Academic literature on the topic 'Nucleotides binding/release'
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Journal articles on the topic "Nucleotides binding/release"
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MISSIAEN, Ludwig, Jan B. PARYS, Humbert DE SMEDT, Ilse SIENAERT, Henk SIPMA, Sara VANLINGEN, Karlien MAES, and Rik CASTEELS. "Effect of adenine nucleotides on myo-inositol-1,4,5-trisphosphate-induced calcium release." Biochemical Journal 325, no. 3 (August 1, 1997): 661–66. http://dx.doi.org/10.1042/bj3250661.
Full textAbstract:
The effects of a whole series of adenine nucleotides on Ins(1,4,5)P3-induced Ca2+ release were characterized in permeabilized A7r5 smooth-muscle cells. Several adenine nucleotides activated the Ins(1,4,5)P3 receptor. It was observed that 3′-phosphoadenosine 5′-phosphosulphate, CoA, di(adenosine-5′)tetraphosphate (Ap4A) and di(adenosine-5′)pentaphosphate (Ap5A) were more effective than ATP. Ap4A and Ap5A also interacted with a lower EC50 than ATP. In order to find out how these adenine nucleotides affected Ins(1,4,5)P3-induced Ca2+ release, we have measured their effect on the response of permeabilized A7r5 cells to a progressively increasing Ins(1,4,5)P3 concentration. Stimulatory ATP and Ap5A concentrations had no effect on the threshold Ins(1,4,5)P3 concentration for initiating Ca2+ release, but they stimulated Ca2+ release in the presence of supra-threshold Ins(1,4,5)P3 concentrations by increasing the co-operativity of the release process. Inhibition of the Ins(1,4,5)P3-induced Ca2+ release at higher ATP concentrations was associated with a further increase in co-operativity and also with a shift in threshold towards higher Ins(1,4,5)P3 concentrations. ATP had no effect on the non-specific Ca2+ leak in the absence of Ins(1,4,5)P3. We conclude that the adenine-nucleotide-binding site can be activated by many different adenine nucleotides. Binding of these compounds to the transducing domain of the Ins(1,4,5)P3 receptor increases the efficiency of transmitting Ins(1,4,5)P3 binding to channel opening. The inhibition by high ATP concentrations is exerted at a different site, related to Ins(1,4,5)P3 binding.
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Volkán-Kacsó, Sándor, and Rudolph A. Marcus. "Theory of single-molecule controlled rotation experiments, predictions, tests, and comparison with stalling experiments in F1-ATPase." Proceedings of the National Academy of Sciences 113, no. 43 (October 10, 2016): 12029–34. http://dx.doi.org/10.1073/pnas.1611601113.
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A recently proposed chemomechanical group transfer theory of rotary biomolecular motors is applied to treat single-molecule controlled rotation experiments. In these experiments, single-molecule fluorescence is used to measure the binding and release rate constants of nucleotides by monitoring the occupancy of binding sites. It is shown how missed events of nucleotide binding and release in these experiments can be corrected using theory, with F1-ATP synthase as an example. The missed events are significant when the reverse rate is very fast. Using the theory the actual rate constants in the controlled rotation experiments and the corrections are predicted from independent data, including other single-molecule rotation and ensemble biochemical experiments. The effective torsional elastic constant is found to depend on the binding/releasing nucleotide, and it is smaller for ADP than for ATP. There is a good agreement, with no adjustable parameters, between the theoretical and experimental results of controlled rotation experiments and stalling experiments, for the range of angles where the data overlap. This agreement is perhaps all the more surprising because it occurs even though the binding and release of fluorescent nucleotides is monitored at single-site occupancy concentrations, whereas the stalling and free rotation experiments have multiple-site occupancy.
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Beslin, A., M. P. Vié, J. P. Blondeau, and J. Francon. "Identification by photoaffinity labelling of a pyridine nucleotide-dependent tri-iodothyronine-binding protein in the cytosol of cultured astroglial cells." Biochemical Journal 305, no. 3 (February 1, 1995): 729–37. http://dx.doi.org/10.1042/bj3050729.
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High-affinity 3,3′,5-tri-iodo-L-thyronine (T3) binding (Kd approximately 0.3 nM) to the cytosol of cultured rat astroglial cells was strongly activated in the presence of pyridine nucleotides. A 35 kDa pyridine nucleotide-dependent T3-binding polypeptide (35K-TBP) was photoaffinity labelled using underivatized [125I]T3 in the presence of pyridine nucleotides and the free-radical scavenger dithiothreitol. Maximum activations of T3 binding and 35K-TBP photolabelling were obtained at approx. 1 x 10(-7) M NADP+ or NADPH, or 1 x 10(-4) M NADH. NAD+ and other nucleotides were without effect. NADPH is the form which activates T3 binding and 35K-TBP photolabelling, since cytosol contains NADP(+)-reducing activity, and the activation of both processes in the presence of NADPH and NADP+ was prevented by an exogenous NADPH oxidation system. NADPH behaved as an allosteric activator of T3 binding. The NADPH oxidation system promoted the release of bound T3 in the absence of any change in the total concentration of the hormone. The 35K-TBP photolabelling and [125I]T3 binding were similarly inhibited by non-radioactive T3 (half-maximum effect at 0.5-1.0 nM T3). The concentrations of iodothyronine analogues that inhibited both processes were correlated (3,3′,5-tri-iodo-D-thyronine > or = T3 > L-thyroxine > tri-iodothyroacetic acid > 3,3′5′-tri-iodo-L-thyronine). Molecular sieving and density-gradient centrifugation of cytosol identified a 65 kDa T3-binding entity, which included the 35K-TBP. These results indicate that 35K-TBP is the cytosolic entity involved in the pyridine nucleotide-dependent T3 binding, and suggest that the sequestration and release of intracellular thyroid hormones are regulated by the redox state of astroglial cell compartment(s).
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Dawicki, D. D., J. McGowan-Jordan, S. Bullard, S. Pond, and S. Rounds. "Extracellular nucleotides stimulate leukocyte adherence to cultured pulmonary artery endothelial cells." American Journal of Physiology-Lung Cellular and Molecular Physiology 268, no. 4 (April 1, 1995): L666—L673. http://dx.doi.org/10.1152/ajplung.1995.268.4.l666.
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Adenosine, ATP, and various nucleotides were examined for their effects on the adherence of leukocytes to bovine pulmonary artery endothelial cells. Extracellular ATP enhanced adherence of HL-60 cells and human neutrophils to endothelial cells in a dose-dependent fashion. Maximal adherence occurred after 15 min coincubation of ATP and HL-60 cells or neutrophils with endothelial cells. ATP stimulation was mediated by direct effects on both HL-60 cells and endothelial cells. The potency profile of various nucleotides was ATP = 2-MeSATP > beta,gamma-CH2ATP, indicative of a P2y receptor. Interestingly, UTP was as potent as ATP in stimulating HL-60 cell adherence, suggesting the presence of a pyrimidine nucleotide receptor. Photoaffinity labeling of endothelial cells with 8-Az-[alpha-32P]ATP showed the presence of two ATP binding proteins of 48 and 87 kDa. ATP and 2-MeSATP inhibited binding by both proteins. Labeling of the 87-kDa protein was inhibited by beta,gamma-CH2ATP, whereas UTP blocked binding by the 48-kDa protein. Thus photoaffinity labeling experiments support the proposal that endothelial cells possess two ATP receptors, one of which is a P2u nucleotide receptor. These findings show that extracellular nucleotides enhance leukocyte adherence to endothelial cells. Nucleotide release into the extracellular space may be one mechanism of exacerbating vascular cell injury relevant to conditions such as adult respiratory distress syndrome and septic shock.
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Jackson, A. P., and C. R. Bagshaw. "Transient-kinetic studies of the adenosine triphosphatase activity of scallop heavy meromyosin." Biochemical Journal 251, no. 2 (April 15, 1988): 515–26. http://dx.doi.org/10.1042/bj2510515.
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Fluorescence stopped-flow experiments were performed to elucidate the elementary steps of the ATPase mechanism of scallop heavy meromyosin in the presence and in the absence of Ca2+. ATP binding and hydrolysis, as monitored by the change in tryptophan fluorescence, appear to be Ca2+-insensitive, whereas both Pi release and ADP release are markedly suppressed in the absence of Ca2+. Rate constants for Pi release are 0.2 s-1 and 0.002 s-1 and for ADP release are 6 s-1 and 0.01 s-1 in the presence and in the absence of Ca2+ respectively. Ca2+ binding to the specific site of the regulatory domain is rapid and its release occurs at 25 s-1, consistent with the time scale of a twitch of the striated adductor muscle. Nucleotide binding is a multi-step process requiring a minimum of three states. In such a model Ca2+ controls the rate of conformational changes at the active site in both the forward and the reverse direction, leading to a large dependence of the rate of nucleotide release, but a lesser effect on the overall equilibrium position. The kinetic trapping of nucleotides and Pi at the active site, in the absence of Ca2+, appears to be a fundamental step in suppressing the interaction of the myosin head with the thin filaments in relaxed molluscan muscle.
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Batasheva, Svetlana, Marina Kryuchkova, Ramil Fakhrullin, Giuseppe Cavallaro, Giuseppe Lazzara, Farida Akhatova, Läysän Nigamatzyanova, Vladimir Evtugyn, Elvira Rozhina, and Rawil Fakhrullin. "Facile Fabrication of Natural Polyelectrolyte-Nanoclay Composites: Halloysite Nanotubes, Nucleotides and DNA Study." Molecules 25, no. 15 (August 4, 2020): 3557. http://dx.doi.org/10.3390/molecules25153557.
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Complexation of biopolymers with halloysite nanotubes (HNTs) can greatly affect their applicability as materials building blocks. Here we have performed a systematic investigation of fabrication of halloysite nanotubes complexes with nucleotides and genomic DNA. The binding of DNA and various nucleotide species (polyAU, UMP Na2, ADP Na3, dATP Na, AMP, uridine, ATP Mg) by halloysite nanotubes was tested using UV-spectroscopy. The study revealed that binding of different nucleotides to the nanoclay varied but was low both in the presence and absence of MgCl2, while MgCl2 facilitated significantly the binding of longer molecules such as DNA and polyAU. Modification of the nanotubes with DNA and nucleotide species was further confirmed by measurements of ζ-potentials. DNA-Mg-modified nanotubes were characterized using transmission electron (TEM), atomic force (AFM) and hyperspectral microscopies. Thermogravimetric analysis corroborated the sorption of DNA by the nanotubes, and the presence of DNA on the nanotube surface was indicated by changes in the surface adhesion force measured by AFM. DNA bound by halloysite in the presence of MgCl2 could be partially released after addition of phosphate buffered saline. DNA binding and release from halloysite nanotubes was tested in the range of MgCl2 concentrations (10–100 mM). Even low MgCl2 concentrations significantly increased DNA sorption to halloysite, and the binding was leveled off at about 60 mM. DNA-Mg-modified halloysite nanotubes were used for obtaining a regular pattern on a glass surface by evaporation induced self-assembly process. The obtained spiral-like pattern was highly stable and resisted dissolution after water addition. Our results encompassing modification of non-toxic clay nanotubes with a natural polyanion DNA will find applications for construction of gene delivery vehicles and for halloysite self-assembly on various surfaces (such as skin or hair).
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MISSIAEN, Ludwig, B. Jan PARYS, Humbert DE SMEDT, Ilse SIENAERT, Henk SIPMA, Sara VANLINGEN, Karlien MAES, Karl KUNZELMANN, and Rik CASTEELS. "Inhibition of inositol trisphosphate-induced calcium release by cyclicADP-ribose in A7r5 smooth-muscle cells and in 16HBE14o- bronchial mucosal cells." Biochemical Journal 329, no. 3 (February 1, 1998): 489–95. http://dx.doi.org/10.1042/bj3290489.
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Ca2+ release from intracellular stores occurs via two families of intracellular channels, each with their own specific agonist: Ins(1,4,5)P3 for the Ins(1,4,5)P3 receptor and cyclic ADP-ribose (cADPR) for the ryanodine receptor. We now report that cADPR inhibited Ins(1,4,5)P3-induced Ca2+ release in permeabilized A7r5 cells with an IC50 of 20 μM, and in permeabilized 16HBE14o- bronchial mucosal cells with an IC50 of 35 μM. This inhibition was accompanied by an increase in specific [3H]Ins(1,4,5)P3 binding. 8-Amino-cADPR, but not 8-bromo-cADPR, antagonized this effect of cADPR. The inhibition was prevented by a whole series of inositol phosphates (10 μM) that did not affect Ins(1,4,5)P3-induced Ca2+ release, and by micromolar concentrations of PPi and various nucleotide di- or triphosphates. We propose that cADPR must interact with a novel regulatory site on the Ins(1,4,5)P3 receptor or on an associated protein. This site is neither the Ins(1,4,5)P3-binding domain, which prefers Ins(1,4,5)P3 and only binds nucleotides and PPi in the millimolar range, nor the stimulatory adenine nucleotide binding site.
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Bokoch, G. M., and L. A. Quilliam. "Guanine nucleotide binding properties of rap1 purified from human neutrophils." Biochemical Journal 267, no. 2 (April 15, 1990): 407–11. http://dx.doi.org/10.1042/bj2670407.
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The guanine nucleotide binding properties of rap1 protein purified from human neutrophils were examined using both the protein kinase A-phosphorylated and the non-phosphorylated forms of the protein. Binding of GTP[S] (guanosine 5′-[gamma-thio]triphosphate) or GDP was found to be slow in the presence of free Mg2+, but very rapid in the absence of Mg2+. The binding of guanine nucleotides was found to correlate with the loss of endogenous nucleotide from the rap1 protein, which was rapid in the absence of Mg2+. The relative affinities of GTP and GDP for the binding site on rap1 were modulated by the presence of Mg2+, with a preferential affinity (approx. 15-fold) for GTP observed only in the absence of this bivalent cation. The dissociation of GDP from rap1 was not affected by the G-protein beta/gamma-subunit complex. Phosphorylation of rap1 in vitro by protein kinase A did not modify any of the observed nucleotide-binding parameters. Furthermore, the ability of a cytosolic rap1 GTPase-activating protein to stimulate neutrophil rap1 GTP hydrolysis was not modified by phosphorylation. These data suggest that the activation of rap in vivo may be regulated by the release of endogenous GDP, but that phosphorylation by protein kinase A does not affect guanine nucleotide binding or hydrolysis.
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Milligan, G., and C. G. Unson. "Persistent activation of the α subunit of Gs promotes its removal from the plasma membrane." Biochemical Journal 260, no. 3 (June 15, 1989): 837–41. http://dx.doi.org/10.1042/bj2600837.
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As assessed both by cholera-toxin-catalysed ADP-ribosylation and by immunoblotting with an anti-peptide antiserum raised against the C-terminal decapeptide of forms of Gs alpha (the alpha subunit of the stimulatory guanine nucleotide-binding protein), rat glioma C6 BU1 cells express two forms of Gs alpha: a major 44 kDa form and a much less prevalent 42 kDa form. We examined the effects of guanine nucleotides on the interaction of the 44 kDa form with the plasma membrane. Incubation of membranes of C6 BU1 cells with poorly hydrolysed analogues of GTP, but not with analogues of either ATP or GDP, caused the release of this Gs alpha from the membrane fraction. Release of Gs alpha was observed within 5 min, and continued throughout the incubation period. After treatment with guanosine 5′-[beta gamma-imido]triphosphate for 60 min, some 75% of this polypeptide had been released from its site of membrane attachment. These experiments demonstrate that Gs alpha need not remain associated invariantly with the plasma membrane.
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Yao, Jia, and Sandra M. Bajjalieh. "Synaptic Vesicle Protein 2 (SV2) does not hydrolyze ATP." F1000Research 2 (October 9, 2013): 209. http://dx.doi.org/10.12688/f1000research.2-209.v1.
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Synaptic vesicle protein 2 (SV2) is a transporter-like protein specifically expressed in endocrine cells and neurons, where it is localized to vesicles that undergo regulated secretion and plays an essential role in regulating neurotransmitter release. SV2 binds adenine nucleotides including ATP. Analysis of ATP transport revealed that SV2 is not an ATP transporter, nor does it affect ATP transport. As a further step toward understanding how ATP binding contributes to SV2 function, we investigated whether SV2 is an ATPase using an in vitro measure of ATPase activity. The study reported here indicates that SV2 does not have ATPase activity. Thus, binding to adenine nucleotides likely modulates other actions of SV2.
Dissertations / Theses on the topic "Nucleotides binding/release"
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Aung-Htut, May Thandar Biotechnology & Biomolecular Sciences Faculty of Science UNSW. "Characterisation of Escherichia coli GTPase Der reveals previously unknown regulation by RNA." Publisher:University of New South Wales. Biotechnology & Biomolecular Sciences, 2008. http://handle.unsw.edu.au/1959.4/41840.
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GTPases are found in all domains of life and are highly conserved. In eukaryotes, they serve as signalling molecules for many cellular processes. However, the prokaryotic GTPases play a very different role and are found to be associated with ribosome function. Among the 11 conserved GTPases, Der is the most interesting in prokaryotes. It possesses a unique structure with two GTPase domains (G-Domains) tethered by a variable length acidic linker and a carboxyl terminal KH-like domain. The exact function of Der is still under investigation and most of the data suggest that it is important for 50S ribosomal assembly or stability. In order to investigate the function of Escherichia coli Der (Ec-Der), expression plasmids for wild-type and mutated proteins were created and the proteins were successfully expressed. The expression of the mutant protein that lacked G-Domain 1 was toxic to the cells and it was found that some large ribosomal proteins were missing from the ribosomes of these cells. In addition, other macromolecular complexes such as the GroEL/GroES chaperonin appeared not to be assembled under these conditions. The activities of both wild-type and mutated proteins were also tested and found to be dependent on potassium ions (K+), which enhanced nucleotide binding. Additionally, intra-molecular control over nucleotide binding and release was also observed for Ec-Der. The in vitro selection of RNA aptamers with nanomolar affinity for Ec-Der produced aptamers that contained short variable sequences. These aptamers affected the growth of the E. coli cells and caused a change in cellular morphology that had been noted previously during Ec-Der over-expression. Ec-Der showed high affinity (nM) to both selected RNA and the unselected RNA library. The activity of Ec-Der and Era was inhibited in the presence of any sequence of RNA that has the length of greater than 16 nucleotides. RNA was also cross-linked to Ec-Der in the presence of GTP, but not GDP, suggesting that RNA was a regulator of the Ec-Der GTPase cycle. Based on these results, it is speculated that Ec-Der might be involved in more than one function. It may be acting at the level of the membrane (based on cellular morphology reported here and by Hwang and Inouye 2001) and may also take part in processes related to ribosome function. Regulation of protein activity by RNA length has not been predicted or described and this may represent a novel mean of regulation of the Era subfamily of GTPases.
Books on the topic "Nucleotides binding/release"
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Stilwell, Shaun Nicholas. The involvement of NADP(H) binding and release in energy transduction by proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli and Rhodospirillum rubrum. Birmingham: University of Birmingham, 1997.
Find full textBook chapters on the topic "Nucleotides binding/release"
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"Crk SH3 Domain Binding Guanine Nucleotide Release Protein (C3G)." In Encyclopedia of Signaling Molecules, 1209. Cham: Springer International Publishing, 2018. http://dx.doi.org/10.1007/978-3-319-67199-4_100838.
Full textConference papers on the topic "Nucleotides binding/release"
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Hardisty, R. M., A. Pannocchia, N. Mahmood, T. J. C. Nokes, D. Pidard, C. Bouillot, C. Legrand, and A. T. Nurden. "PARTIAL PLATELET FUNCTION DEFECT IN A VARIANT OF GLANZMANN'S THROMBASTHENIA WITH INTERMEDIATE LEVELS OF GP IIb/IIIa." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644743.
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A 17-year-old Italian boy has had a lifelong bleeding tendency, with frequent epistaxes and gum bleeding. The bleeding time is prolonged and the platelet count low normal. Electron microscopy showed a wide diversity of platelet size with many giant forms. In citrated PRP, ADP and other agonists induce slow and incomplete aggregation. The response of washed platelets varied with the agonist but ranged from subnormal to almost normal. Fibrinogen binding to washed platelets occurred slowly in response to ADP but eventually approached normal levels. No significant abnormality was observed of 5HT uptake, adenine nucleotide content, platelet factor-3 availability, β-thromboglobulin content or release, or malonyldialdehyde production. Clot retraction was normal. SDS-PAGE showed reduced amounts of GPIIb and GPU Ia. Crossed immunoelectrophoresis of Triton X-100 extracts of washed platelets showed the presence of GPIIb/IIIa complexes at 25-50% of normal levels. SDS-PAGE combined with an immunoblot procedure confirmed unchanged mobilities of GPIIb and GPIIIa and a normal proportion of GPIIb to GPIIIa. However, binding studies with radiolabelled monoclonal antibodies showed that intact washed platelets expressed only 12-20% of the normal binding sites for M148, AP-2 and Tab. These antibodies recognize different epitopes on GPIIb/lIIa complexes. Similar levels of these glycoproteins were detected by autoradiography after SDS-PAGE of radio-iodinated patient's platelets. GP lb was normally present. A possible defect in the exposure of fibrinogen binding sites might contribute to the altered platelet function. Meanwhile, the patient appears to be a unique variant of Glanzmann's thrombasthenia with GP IIb/IIIa complexes at the borderline of those able to support platelet aggregation.
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Kienast, J., J. Arnout, G. Pfliegler, H. Deckmyn, E. Van Houtte, and J. Vermylen. "DUAL EFFECT OF FLUORIDE ON ENDOTHELIAL PROSTACYCLIN PRODUCTION." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643377.
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The effects of fluoride at mM concentrations on prostacyclin (PGI2) production by human umbilical vein endothelial cells in culture were investigated. At lower concentrations, with a peak activity around 20-30 mM, sodium fluoride (NaF) caused a slow rise in PGI2 release reaching a maximum of 6 to 8 × control values after 1 hour. With increasing concentrations, the NaF-induced PGI2 production progressively decreased to almost control levels at 100 mM. Preincubation of the cells with 100 mM NaF inhibited thrombin— and A23187-stimulated PGI2 production by 46 % and 64 % respectively. The response to NaF was not significantly enhanced by AlCl3 nor was it inhibited by pertussis toxin.The dual, stimulatory and inhibitory, effect of NaF on endothelial PGI2 production as well as its slow mode of action resemble the stimulus-response pattern observed in platelets upon exposure to NaF as it has previously been described by our grcup. Therefore, NaF might be an interesting tool for the investigation of intracellular regulatory processes triggering endothelial PGI2 production, particularly in view of the known capacity of fluoride to activate guanine nucleotide—binding regulatory proteins.
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Lapetina, Eduardo G. "THE ROLE OF INOSITIDES, PHOSPHOLIPASE C AND G-PROTEINS IN RECEPTOR TRANSDUCTION." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644775.
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It is now widely recognized that the activation of phospholipase C by specific agonists leads to the formation of two second messengers: (1) inositol trisphosphate, which releases Ca2+ from the endoplasmic reticulum to the cytosol and (2) 1,2- diacylglycerol, which stimulates protein kinase C. In the past few years, GTP-binding proteins have been associated with the regulation of phospholipase C. However, the identity of the GTP-binding protein involved and the type of association with phospholipase C is not yet known. It is now recognized that there are two types of phospholipase C enzymes: (a) a soluble enzyme that has been characterized in several tissues and does not preferentially hydrolyze polyphospholinositides and (b) membrane-bound enzymes that are coupled to the receptors, specifically hydrolyzing polyphosphoinositides and activated by membrane guanine nucleotide-binding proteins. Recent reports have tried to assess the involvement of GTP-binding proteins in the agonist-induced stimulation of phospholipase C, and various related aspects have been reported. These are concerned with: (a) detection of various GTP-binding proteins in platelets, (b) the effects of known inhibitors of GTP-binding proteins such as GDPgS or pertussis toxin on the agonist-induced stimulation of phospholipase C, (c) the direct effects of stimulators of GTP-binding proteins such as GTP, GTP-analogs and fluoride on phospholipase C activity, (d) the possible association of GTP-binding proteins to cytosolic phospholipase C that would then lead to degradation of the membrane-bound inositides and (e) cytosolic phospholipase C response to the activation of cell surface receptors. The emerging information has had contradictory conclusions. (1) Pretreatment of saponin-permeabilized platelets with pertussis toxin has been shown to enhance and to inhibit the thrombin-induced activation of phospholipase C. Therefore, it is not clear if a G protein that is affected by pertussis toxin in a manner similar to Gi or Go plays a central role in activation of phospholipase C. (2) Studies on the effect of GDPβ;S are also conflicting indicating that there may be GTP-independent and/or -dependent pathways for the activation of phosphoinositide hydrolysis. (3) A cytosolic phospholipase C is activated by GTP, and it has been advanced that this activity might trigger the hydrolysis of membrane-bound inositides. A cytosolic GTP-binding protein might be involved in this action, and it is speculated that an α-subunit might be released to the cytoplasm by a receptor-coupled mechanism to activate phospholipase C. However, no direct evidence exists to support this conclusion. Moreover, the exact contribution of phospholipase C from the membranes or the cytosol to inositide hydrolysis in response to cellular agonists and the relationship of those activites to membrane-bound or soluble GTP-binding proteins are unknown. Our results indicate that the stimulation of phospholipase C in platelets by GDPβS and thrombin are affected differently by GDPβS. GDPgSinhibits the formation of inositol phosphates produced by GTPγS but not that induced by thrombin. Thrombin, therefore, can directly stimulate phospholipase C without the involvement of a “stimulatory” GTP-binding protein, such as Gs, for the agonist stimulation of adenylate cyclase. However, an “inhibitory” GTP-binding protein might have some influence on thrombin-stimulated phospholipase C, since in the presence of GDPγS thrombin produces a more profound stimulation of phospholipase C.This “inhibitory” GTP-binding protein might be ADP-ribosylated by pertussis toxin because pertussis toxin can also enhance thrombin action on phospholipase C activity. Therefore, phospholipase C that responds to thrombin could be different from the one that responds to GTPγS. Cytosolic phospholipase C can be activated by GTP or GTP analogs, and the one that responds to thrombin should be coupled to the receptors present in the plasma membrane. The initial action of thrombin is to directly activate the plasma membrane-bound phospholipase C and the mechanism of this activation is probably related to the proteolytic action of thrombin or the activation of platelet proteases by thrombin. In agreement with this, trypsin can also directly activate platelet phospholipase C and, subsequently, GTPyS produces further activation of phospholipase C. If these two mechanisms are operative in platelets, the inhibition of cytosolic phospholipase C by GDPβS would allow a larger fraction of inositides for degradation of the thrombin-stimulated phospholipase C, as our results show.
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Ortega, M. P., C. Sunkel, and J. G. Priego. "INHIBITION OF HUMAN PLATELET FUNCTION BY PCA-4230." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643431.
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PCA-4230 (3-{2-(N-l,2-benzisothiazolyl-3(2H)one-1,1-dioxide) ethoxycarbonyl}-2,6-dimethyl-5-ethoxycarbonyl-4-methyl-l,4-dihy-dropyridine) is a new synthetic compound which has been selected after evaluation of .several series of molecules included in an ex tensive program of synthesis and biological screening.The purpose of this study was to investigate the In vUjiO effects of PCA-4230 on human platelet function.Platelet aggregation (PA) was measured, in platelet rich plasma (PRP) or washed platelets, according to Born’s technique. Release reaction (RR) was measured by the luminiscence method as adenosine triphosphate (ATP) release in response to stimulation. Aggregating agents were adenosine diphosphate (ADP), epinephrine (Epi), collagen (Col), thrombin (Thr), calcium ionophore (A23187) arachidonic acid (AA), thromboxane agonist (U46619) or platelet activating factor (PAF). Incubation with PCA-4230 (1 to 10 μM) we re carried out at 37°C for 15 minutes. PCA-4230 potentiation of Prostacyclin (PGI2) anti-aggregatory activity was also studied by addition to PRP of PGI2, PCA-4230 or both, and PA by ADP.PCA-4230 inhibited PA and RR in PRP in a concentration-dependent fashion when Col, Epi, U46619 or PAF were used as agonists. AA-and Thr-induced aggregation were only slightly impaired and no in hibition was shown on ADP or A23187-triggered activation. A23187-induced aggregation and RR were inhibited only in the absence of extracellular Ca++ in washed platelets. This effect was overcome by addition of Ca++ 1 mM. Additionally, the inhibitory effect of PGI2 on ADP-induced PA, was synergistically potentiated by PCA-4230, suggesting inhibitory activity of the compound on cjy clic nucleotide phosphodiesterase.Since PCA-4230 inhibited PA and RR induced by Epi, U46619 and PAF, mediated viareceptor-agonist binding, and Col-inducedactivation, it is reasonable to suspect that common process(es) may be involved. Recently, it has been suggested that intraplatelet Ca++ levels, actingas a second messenger, are directly linked to the d<2 gree of platelet activation.Therefore, the ability of PCA-4230 to modulate platelet function appears, at leastin part, to be due to regulation of cytosolic Ca++ levels. This hypothesis is confii: med by the results with A23187-induced aggregation in absence of extracellular Ca++.
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Authi, K. S., B. J. Evenden, and N. Crawford. "ACTION OF GTPγS [GUANOSINE 5∲-0-(3-THIOPHOSPHATE)] ON SAPONIN-PERMEABILISED PLATELETS: INVOLVEMENT OF 'G' PROTEINS IN PLATELET ACTIVATION." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644514.
Full textAbstract:
Certain ligand-receptor interactions at cell surfaces lead to the phospholipase-C (PLC) hydrolysis of phosphatidyl inositol (4.5) bisphosphate (PIP2). The products serve as intracellular second messengers, e.g. inositol (1.4.5) trisphosphate (IP3) releases Ca2+ from intracellular stores and diacylglycerol activates protein kinase-C. From studies using GTP and analogues (e.g. GTPγS) there is evidence of a key role for a guanine nucleotide binding protein(s) as a link between receptors and PIP2 hydrolysis. We report the actions of GTPγS on washed human platelets permeabilised with saponin (12-14 μg/ml) to allow penetration of low MWt polar substances. The responses to GTPγS are dose dependent (range 9-60 μM) and at 60 μM the agent induces shape change, aggregation and the secretion of 50% of previously incorporated [14C]-5HT. No effect of GTPγS is seen with intact cells. Shape change occurs 25-30 sec after GTPγS; aggregation and secretion is complete after 3 min. When GTP was used (up to 135 μM) with similarly permeabilised platelets no responses were initiated. Phosphatidylinositol turnover was monitored using 32P-labelling before permeabilisation. The addition of 90 μM GTPγS resulted in a 143 ± 23% (n=4) increase in 32P-phosphatidic acid (PA) with respect to the basal levels of “saponised control” cells. These findings suggest that GTPγS stimulates PLC activity through a ‘G’ protein interaction. The GDP analogue (GDPβS) produced no activation responses in saponised platelets but inhibited responses induced by GTPγS in a dose dependent manner (0-480 μM, max inhibition 480 μM). At 960 μM, GDPβS totally inhibited aggregation and secretion initiated by low doses of thrombin (0.1 U/ml) and collagen (1 μg/ml). Identical inhibition by GDPβS of thrombin and collagen-induced activation of intact platelets was observed indicating membrane penetration of this analogue. Shape change effects were not inhibited by GDPSS. The inhibitory effects of GDPSS towards thrombin and collagen induced secretion could be progressively overcome at higher doses of thrombin (0.2 U/ml - 2 U/ml) and collagen (5 μg/ml - 60 μg/ml) suggesting that at higher concentrations these agonists may exert effects through 'G' protein-independent mechanisms.