosphorylation. 131 26

Antral gastrin secretion and gene expression is inhibited by the paracrine release of somatostatin from antral D cells. Transforming growth factor-alpha and epidermal growth factor (EGF) stimulate gastrin reporter gene constructs when transfected into pituitary GH4 cells. Somatostatin inhibits EGF stimulation of gastrin gene expression, which is in part mediated at the level of transcriptional regulation as somatostatin inhibits EGF stimulation of gastrin reporter gene constructs. Somatostatin inhibition was abolished by pertussis toxin, indicating somatostatin inhibits transcription through the inhibitory G protein Gi. Somatostatin inhibition was unaffected by vanadate and okadaic acid, implying this inhibitory pathway is mediated neither through phosphotyrosine phosphatases nor serine/threonine phosphatases, respectively. Gastrin reporter genes containing 82 base pairs of the 5'-flanking DNA were sufficient to confer both EGF responsiveness and inhibition by somatostatin in GH4 cells. However, transcription of a gastrin reporter gene construct containing only the EGF response element (GGGGCGGGGTGGGGGG), located at -68 to -53, was stimulated by EGF but was not inhibited by somatostatin. Thus, somatostatin inhibits EGF-stimulated gastrin gene transcription by a mechanism other than by interfering with cell signals elicited by the EGF receptor. Since the 82 GASCAT is inhibited by somatostatin, this result also implies that sequences adjacent to the EGF response element contain a cis-regulatory element mediating transcriptional inhibition by somatostatin. This cis-element was located using gastrin reporter genes comprising sequential segments of the human gastrin promoter sequence from the transcriptional start site to -82 in the 5'-flanking DNA. Gastrin oligonucleotide constructs lacking the D oligonucleotide (gatcCATATGGCAGGGTA), located at -82 to -69 in the 5'-flanking DNA, were not inhibited by somatostatin, indicating that a somatostatin inhibitory cis-element is located between -82 and -69 in the 5'-flanking DNA of the human gastrin promoter.
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PMID:Identification of a cis-regulatory element mediating somatostatin inhibition of epidermal growth factor-stimulated gastrin gene transcription. 135 47

The intracellular signaling pathways regulating the synthesis of leukotrienes by myeloid cells are largely unknown. In addition, the signal transduction mechanisms utilized by the cytokine receptor family are still poorly understood. The fact that in mature human basophils the synthesis of leukotriene C4 (LTC4) induced by C5a is strictly dependent on a short preincubation with the cytokine interleukin-3 (IL-3), allowed us to investigate the metabolic requirements for LTC4 synthesis, and also to provide some information on early signal transduction mechanisms of IL-3 in these differentiated, non-dividing blood leukocytes. IL-3 itself does not alter intracellular free calcium concentration ([Ca2+]i) in basophils, whereas C5a induces a transient rise independent of IL-3 pretreatment, indicating that the priming effect of IL-3 cannot be explained by alterations in [Ca2+]i changes. The protein kinase C inhibitor staurosporine did not inhibit C5a-induced histamine release nor IL-3-dependent LTC4 formation in contrast to the IgE receptor-dependent basophil response. Activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA) induced histamine release without leukotriene formation. PMA-treated basophils did not produce LTC4 in response to C5a. Rather, PMA blocked the IL-3 effect on C5a-induced LTC4 synthesis. Only the C5a signal but not the IL-3 effect was pertussis toxin sensitive. Two unrelated tyrosine kinase inhibitors, tyrphostin RG-50864 and herbimycin A, were both very efficient blockers of IL-3-dependent lipid mediator formation whereas C5a-induced histamine release was preserved. Thus LTC4 formation does not require activation of a staurosporine-sensitive serine/threonine kinase. To the contrary, IL-3-dependent LTC4 formation appears to be regulated by serine/threonine and tyrosine phosphorylation in an antagonistic manner.
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PMID:Signal transduction for interleukin-3-dependent leukotriene synthesis in normal human basophils: opposing role of tyrosine kinase and protein kinase. 142 16

ADP-ribosylation is a posttranslational modification of proteins by amino acid-specific ADP-ribosyltransferases. Both pertussis toxin and eukaryotic enzymes ADP-ribosylate cysteine residues in proteins and also, it has been suggested, free cysteine. Analysis of the reaction mechanisms of cysteine-specific ADP-ribosyltransferases revealed that free ADP-ribose combined nonenzymatically with cysteine. L- and D-cysteine, L-cysteine methyl ester, and cysteamine reacted with ADP-ribose, but alanine, serine, lysine, arginine, N-acetyl-L-cysteine, 2-mercaptoethanol, dithiothreitol, and glutathione did not. The 1H NMR spectrum of the product, along with the requirement for both free sulfhydryl and amino groups of cysteine, suggested that the reaction produced a thiazolidine linkage. ADP-ribosylthiazolidine was labile to hydroxylamine and mercuric ion, unlike the ADP-ribosylcysteine formed by pertussis toxin and NAD in guanine nucleotide-binding (G-) proteins, which is labile to mercuric ion but stable in hydroxylamine. In the absence of G-proteins but in the presence of NAD and cysteine, pertussis toxin generated a hydroxylamine-sensitive product, suggesting that a free ADP-ribose intermediate, expected to be formed by the NADase activity of the toxin, reacted with cysteine. Chemical analysis, or the use of alternative thiol acceptors lacking a free amine, is necessary to distinguish the enzymatic formation of ADP-ribosylcysteine from nonenzymatic formation of ADP-ribosylthiazolidine, thereby differentiating putative NAD:cysteine ADP-ribosyltransferases from NAD glycohydrolases.
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PMID:Amino acid-specific ADP-ribosylation: structural characterization and chemical differentiation of ADP-ribose-cysteine adducts formed nonenzymatically and in a pertussis toxin-catalyzed reaction. 144 18

Activation of T lymphocytes leads to the production of the T cell growth factor IL-2 that regulates T cell proliferation. This activation is associated with several potential intracellular signalling events including increased activity of phospholipase C (PLC) and resultant increases in production of inositol phosphates and diacylglycerols. In addition, phosphorylation of specific intracellular proteins on serine, threonine, and tyrosine residues increases. The role of each of these events in IL-2 production is unclear. Using Western blotting with antiphosphotyrosine antibodies, we demonstrate that activation of murine T cells with mitogenic lectins or anti-CD3 antibodies leads to a rapid increase in tyrosine phosphorylation of proteins of 120, 72, 62, 55, and 40 kDa. Similar patterns of antiphosphotyrosine antibodies reactivity were observed in splenocytes, a T cell hybridoma, and a T lymphoma. Tyrosine phosphorylation was detectable within minutes of addition of mitogenic lectins and persisted for at least 6 h. Pretreatment of the cells with pertussis toxin did not inhibit tyrosine phosphorylation indicating that a pertussis toxin-sensitive G protein is not involved in signal transduction. Neither increasing cytosolic-free calcium nor activating protein kinase C mimicked the effects of mitogenic lectins suggesting that tyrosine phosphorylation was not a consequence of activation of PLC. This was confirmed by demonstrating that mitogenic lectins induced similar patterns of tyrosine phosphorylation in cells in which activation of the TCR leads to increased PLC activity and in cells in which PLC is not stimulated. To test whether tyrosine phosphorylation is linked to IL-2 secretion, we determined the effect of three specific tyrosine kinase inhibitors (tyrphostins) on tyrosine phosphorylation, IL-2 secretion, and cellular proliferation. The concentration dependence of inhibition of tyrosine phosphorylation and IL-2 production were similar. However, higher concentrations of the tyrphostins were required to inhibit constitutive proliferation of the T cell line indicating that inhibition of IL-2 secretion was not secondary to nonspecific toxic effects of the tyrphostins. Addition of the tyrphostins after mitogenic lectin decreased the amount of tyrosine phosphorylation and IL-2 secretion in parallel. This indicates that both tyrosine kinases and phosphatases are activated and that continuous tyrosine phosphorylation is likely required for IL-2 secretion. Therefore, tyrosine phosphorylation appears to represent an obligatory event in the transmembrane signaling processes that lead to IL-2 secretion.
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PMID:Tyrosine phosphorylation is an obligatory event in IL-2 secretion. 169 78

Pertussis toxin is an ADP-ribosyltransferase which alters the function of some of the GTP-binding proteins and inhibits some actions of insulin. In vivo, pertussis toxin (2 micrograms/ml/2h) inhibited insulin-stimulated tyrosyl autophosphorylation of the insulin receptor by 50% in FaO cells, and nearly completely inhibited phosphorylation of the cellular insulin receptor substrate pp185. Similarly, insulin-stimulated autophosphorylation and kinase activity of the insulin receptor purified on wheat germ agglutinin-agarose from pertussis toxin-treated FaO cells was diminished 50%; however, treatment of cells with the catalytically inactive B-oligomer of the toxin had no effect on receptor tyrosine kinase activity in vitro. Pertussis toxin did not alter insulin binding or the cellular levels of ATP, cAMP, and cGMP. Furthermore, immunoprecipitation of the insulin receptor from intact cells with anti-insulin receptor antibodies showed that pertussis toxin did not increase the phosphorylation of serine or threonine residues in the insulin receptor. These results suggest that pertussis toxin can modulate signal transduction of insulin at the level of the insulin receptor kinase.
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PMID:Pertussis toxin inhibits autophosphorylation and activation of the insulin receptor kinase. 172 5

Mast cells appear to promote fibroblast proliferation, presumably through secretion of growth factors, although the molecular mechanisms underlying this mitogenic potential have not been explained fully by known mast cell-derived mediators. We report here that tryptase, a trypsin-like serine proteinase of mast cell secretory granules, is a potent mitogen for fibroblasts in vitro. Nanomolar concentrations of dog tryptase strongly stimulate thymidine incorporation in Chinese hamster lung and Rat-1 fibroblasts and increase cell density in both subconfluent and confluent cultures of these cell lines. Tryptase-induced cell proliferation appears proteinase-specific, as this response is not mimicked by pancreatic trypsin or mast cell chymase. In addition, low levels of tryptase markedly potentiate DNA synthesis stimulated by epidermal growth factor, basic fibroblast growth factor, or insulin. Inhibitors of catalytic activity decrease the mitogenic capacity of tryptase, suggesting, though not proving, the participation of the catalytic site in cell activation by tryptase. Differences in Ca++ mobilization and sensitivity to pertussis toxin suggest that tryptase and thrombin activate distinct signal transduction pathways in fibroblasts. These data implicate mast cell tryptase as a potent, previously unrecognized fibroblast growth factor, and may provide a molecular link between mast cell activation and fibrosis.
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PMID:Mast cell tryptase is a mitogen for cultured fibroblasts. 186 60

We transfected COS cells with expression vectors for the wild-type G protein alpha i1 subunit (pWT) and for mutated alpha i1 subunits, including the nonmyristylated glycine 2 to alanine mutant (pGA) and mutants in which the carboxyl termini of pWT and pGA were changed from CGLF to CVLS (pCVLS and pGA-CVLS, respectively). Immunoblot analysis of transfected COS cells with an antibody to residues 159-168 of the alpha i1 protein indicated that all four proteins were expressed. Unlike the WT and GA proteins, both CVLS mutant proteins failed to react with an antibody specific for the carboxyl terminus and failed to undergo pertussis toxin-catalyzed ADP-ribosylation. Analysis of COS cell lysates after [3H]mevalonic acid labeling indicated that specific incorporation of radioactivity occurred only in the alpha i1 subunits with the CVLS mutation. Immuno-precipitation of COS cell fractions after labeling with [35S]methionine indicated that both WT and CVLS mutant proteins were localized predominantly in the particulate fraction, whereas GA and GA-CVLS mutant proteins were found primarily in the soluble fraction. These results directly demonstrate that the carboxyl-terminal sequence, CGLF, is incapable of leading to isoprenylation but that alteration of two residues (glycine to valine, phenylalanine to serine) is sufficient to promote isoprenylation.
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PMID:Isoprenylation of an inhibitory G protein alpha subunit occurs only upon mutagenesis of the carboxyl terminus. 212 86

The S1 subunit (Mr 28,000) of pertussis toxin expresses thiol-dependent enzymatic ADP-ribosyltransferase and NAD-glycohydrolase activities. Site-directed mutagenesis experiments were performed on the codon for Cys-41 of this subunit to investigate the role of this residue in both enzymatic activities. Deletion of Cys-41 caused a decrease in both activities below detectable levels, whereas replacement of this residue by serine, glycine, proline, or asparagine only slightly reduced the activities. The enzymatic activities of these mutants were thiol-independent. The deletion of Ser-40, adjacent to Cys-41, again caused reduction of the enzymatic activities to undetectable levels. Steady-state kinetic experiments showed that the kcat of the mutant protein in which Cys-41 was replaced by glycine was nearly identical to the kcat of the parent version. However, the Km for NAD of the mutant was significantly higher relative to that of the wild type version. These results indicate that the side-chain of Cys-41 is not essential for enzymatic activities and that Cys-41 is not involved in the rate of catalysis but is probably located at or close to the NAD-binding site. The introduction of a negative charge at position 41 through the replacement of Cys-41 by either aspartate or glutamate reduced the enzymatic activities to very low but measurable levels, suggesting a charge-charge repulsive interaction between these residues and possibly one or both of the phosphates of NAD. Cys-41 may therefore be located close to the phosphate subsite of the NAD-binding site.
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PMID:The role of cysteine 41 in the enzymatic activities of the pertussis toxin S1 subunit as investigated by site-directed mutagenesis. 215 32


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