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)

Guanine nucleotide-binding regulatory proteins (G proteins) transduce a remarkably diverse group of extracellular signals to a relatively limited number of intracellular target enzymes. In the neutrophil, transduction of the signal following fMet-Leu-Phe receptor-ligand interaction is mediated by a pertussis toxin substrate (Gi) that activates inositol-specific phospholipase C. We have utilized a plasma membrane-containing fraction from unstimulated human neutrophils as the target enzyme to explore the role of G proteins in arachidonate and cytosolic cofactor-dependent activation of the NADPH-dependent O-2-generating oxidase. When certain guanine nucleotides or their nonhydrolyzable analogues were present during arachidonate and cytosolic cofactor-dependent activation, they exerted substantial dose-dependent effects. The GTP analogue, GTP gamma S, caused a 2-fold increase in NADPH oxidase activation (half-maximal stimulation, 1.1 microM). Either GDP or its nonhydrolyzable analogue, GDP beta S, inhibited up to 80% of the basal NADPH oxidase activation (Ki GDP = 0.12 mM, GDP beta S = 0.23 mM). GTP caused only slight and variable stimulation, whereas F-, an agent known to promote the active conformation of G proteins, caused a 1.6-fold stimulation of NADPH oxidase activation. NADPH oxidase activation in the cell-free system was absolutely and specifically dependent on Mg2+. Although O2- production in response to fMet-Leu-Phe was inhibited greater than 90% in neutrophils pretreated with pertussis toxin, cytosolic cofactor and target oxidase membranes from neutrophils treated with pertussis toxin showed no change in basal- or GTP gamma S-stimulated NADPH oxidase activation. Cholera toxin treatment of neutrophils also had no effect on the cell-free activation system. Our results suggest a role for a G protein that is distinct from Gs or Gi in the arachidonate and cytosolic cofactor-dependent NADPH oxidase cell-free activation system.
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PMID:Regulation of neutrophil NADPH oxidase activation in a cell-free system by guanine nucleotides and fluoride. Evidence for participation of a pertussis and cholera toxin-insensitive G protein. 302 97

We have studied the interaction of guanine nucleotides with alpha 1-adrenergic receptors of two cloned cell lines, the Madin Darby canine kidney (MDCK-D1) cells and BC3H-1 muscle cells. Although guanylylimidodiphosphate, Gpp(NH)p, had no effect on the affinity or the total number of [3H]prazosin-binding sites in membranes prepared from these cells, the nucleotide decreased the apparent affinity of the agonists (-)-epinephrine and (-)-norepinephrine in competing for [3H]prazosin-binding sites in both cell types. A maximal effect of Gpp(NH)p occurred at 10 microM. Guanine nucleotides were significantly more effective in shifting agonist affinity for the alpha 1 receptor than adenine nucleotides, and Mg2+ was required to observe a maximal effect. Binding of agonist to alpha 1-adrenergic receptors activated phosphatidylinositol (PI) hydrolysis in both cell types but had no effect on membrane adenylate cyclase activity. Incubation of MDCK cells for 19 hr with 100 ng/ml pertussis toxin, which eliminated the ability of pertussis toxin added to membranes to ADP-ribosylate 39-41-KDa substrate(s), failed to alter binding of agonists to alpha 1-adrenergic receptors, the ability of Gpp(NH)p to regulate agonist binding to these receptors, or epinephrine-stimulated PI hydrolysis and prostaglandin E2 production. Incubation of BC3H1 cells with pertussis toxin had no effect on the ability of epinephrine to stimulate PI turnover. These results show that binding of agonists to alpha 1-adrenergic receptors in mammalian kidney and muscle cells is regulated by guanine nucleotides, presumably by interaction with a guanine nucleotide-binding (G) protein. The failure of the G-protein to regulate adenylate cyclase activity and the lack of effect of pertussis toxin to alter receptor-mediated binding or functional activity suggests that a G-protein other than Gs, Gi, or Go interacts with alpha 1-adrenergic receptors in kidney and smooth muscle.
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PMID:Alpha 1-adrenergic receptor-linked guanine nucleotide-binding protein in muscle and kidney epithelial cells. 302 23

Alterations in receptor-independent activation of adenylate cyclase during proliferation and differentiation of L6E9 myoblasts were studied using Mn2+, forskolin, and Gpp(NH)p. Analyses were performed 3, 6, and 10 days following subculture, corresponding to onset of proliferation, end of proliferation with start of differentiation, and completion of differentiation, respectively. The apparent activation constant for Mn2+ decreases with the age of the culture; the apparent activation constant for Mg2+ does not. Bimodal activation by Mn2+, i.e., at concentrations greater than 10 mM, results in total adenylate cyclase activity less than the Vmax and occurs exclusively in differentiated cultures. Independent of the presence of Mg2+, forskolin activation occurs with low-and high-affinity constants in differentiated cultures and with a low affinity constant in youngest cultures; intermediate cultures (day 6) demonstrate low- and high-affinity activation only in the presence of high Mg2+. In contrast, the Vmax for forskolin increases with increasing Mg2+ in all culture ages. Although Gpp(NH)p-dependent adenylate cyclase activation occurs with an apparent activation constant independent of culture age and Mg2+, low Mg2+ fosters bimodal activation by Gpp(NH)p, i.e., above 100 microM nucleotide, total adenylate cyclase activity is less than the Vmax. The loss of stimulatory capacity by high Gpp(NH)p is greatest in differentiated cultures. Additional experiments are presented to substantiate that bimodal activation by Gpp(NH)p is specific. Cholera- and pertussis toxin-dependent ADP ribosylation patterns demonstrate a marked decrease in both Ns and Ni in differentiated cultures. The data suggest that alterations in postreceptor activation of adenylate cyclase during the course of differentiation and proliferation are mediated by guanine nucleotide binding proteins as well as by allosteric cation regulatory units.
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PMID:Modifications of the adenylate cyclase complex during differentiation of cultured myoblasts. 308 96

Two proteins serving as substrates for ADP-ribosylation catalyzed by islet-activating protein (IAP), pertussis toxin, and binding guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) with high affinities were purified from the cholate extract of rat brain membranes. The purified proteins had the same heterotrimeric structure (alpha beta gamma) as the IAP substrates previously purified from rabbit liver and bovine brain and differed from each other in alpha only; the molecular weight of alpha was 41,000 (alpha 41 beta gamma) and 39,000 (alpha 39 beta gamma). Both were further resolved into alpha (alpha 41 or alpha 39) and beta gamma which were also purified to homogeneity to compare the activities of alpha-monomers with the original trimers. The maintenance of the rigid trimeric structure by combining alpha 41 or alpha 39 with beta gamma in the absence of Mg2+ was essential for the alpha-subunit to be ADP-ribosylated by IAP. The alpha-subunit was very stable but displayed the only partial GTP gamma S-binding activity under these conditions. Isolated alpha-monomers exhibited high GTPase activities when assayed in the presence of submicromolar Mg2+ but were very unstable at 30 degrees C and not ADP-ribosylated by IAP. The most favorable conditions for the GTP gamma S binding to alpha-subunits were achieved by combining alpha 41 or alpha 39 with beta gamma in the presence of millimolar Mg2+, probably due to the increase in stability and unmasking of the GTP-binding sites. There was no qualitative difference in these properties between alpha 41 beta gamma (alpha 41) and alpha 39 beta gamma (alpha 39). But alpha 39 beta gamma (or alpha 39) was usually more active than alpha 41 beta gamma (or alpha 41), at least partly due to its higher affinity for Mg2+ and lower affinity for beta gamma. Relation of these differences in activity between alpha 41 beta gamma and alpha 39 beta gamma to their physiological roles in signal transduction is discussed.
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PMID:Two guanine nucleotide-binding proteins in rat brain serving as the specific substrate of islet-activating protein, pertussis toxin. Interaction of the alpha-subunits with beta gamma-subunits in development of their biological activities. 308 70

The interaction of nucleotides with pertussis toxin (PT), and their effects on the ability of the toxin to ADP-ribosylate pure Ni, were evaluated. [32P]ATP (10 nM) bound directly to dithiothreitol-activated PT. This binding was competitively inhibited by nucleotides and anions with the following IC50 concentrations in order of decreasing potency: ATP = ATP gamma S (adenosine-5'-O-(3-thiotriphosphate)) = 0.2-0.3 microM, GDP beta S (guanosine-5'-O-(2-thiodiphosphate)) = 2-3 microM, GTP gamma S (guanosine-5'-O-(3-thiotriphosphate)) = 10-15 microM, ADP = 20-25 microM, GTP = 30-40 microM, GMP-P(NH)P (guanyl-5'-yl imidodiphosphate) = 100-150 microM, GDP = 150-200 microM, Pi = SO4(2-) = 20 mM and Cl- = acetate = 30-35 mM. Treatment of PT with ATP, AMP-P(NH)P, GTP, GDP, or GDP beta S, resulted in a stimulated state of NAD+-Ni ADP-ribosyltransferase activity. Addition of ATP, AMP-P(NH)P (adenyl-5'-yl imidodiphosphate), GTP, GDP, and GDP beta S to the ADP-ribosylation reactions resulted in increased rates of ADP-ribosyl-Ni formation. It is concluded that these effects on the nucleotides are due to their action to stimulate the activity of PT. At concentrations of PT between 0.04 and 0.4 microgram/ml, the stimulation of ADP-ribosylation of Ni effected by nucleotides was hysteretic in nature, exhibiting an approximately 25-min long lag when GDP was used as the activating nucleotide. These lags decreased with increasing concentrations of PT, and were abolished by pretreatment of the toxin with GDP or ATP. Preliminary incubation of Ni with GDP had no effect on the lag in its ADP-ribosylation by non-nucleotide treated PT. Addition of divalent cations (Mg2+, Mn2+, and Ca2+) inhibited formation of ADP-ribosyl-Ni, possibly by causing aggregation and denaturation of Ni. This is the first demonstration that both adenine and guanine nucleotides interact directly with PT and act to stimulate its activity to ADP-ribosylate Ni, and that guanine nucleotides do so regardless of whether they are nucleoside di- or triphosphates.
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PMID:The interaction of nucleotides with pertussis toxin. Direct evidence for a nucleotide binding site on the toxin regulating the rate of ADP-ribosylation of Ni, the inhibitory regulatory component of adenylyl cyclase. 309 44

Intact neutrophils exhibit interconverting active and inactive receptor states with half-times for dissociation of 10 s and 2 min, respectively. We examined the effect of guanine nucleotides on ligand-receptor dynamics at 37 degrees C in neutrophils permeabilized with digitonin using continuous fluorometric measurements. The permeabilized cells exhibit a single class of slowly dissociating receptors with a half-time similar to the inactive state. The slowly dissociating state is lengthened in the presence of 10 mM by Mg2+ about two-fold but is relatively insensitive to substitutions of Na+ or K+. When guanine nucleotide is added the receptors dissociate uniformly with a half-time similar to the active state but are sensitive to the substitution of Na+ or K+ (K+ or K+/Mg2+ approximately 10 s; Na+ or Na+/Mg2+ approximately 4 s). When receptors in permeabilized cells are ADP-ribosylated with pertussis toxin the rapidly dissociating state is detected. In the presence of nonsaturating nucleotide or incomplete ribosylation, complex rates of ligand dissociation intermediate between the active and inactive forms are observed. Micromolar concentrations of Ca2+ block the effect of guanine nucleotide on the receptor. The relationships between ligand-receptor dynamics in intact neutrophils and interconverting states regulated by guanine nucleotides and ions in permeabilized cells are discussed.
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PMID:Regulation of ligand-receptor dynamics by guanine nucleotides. Real-time analysis of interconverting states for the neutrophil formyl peptide receptor. 309 37

In purified preparations of human erythrocyte GTP-binding proteins, we have identified a new substrate for pertussis toxin, which has an apparent molecular mass of 43 kDa by silver and Coomassie Blue staining. Pertussis toxin-catalyzed ADP-ribosylation of the 43-kDa protein is inhibited by Mg2+ ion and this inhibition is relieved by the co-addition of micromolar amounts of guanine nucleotides. GTP affects the ADP-ribosylation with a K value of 0.8 microM. Addition of a 10-fold molar excess of purified beta gamma subunits (Mr = 35,000 beta; and Mr = 7,000 gamma) of other GTP-binding proteins results in a significant decrease in the pertussis toxin-mediated ADP-ribosylation of the 43-kDa protein. Treatment of the GTP-binding proteins with guanosine 5'-O-(thiotriphosphate) and 50 mM MgCl2 resulted in shifting of the 43-kDa protein from 4 S to 2 S on sucrose density gradients. Immunoblotting analysis of the 43-kDa protein with the antiserum A-569, raised against a peptide whose sequence is found in the alpha subunits of all of the known GTP-binding, signal-transducing proteins (Mumby, S. M., Kahn, R. A., Manning, D. R., and Gilman, A. G. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 265-259) showed that the 43-kDa protein is specifically recognized by the common peptide antiserum. A pertussis toxin substrate of similar molecular weight was observed in human erythrocyte membranes, bovine brain membranes, membranes made from the pituitary cell line GH4C1, in partially purified GTP-binding protein preparations of rat liver, and in human neutrophil membranes. Treatment of neutrophils with pertussis toxin prior to preparation of the membranes resulted in abolishment of the radiolabeling of this protein. From these data, we conclude that we have found a new pertussis toxin substrate that is a likely GTP-binding protein.
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PMID:Identification of a new GTP-binding protein. A Mr = 43,000 substrate for pertussis toxin. 311 Jan 56

The role of Mg2+ in the GTP hydrolytic cycle was investigated by using purified subunits (G alpha and G beta, gamma) of the GTP-binding protein isolated from Bufo marinus rod outer segments (ROS). Mg2+ markedly stimulated the rate of GTP and guanosine-5'-O-(3-thiotriphosphate) (GTP gamma-s) binding to G alpha. This effect was especially striking in the presence of very small quantities of illuminated ROS disc membranes. GTP hydrolysis could occur in the absence of Mg2+, and Mg2+ increased the rate of GTP hydrolysis only about 50%. These data indicate that Mg2+ plays a fundamental role in amplification of the photon signal by markedly stimulating the rate of formation of GTP X G alpha complexes by very small amounts of illuminated rhodopsin while producing only a modest increase in the rate of GTP hydrolysis. Following hydrolysis of GTP, GDP X G alpha could reassociate with illuminated or unilluminated ROS disc membranes in the presence or absence of Mg2+. In the absence of guanine nucleotides, release of GDP from G alpha bound to illuminated disc membranes was detected in the presence or absence of Mg2+. Moreover, Mg2+ did not affect the rate of GDP release from membrane-bound G alpha. Illumination of B. marinus crude ROS disc membrane preparations markedly reduced pertussis toxin-mediated ADP-ribosylation of a 39,000 Mr (G alpha) protein in the presence but not in the absence, of Mg2+. Moreover, extensive dialysis of illuminated (but not unilluminated) crude ROS disc membranes against a Mg2+-containing buffer caused a marked reduction in the subsequent ADP-ribosylation of G alpha, even when Mg2+ was not present during the ADP-ribosylation step. This reduction was reversed by the addition of GDP or a GDP analogue (but not GMP or hydrolysis-resistant GTP analogues) during the ADP-ribosylation step. Dialysis of crude ROS disc membrane preparations (illuminated or unilluminated) against a Mg2+ -free buffer did not reduce the subsequent ADP-ribosylation of G alpha. These data indicate that Mg2+, in the presence of photolysed rhodopsin, can stimulate the release of GDP from crude preparations of ROS disc membranes. Four lines of evidence suggest that G alpha and G beta, gamma have Mg2+-binding site(s). When stored at 4 degrees C, in the absence of glycerol, G beta, gamma was more stable in the absence than in the presence of Mg2+.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The GTP-binding protein of rod outer segments. II. An essential role for Mg2+ in signal amplification. 311 Jan 57

The effect of the addition of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), the GTP analog which activates the inhibitory guanine nucleotide-binding regulatory protein of adenylyl cyclase (Ni), on the pertussis toxin-mediated ADP-ribosylation reaction was studied in detail. Two effects were discerned: a stimulation of the ADP-ribosyltransferase activity of the toxin, akin to what was described for ATP and GDP in a previous report (Mattera, R., Codina, J., Sekura, R., and Birnbaumer, L. (1986) J. Biol. Chem. 261, 11173-11179), and a decrease in the ability of Ni to be a substrate for the activated toxin. Both effects were time-dependent with activation of the toxin being somewhat faster than inactivation of Ni. The effect of the addition of GTP gamma S on Ni was readily reversed by excess GDP and attenuated by increasing EDTA in the medium from 0.35 to 10 mM, suggesting dependence on trace concentrations of a divalent cation. It is suggested that this cation is Mg2+ on the basis that low (5-10 nM) concentrations of Mg2+ are needed for the endogenous GTPase activity of Ni (Sunyer, T., Codina, J., and Birnbaumer, L. (1984) J. Biol. Chem. 259, 15447-15451). Sucrose density gradient analysis of the Ni X GTP gamma S complexes with decreased susceptibility to ADP-ribosylation by pertussis toxin showed the same sedimentation parameters as Ni or Ni X GDP complexes, indicating that the molecule of Ni with GTP gamma S bound is heterotrimetric as opposed to dissociated into alpha i X GTP gamma S plus beta X gamma. Thus, these experiments define two conformations of heterotrimeric Ni: one -pt+, ADP-ribosylated by pertussis toxin, and the other pt-, poorly or not ADP-ribosylated by pertussis toxin. This latter, hitherto unrecognized conformation, is stabilized by the addition of strongly activating guanine nucleotides such as GTP gamma S and guanyl-5'-yl imidodiphosphate and should be important in the train of events that lead from an inactive heterotrimeric Ni to a fully active and dissociated Ni.
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PMID:Guanosine 5'-O-(3-thiotriphosphate) reduces ADP-ribosylation of the inhibitory guanine nucleotide-binding regulatory protein of adenylyl cyclase (Ni) by pertussis toxin without causing dissociation of the subunits of Ni. Evidence of existence of heterotrimeric pt+ and pt- conformations of Ni. 311 55

Mastoparan, a peptide toxin from wasp venom, is a nonspecific secretagogue. We show here that mastoparan increases the GTPase activity and the rate of nucleotide binding of several purified GTP-binding regulatory proteins (G proteins) whose function is to couple cell-surface receptors to intracellular mediators. Mastoparan accelerated guanosine-5'-(3-O-thiotriphosphate binding and consequent G protein activation in part by promoting the dissociation of bound GDP, the mechanism by which receptors regulate G proteins. ADP-ribosylation by pertussis toxin, which uncouples receptors from G proteins, selectively inhibited mastoparan-stimulated activation. Like receptors, mastoparan was more potent if the G protein was reconstituted in phospholipid vesicles and was active at micromolar concentrations of Mg2+. The structure of mastoparan in a lipid bilayer is similar to that predicted for a cationic intracellular loop of G protein-coupled receptors. Mastoparan thus displays a novel mode of toxicity by acting directly on G proteins to mimic the role normally played by agonist-liganded receptors.
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PMID:Mastoparan, a peptide toxin from wasp venom, mimics receptors by activating GTP-binding regulatory proteins (G proteins). 312 26

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