Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The addition of bradykinin to NG108-15 cells resulted in an increase in the intracellular Ca2+ concentration [( Ca2+]i) and the formation of inositol monophosphate, inositol bisphosphate, and inositol trisphosphate in these cells. The bradykinin-stimulated formation of inositol polyphosphates in plasma membrane preparations was dependent on the presence of GTP or guanosine-5'-O-thiotriphosphate (GTP gamma S) but not of GDP. GTP gamma S, unlike GTP, increased the basal formation of inositol polyphosphate in NG108-15 membranes. Iontophoretic injection of GTP gamma S into single cells induced increases in [Ca2+]i. These effects of bradykinin and GTP gamma S on [Ca2+]i and the formation of inositol phosphates in the intact cells and membranes were not affected by treatment of the cells with pertussis toxin or cholera toxin. Data on binding of bradykinin to membrane preparations indicated the presence of two classes of binding sites with Kd values of 0.80 +/- 0.26 and 9.63 +/- 0.13 nM. Approximately 74% of the receptors were in the high affinity state. In the presence of guanyl-5'-yl-imidodiphosphate [Gpp(NH)p], the high affinity sites in the membrane preparations were converted to low affinity sites with no change in the total receptor number. These toxin treatments had no effect on binding of bradykinin to its receptors. Thus, these results indicate that a guanine nucleotide regulatory protein, which is not a substrate of pertussis toxin or cholera toxin, is involved in mediating the effects of bradykinin on membrane-bound phosphoinositide-specific phospholipase C to induce the increase of cytosolic calcium.
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PMID:Role of a protein regulating guanine nucleotide binding in phosphoinositide breakdown and calcium mobilization by bradykinin in neuroblastoma X glioma hybrid NG108-15 cells: effects of pertussis toxin and cholera toxin on receptor-mediated signal transduction. 288 51

Membranes prepared from [3H]inositol-labeled turkey erythrocytes express a phospholipase C that is markedly stimulated by stable analogs of GTP (Harden, T. K., Stephens, L., Hawkins, P. T., and Downes, C. P. (1987) J. Biol. Chem. 262, 9057-9061). We now report that P2-purinergic receptor-mediated regulation of the enzyme occurs in the membrane preparation. The order of potency of a series of ATP and ADP analogs for stimulation of inositol phosphate formation, i.e. 2-methylthioadenosine 5'-triphosphate (2MeSATP) greater than adenosine 5'-O-(2-thiodiphosphate) greater than adenosine 5'-O-(3-thiotriphosphate) greater than ATP greater than 5'-adenylyl imidodiphosphate approximately ADP greater than alpha, beta-methyleneadenosine 5'-triphosphate greater than beta, gamma-methyleneadenosine 5'-triphosphate, was consistent with that for the P2Y-purinergic receptor subtype. Agonist-stimulated effects were completely dependent on the presence of guanine nucleotide. Activation of phospholipase C by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) occurred with a considerable time lag. The rate of activation followed first order kinetics and was markedly increased by increasing concentrations of a P2Y receptor agonist; in contrast, the rate of activation at a fixed agonist concentration was independent of guanine nucleotide concentration. Addition of guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) prior to addition of agonist and GTP, 5'-guanylyl imidodiphosphate (Gpp(NH)p), or GTP gamma S blocked in a concentration-dependent manner the stimulatory effect of guanine nucleotide. GDP beta S, added subsequent to preactivation of membranes with 2MeSATP and GTP gamma S or Gpp(NH)p had only small inhibitory effects on the rate of inositol phosphate production observed over the subsequent 10 min. In contrast, addition of GDP beta S to GTP-preactivated membranes resulted in a rapid return of enzyme activity to the basal state within 60 s. Taken together, the data are consistent with the idea that P2Y receptor activation increases the rate of exchange of GTP and GTP analogs for GDP on the relevant guanine nucleotide regulatory protein. Once the active enzymic species is formed, hydrolysis of guanine nucleotide reverts the enzyme to the inactive state.
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PMID:Kinetics of activation of phospholipase C by P2Y purinergic receptor agonists and guanine nucleotides. 291 Aug 69

The guanine nucleotides guanosine 5'[beta, gamma-imido]triphosphate (Gpp[NH]p), guanosine 5'-[gamma-thio]-triphosphate (GTP gamma S), GMP, GDP and GTP stimulated the hydrolysis of inositol phospholipids by a phosphodiesterase in rat cerebral cortical membranes. Addition of 100 microM-Gpp[NH]p to prelabelled membranes caused a rapid accumulation of [3H )inositol phosphates (less than 30 s) for up to 2 min. GTP gamma S and Gpp [NH]p caused a concentration-dependent stimulation of phosphoinositide phosphodiesterase with a maximal stimulation of 2.5-3-fold over control at concentrations of 100 microM. GMP was as effective as the nonhydrolysable analogues, but much less potent (EC50 380 microM). GTP and GDP caused a 50% stimulation of the phospholipase C at 100 microM and at higher concentrations were inhibitory. The adenine nucleotides App[NH]p and ATP also caused small stimulatory effects (64% and 29%). The guanine nucleotide stimulation of inositide hydrolysis in cortical membranes was selective for inositol phospholipids over choline-containing phospholipids. Gpp[NH]p stimulated the production of inositol trisphosphate and inositol bisphosphate as well as inositol monophosphate, indicating that phosphoinositides are substrates for the phosphodiesterase. EGTA (33 microM) did not prevent the guanine nucleotide stimulation of inositide hydrolysis. Calcium addition by itself caused inositide phosphodiesterase activation from 3 to 100 microM which was additive with the Gpp[NH]p stimulation. These data suggest that guanine nucleotides may play a regulatory role in the modulation of the activity of phosphoinositide phosphodiesterase in rat cortical membranes.
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PMID:Guanine nucleotides stimulate production of inositol trisphosphate in rat cortical membranes. 300 20

The metabolism of phosphatidylinositol (PtdIns) was studied in a mink lung epithelial cell line and its subclones transformed by feline sarcoma viruses containing either the v-fms or v-fes oncogenes. The transformed cell lines had a higher rate of PtdIns turnover but did not have elevated levels of phosphorylated PtdIns species or PtdIns kinase activity. Significantly higher specific activities of a guanine nucleotide-activated PtdIns-4,5-diphosphate phospholipase C were detected in both transformed cell lines (F3CL7(v-fes), 55 pmol/min/mg of protein and G2M(v-fms), 18 pmol/min/mg of protein) as compared to the nontransformed parental cell line (CCL64, 2 pmol/min/mg of protein). The guanine nucleotide-stimulated phospholipase C activity was specific for PtdIns-4,5-diphosphate, and the water-soluble hydrolysis product was inositol 1,4,5-triphosphate. Both GTP and nonhydrolyzable GTP analogs activated the phospholipase C, whereas ATP was weakly effective and GDP was inactive. The phospholipase C activity was maximally active in the presence of 9 mM sodium cholate, had a sharp pH optimum of pH 6.5, and was not activated by calcium although hydrolysis was inhibited by high concentrations of EDTA. These data point to enhanced production of diacylglycerol and inositol 1,4,5-triphosphate second messengers in transformed cells due to the activation of guanine nucleotide-dependent PtdIns-4,5-diphosphate-specific phospholipase C and suggest that the generation of aberrant hormonally independent signals is associated with cell transformation by oncogenes encoding tyrosine-specific protein kinases.
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PMID:A guanine nucleotide-dependent phosphatidylinositol 4,5-diphosphate phospholipase C in cells transformed by the v-fms and v-fes oncogenes. 300 85

The effect of guanine nucleotides on platelet and calf brain cytosolic phospholipase C was examined in the absence of membranes or detergents in an assay using labeled lipid vesicles. Guanine nucleotides stimulate hydrolysis of [3H]phosphatidylinositol 4,5-bisphosphate [( 3H]PtdIns-4,5-P2) catalyzed both by enzyme from human platelets and by partially purified enzyme from calf brain. Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) was the most potent guanine nucleotide with a half-maximal stimulation at 1-10 microM, followed by guanosine 5'-(beta, gamma-imido)triphosphate greater than GTP greater than GDP = guanosine 5'-O-(2-thiodiphosphate). Guanosine 5'-O-(2-thiodiphosphate) was able to reverse the GTP gamma S-mediated stimulation. NaF also stimulated phospholipase C activity, further implying a role for a guanine nucleotide-binding protein. In the presence of GTP gamma S, the enzyme cleaved PtdIns-4,5-P2 at higher pH values, and the need for calcium ions was reduced 100-fold. The stimulation of PtdIns-4,5-P2 hydrolysis by GTP gamma S ranged from 2 to 25-fold under various conditions, whereas hydrolysis of [3H]phosphatidylinositol was only slightly affected by guanine nucleotides. We propose that a soluble guanine nucleotide-dependent protein activates phospholipase C to hydrolyze its initial substrate in the sequence of phosphoinositide-derived messenger generation.
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PMID:Guanine nucleotides stimulate soluble phosphoinositide-specific phospholipase C in the absence of membranes. 302 54

In platelets activated by thrombin, the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C produces inositol 1,4,5-triphosphate (IP3) and diacylglycerol, metabolites which are known to cause Ca2+ release from the platelet dense tubular system and granule secretion. Previous studies suggest that phospholipase C activation is coupled to platelet thrombin receptors by a guanine nucleotide-binding protein or G protein. The present studies examine the contribution of this protein to thrombin-induced platelet activation and compare its properties with those of Gi, the G protein which mediates inhibition of adenylate cyclase by thrombin. In platelets permeabilized with saponin, nonhydrolyzable GTP analogs reproduced the effects of thrombin by causing diacylglycerol formation, Ca2+ release from the dense tubular system and serotonin secretion. In intact platelets, fluoride, which by-passes the thrombin receptor and directly activates G proteins, caused phosphoinositide hydrolysis and secretion. Fluoride also caused an increase in the platelet cytosolic free Ca2+ concentration that appeared to be due to a combination of Ca2+ release from the dense tubular system and increased Ca2+ influx across the platelet plasma membrane. Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which inhibits G protein function, inhibited the ability of thrombin to cause IP3 and diacylglycerol formation, granule secretion, and Ca2+ release from the dense tubular system in saponin-treated platelets. Increasing the thrombin concentration overcame the effects of GDP beta S on secretion without restoring diacylglycerol formation. The effects of GDP beta S on platelet responses to thrombin which had been subjected to partial proteolysis (gamma-thrombin) were similar to those obtained with native alpha-thrombin despite the fact that gamma-thrombin is a less potent inhibitor of adenylate cyclase than is alpha-thrombin. Thrombin-induced diacylglycerol formation and 45Ca release were also inhibited when the saponin-treated platelets were preincubated with pertussis toxin, an event that was associated with the ADP-ribosylation of a protein with Mr = 41.7 kDa. At each concentration tested, the inhibition of thrombin-induced diacylglycerol formation by pertussis toxin paralleled the inhibition of thrombin's ability to suppress PGI2-stimulated cAMP formation.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Regulation of the phosphoinositide hydrolysis pathway in thrombin-stimulated platelets by a pertussis toxin-sensitive guanine nucleotide-binding protein. Evaluation of its contribution to platelet activation and comparisons with the adenylate cyclase inhibitory protein, Gi. 302 67

Binding of chemoattractants to their receptors on phagocytes activates a guanine nucleotide regulatory (N) protein through the substitution of GTP for GDP on N. The activated N protein in turn stimulates a membrane-associated phospholipase C by lowering the Ca2+ concentration required to activate this enzyme from supraphysiologic levels to ambient intracellular concentrations. The phospholipase C hydrolyzes phosphatidylinositol 4,5-bisphosphate into the Ca2+ mobilizer inositol 1,4,5-trisphosphate and the protein kinase C activator 1,2-diacylglycerol. In addition to promoting cellular activation, the products of this hydrolysis initiate processes which feed back to inhibit poly-phosphoinositide breakdown. The regulatory model proposed herein may be relevant to other receptors which stimulate polyphosphoinositide metabolism.
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PMID:Model for leukocyte regulation by chemoattractant receptors: roles of a guanine nucleotide regulatory protein and polyphosphoinositide metabolism. 302 15

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

In a crude membrane preparation of rat 7315c cells, GTP was found to enhance thyrotropin-releasing hormone- (TRH) stimulated inositol triphosphate (IP3) formation with a potency of 0.97 +/- 0.1 microM. TRH stimulation of IP3 formation was inhibited by high GDP concentrations. Neither nucleotide had any effect in the absence of TRH. 5'-Guanosine gamma-thiotriphosphate (GTP gamma S) stimulated IP3 formation in the absence of TRH; the apparent affinity of GTP gamma S was 0.16 +/- 0.05 microM. GTP blocked GTP gamma S stimulation of IP3 formation in a concentration-dependent manner. The apparent affinity of GTP for the site of action shared by GTP gamma S was calculated to be 0.98 +/- 0.3 microM. TRH was able to reverse inhibition of GTP gamma S-stimulated IP3 formation by GTP but could not reverse inhibition by GDP. A lag in the rate of IP3 formation in response to GTP gamma S was abolished by addition of TRH. These data support the proposal that activation of the TRH receptor enhances turnover of guanine nucleotides at the binding protein coupling the receptor to phospholipase C. In addition, GTP gamma S diminished high affinity [3H]Me-TRH binding. The potency of GTP gamma S at decreasing [3H]Me-TRH binding was 0.092 +/- 0.03 microM. GTP gamma S (0.1 microM) decreased the affinity of the TRH receptor for [3H]Me-TRH from 2 to 100 nM. Maximally effective concentrations of GTP gamma S, Gpp(NH)p, GTP, and GDP decreased specific [3H]Me-TRH binding by 80%. Pretreatment of cells with pertussis toxin (30 ng/ml for 24 h) failed to affect TRH receptor affinity or the potency or efficacy of GTP gamma S in diminishing [3H]Me-TRH binding, supporting the identification of Gp (a GTP-binding protein associated with phospholipase C and Ca2+-mobilizing receptors) as distinct from Gi (an inhibitory GTP-binding protein). In contrast to its lack of effect on TRH receptor binding, 3-h pertussis toxin treatment decreased agonist affinity of the mu-opiate receptor and abolished the ability of GTP gamma S to shift the affinity of the mu-opiate receptor for its agonist. The affinities calculated for GTP, GDP, GTP gamma S, and Gpp (NH)p for the G-protein regulating receptor affinity and IP3 formation are nearly identical for each guanine nucleotide tested, suggesting the same G-protein regulates both activities.
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PMID:Regulation of thyrotropin-releasing hormone receptor binding and phospholipase C activation by a single GTP-binding protein. 303 63

The mode of phospholipase C activation initiated with platelet-derived growth factor (PDGF) has been studied in comparison with that initiated with vasopressin and bombesin in a rat fibroblast line, WFB. Stimulation of WFB cells by PDGF, vasopressin, and bombesin elicites rapid hydrolysis of polyphosphoinositides and an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i). On stimulation by PDGF, there was a lag period of about 10 s before an increase in [Ca2+]i. No measurable lag period was observed in the [Ca2+]i response induced by vasopressin or bombesin. Pretreatment of WFB cells with phorbol 12-myristate 13-acetate profoundly inhibited inositol phosphate formation evoked by vasopressin and bombesin, but enhanced to some extent inositol phosphate formation stimulated by PDGF. In membranes prepared from WFB cells, GTP markedly augmented inositol polyphosphate formation induced by vasopressin and bombesin. It was not successful in showing the PDGF-stimulated formation of inositol phosphates in the membrane preparation. The effects of GTP, guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), and guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) on polyphosphoinositide hydrolysis stimulated by growth factors were studied in WFB cells made permeable to nucleotides by treatment with either saponin or Pseudomonas aeruginosa cytotoxin. PDGF, vasopressin, and bombesin elicited inositol phosphate production in the permeabilized WFB cells in the absence of added GTP. GDP beta S, a competitive inhibitor of GTP-binding proteins (G-proteins), markedly reduced the bombesin- and vasopressin-stimulated production of inositol phosphates. However, the PDGF-stimulated production of inositol phosphates was not affected by the addition of GDP beta S. GTP gamma S, an agonist of G-proteins, largely enhanced the vasopressin- and bombesin-stimulated hydrolysis of inositol lipids when added at 10-100 microM. In the presence of GTP gamma S, the PDGF-stimulated hydrolysis of inositol lipids was not enhanced, but was reduced: 100 microM GTP gamma S reduced the stimulated hydrolysis to about a half of the control level. Only GTP gamma S, and no other nucleoside triphosphates, was found to have these effects. Activation of G-proteins in WFB cells by fluoroaluminate resulted in the inhibition of inositol phosphate production elicited with not only PDGF, but also with vasopressin and bombesin. These results indicate that a G-protein couples vasopressin and bombesin receptors to the activation of phospholipase C. Moreover, these results suggest that coupling of the PDGF receptor to phospholipase C is not mediated through a G-protein.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Pathway of phospholipase C activation initiated with platelet-derived growth factor is different from that initiated with vasopressin and bombesin. 304 15


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