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)

Hormones have been demonstrated to activate phosphoinositide hydrolysis in plasma membranes in a manner dependent upon or potentiated by GTP. For thyrotropin-releasing hormone activation in GH3 cell membranes, stimulation persisted in membranes from pertussis toxin-treated cells. These observations indicate the presence of a membrane phospholipase C (PL C) and a novel GTP-binding protein (Gp); however, neither of these proteins has been characterized. In this paper, we report studies of GH3 membrane PL C utilizing [3H]phosphatidylinositol 4,5-bisphosphate liposome substrate. Guanosine 5'-O-(3-thiotriphosphate) (GTP[S]), but not other nucleotides, was found to stimulate PL C activity and required greater than 1 nM Ca2+. High concentrations of Ca2+ (10 microM) also activated the membrane PL C. Treatment of membranes with N-ethylmaleimide inhibited Ca2+-activated but not GTP[S]-activated PL C. Extraction of membranes with 1 M KCl solubilized the membrane PL C; however, the solubilized PL C was not GTP[S]-stimulated. N-ethylmaleimide-treated, KCl-extracted membranes were markedly deficient in GTP[S]-stimulated PL C activity; however, activity could be restored by incubation with the desalted extracted PL C. Reconstitution appeared to involve the recoupling of membrane-associated Gp with soluble 330- and 110-kDa forms of the PL C. Cytosolic PL Cs failed to substitute for the solubilized membrane PL C. These results indicate that the Gp-regulated PL C in GH3 cell membranes is an extrinsic membrane protein that can be extracted reversibly at high ionic strength. Moreover, the membrane PL C can be distinguished from cytosolic PL C isoenzymes.
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PMID:Reconstitution of a solubilized membrane but not cytosolic phospholipase C with membrane-associated Gp from GH3 cells. 251 86

Release of arachidonic acid (AA) from 1-stearoyl-2-[14C]arachidonyl-glycerophosphoinositol (PI) by plasma membrane-bound enzyme(s) is a calcium-dependent reaction and is markedly activated at 4 x 10(-4) M CaCl2. In the presence of Ca2+, the agonist of the cholinergic receptor (carbachol) enhances, in a dose-related manner, AA release. Moreover, GTP and its non-hydrolysable analogs GTP gamma S and GppNHp and also NaF additionally increase the carbachol-mediated liberation of AA from PI. On the contrary, in the absence of Ca2+ carbachol and GTP gamma S have no stimulatory effect on AA release. Guanosine-5'-O-2-thiodiphosphate GDP gamma S, which inhibits the function of GTP-binding proteins, also suppresses carbachol-mediated activation of AA release from PI. The stimulatory effect of carbachol and guanine nucleotides was observed exclusively in the brain plasma membrane (there was no effect on mitochondria, microsome and cytosolic enzymes). Quinacrine, the inhibitor of phospholipase A2, completely inhibits carbachol- and guanine nucleotide-activated AA release and greatly (by about 60-70%) decreases Ca(2+)-dependent AA liberation from phosphatidylinositol. These results indicate that GTP-binding protein(s) are involved in the regulation of carbachol-mediated AA release. The main pool of this acid is liberated from phosphatidylinositol by phospholipase A2 and only a small pool of AA may be released indirectly as the result of PI hydrolysis by sequential action of phospholipase C and diacylglycerol lipase.
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PMID:Guanine nucleotides and fluoride enhance carbachol-mediated arachidonic acid release from phosphatidylinositol. Evidence for involvement of GTP-binding protein in phospholipase A2 activation. 251 94

The ability of alcohols to regulate inositol lipid-specific phospholipase C (phosphoinositidase C) was examined in turkey erythrocyte ghosts prepared by cell lysis of erythrocytes which were prelabeled with [3H] inositol. Guanosine 5'-[gamma-thiotriphosphate] GTP[S] stimulated the production of both [3H]inositol bisphosphate (18-fold) and [3H]inositol trisphosphate (6-fold) in this system. The accumulation of [3H]inositol bisphosphate and [3H]inositol trisphosphate was linear up to 8 min following an initial lag period of 1-2 min. Ethanol (300 mM) reduced the lag period for [3H]inositol phosphate accumulation at submaximal GTP[S] concentrations and caused a shift to the left (3-fold) in the dose-response curve. Other short chain alcohols, methanol (300 mM), 1-propanol (200 mM), and 1-butanol (50 mM) also enhanced the accumulation of [3H] inositol phosphates in the presence of submaximal GTP[S] concentrations. Receptor activation by the purinergic agonist adenosine 5'-[beta-thio]disphosphate (ADP[S]) (10 microM) also reduced the lag period for [3H] inositol phosphate formation and shifted the GTP[S] dose response to the left (10-fold). In addition, ADP[S] increased the response to maximal GTP[S] concentrations. The formation of [3H]inositol phosphates induced by GTP[S] was associated with a concomitant decrease in labeling of both [3H]phosphatidylinositol monophosphate and [3H]phosphatidylinositol bisphosphate, but no decrease in [3H]phosphatidylinositol was observed. All of the alcohols tested enhanced the breakdown of [3H]polyphosphoinositides in the presence of GTP[S]. The dose response to guanosine 5'-[beta gamma-imino]triphosphate for [3H]inositol phosphate formation was displaced to the left by ethanol (300 mM) and ADP[S] (10 microM) (2- and 7-fold), respectively. ADP[S] also enhanced the maximal response to guanosine 5'-[beta gamma-imino]triphosphate. The [3H]inositol phosphate formation produced in response to NaF was unaffected by either ethanol or receptor activation. These results indicate that alcohols initiate an activation of phosphoinositidase C, mediated at the level of the regulatory guanine nucleotide-binding protein.
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PMID:Short chain alcohols activate guanine nucleotide-dependent phosphoinositidase C in turkey erythrocyte membranes. 254 Jan 62

The light-stimulated production of inositol triphosphate (IP3), via hydrolysis of phosphatidylinositol bisphosphate (PIP2), can be demonstrated in an in vitro preparation of isolated distal segments of squid photoreceptors. The retina is labeled with [3H]inositol (Szuts, E. Z., Wood, S. F., Reid, M. S., and Fein, A. (1986) Biochem. J. 240, 929-932), and the rhodopsin-containing distal segments are isolated in artificial cytosol. Within 2 s after a flash, IP3 levels increase 200% (corresponding to an intracellular increase of approximately 5 microM), and the lipid precursor PIP2 decreases by 50%. Inositol bisphosphate (IP2) levels increase later, as a breakdown product of IP3. IP3 response is light-dependent, saturating when 0.5% of the rhodopsin is photoactivated. Guanosine-5'-O-(3-thiotriphosphate (GTP gamma S) binding demonstrates that the plasma membrane of most of the photoreceptor distal segments is intact or only transiently permeable. Membrane permeabilization enhances light-activated GTP gamma S binding but abolishes the light-activated IP3 production. Receptor-mediated production of IP3 is believed to be the result of a receptor-G-protein-phospholipase C cascade (i.e. Cockcroft, S., and Gomperts, B. D. (1985) Nature 314, 534-536). To test for G-proteins, we incubated the photoreceptors in AlF4- (an activator of G-proteins) in the dark. IP3 and IP2 were produced with a corresponding decrease in PIP2. Incubation with GTP or GTP gamma S, in hypotonic buffer, which causes transient leakiness, increased dark levels by IP3 by 50%. Addition of GTP in isotonic buffer enhanced the light-induced increase of IP3. These results localize the light-stimulated phospholipase C activity to the distal segments and suggest that a G-protein couples rhodopsin to phospholipase C.
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PMID:Inositol trisphosphate production in squid photoreceptors. Activation by light, aluminum fluoride, and guanine nucleotides. 266 14

Rabbit platelets were labelled with [3H]inositol and a membrane fraction was isolated in the presence of ATP, MgCl2 and EGTA. Incubation of samples for 10 min with 0.1 microM-Ca2+free released [3H]inositol phosphates equivalent to about 2.0% of the membrane [3H]phosphoinositides. Addition of 10 microM-guanosine 5'-[gamma-thio]triphosphate (GTP[S]) caused an additional formation of [3H]inositol phosphates equivalent to 6.6% of the [3H]phosphoinositides. A half-maximal effect was observed with 0.4 microM-GTP[S]. The [3H]inositol phosphates that accumulated consisted of 10% [3H]inositol monophosphate, 88% [3H]inositol bisphosphate ([3H]IP2) and 2% [3H]inositol trisphosphate ([3H]IP3). Omission of ATP and MgCl2 led to depletion of membrane [3H]polyphosphoinositides and marked decreases in the formation of [3H]inositol phosphates. Thrombin (2 units/ml) or GTP (4-100 microM) alone weakly stimulated [3H]IP2 formation, but together they acted synergistically to exert an effect comparable with that of 10 microM-GTP[S]. The action of thrombin was also potentiated by 0.1 microM-GTP[S]. Guanosine 5'-[beta-thio]diphosphate not only inhibited the effects of GTP[S], GTP and GTP with thrombin, but also blocked the action of thrombin alone, suggesting that this depended on residual GTP. Incubation with either GTP[S] or thrombin and GTP decreased membrane [3H]phosphatidylinositol 4-phosphate ([H]PIP) and prevented an increase in [3H]phosphatidylinositol 4,5-bisphosphate ([3H]PIP2) observed in controls. Addition of unlabelled IP3 to trap [3H]IP3 before it was degraded to [3H]IP2 showed that only about 20% of the additional [3H]inositol phosphates that accumulated with GTP[S] or thrombin and GTP were derived from the action of phospholipase C on [3H]PIP2. The results provide further evidence that guanine-nucleotide-binding protein mediates signal transduction between the thrombin receptor and phospholipase C, and suggest that PIP may be a major substrate of this enzyme in the platelet.
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PMID:Activation of phospholipase C associated with isolated rabbit platelet membranes by guanosine 5'-[gamma-thio]triphosphate and by thrombin in the presence of GTP. 282 Mar 81

One of the earliest actions of thrombin in fibroblasts is stimulation of a phospholipase C (PLC) that hydrolyses phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-trisphosphate (IP3) and diacylglycerol. In membranes prepared from WI-38 human lung fibroblasts, thrombin activated an inositol-lipid-specific PLC that hydrolysed [32P]PIP2 and [32P]phosphatidylinositol 4-monophosphate (PIP) to [32P]IP3 and [32P]inositol 1,4-bisphosphate (IP2) respectively. Degradation of [32P]phosphatidylinositol was not detected. PLC activation by thrombin was dependent on GTP, and was completely inhibited by a 15-fold excess of the non-hydrolysable GDP analogue guanosine 5'-[beta-thio]diphosphate (GDP[S]). Neither ATP nor cytosol was required. Guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) also stimulated polyphosphoinositide hydrolysis, and this activation was inhibited by GDP[S]. Stimulation of PLC by either thrombin or p[NH]ppG was dependent on Ca2+. Activation by thrombin required Ca2+ concentrations between 1 and 100 nM, whereas stimulation of PLC activity by GTP required concentrations of Ca2+ above 100 nM. Thus the mitogen thrombin increased the sensitivity of PLC to concentrations of free Ca2+ similar to those found in quiescent fibroblasts. Under identical conditions, another mitogen, platelet-derived growth factor, did not stimulate polyphosphoinositide hydrolysis. It is concluded that an early post-receptor effect of thrombin is the activation of a Ca2+- and GTP-dependent membrane-associated PLC that specifically cleaves PIP2 and PIP. This result suggests that the cell-surface receptor for thrombin is coupled to a polyphosphoinositide-specific PLC by a GTP-binding protein that regulates PLC activity by increasing its sensitivity to Ca2+.
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PMID:Stimulation of polyphosphoinositide hydrolysis by thrombin in membranes from human fibroblasts. 282 18

Vasopressin (VP) and angiotensin II (AT II) stimulate the production of inositol phosphates (IP) in rat glomerulosa cells. Guanosine 5'-[gamma-thio]triphosphate (GTP[S]), but not VP or AT II, stimulates IP production in a myo-[3H]inositol-prelabelled glomerulosa-cell membrane preparation. In combination with GTP[S], these hormones potentiate the response to GTP[S], indicating the existence of a G-protein involved in the coupling of the VP and AT II receptor with the phospholipase C. ADP-ribosylation with pertussis toxin (IAP) revealed the specific labelling of a single molecule of 41 kDa. No significant inhibition of VP- or AT II-stimulated IP accumulation was detected in intact cells when the whole 41 kDa molecule was endogenously ADP-ribosylated by IAP treatment. On the contrary, when glomerulosa cells were infected with cholera toxin (CT), both the VP- and AT II-stimulated IP accumulations were inhibited in a dose-dependent manner. Yet these effects were partial even at high concentrations of CT, and could not be related to the ADP-ribosylation of 'alpha s' molecules. Similarly, when the cells were infected with 1 microgram of CT/ml, the specific binding of VP and AT II decreased by 50-60%. Such results may signify that the treatment primarily affects the densities of the hormone receptors. When glomerulosa cells were incubated for 15 h in the presence of 10 nM-corticotropin (ACTH), a condition in which the intracellular concentration of cyclic AMP was increased 3-fold, the maximum IP response to 0.1 microM-VP or -AT II was decreased by 50%. When similar experiments were carried out only after a 15 min incubation period with the same concentration of ACTH, the increase in cyclic AMP was more pronounced, but no inhibition of hormone-induced IP accumulation was observed. Altogether, these results may suggest that CT exerts its action on the VP- or AT II-sensitive phospholipase C systems via a prolonged increase in intracellular cyclic AMP.
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PMID:Cholera-toxin and corticotropin modulation of inositol phosphate accumulation induced by vasopressin and angiotensin II in rat glomerulosa cells. 284 33

In Swiss 3T3 fibroblasts bradykinin stimulated inositol phosphate (InsP) formation and prostaglandin E2 (PGE2) synthesis. The EC50 values for stimulation of PGE2 synthesis and InsP formation by bradykinin were similar, 200 pM and 275 pM, respectively. Guanosine-5'-[gamma-thio]triphosphate stimulated PGE2 synthesis and InsP formation, and guanosine-5'-[beta-thio]diphosphate inhibited both PGE2 synthesis and InsP formation stimulated by bradykinin. Neither bradykinin-stimulated PGE2 synthesis nor InsP formation was sensitive to pertussis toxin. Phorbol ester, dexamethasone, and cycloheximide distinguished between bradykinin-stimulated PGE2 synthesis and InsP formation. Phorbol 12-myristate 13-acetate enhanced bradykinin-stimulated PGE2 synthesis but inhibited bradykinin-stimulated InsP formation. Pretreatment of cells with dexamethasone for 24 hr inhibited bradykinin-stimulated PGE2 synthesis but was without effect on bradykinin-stimulated InsP formation. Cycloheximide inhibited bradykinin-stimulated PGE2 synthesis but was without effect on bradykinin-stimulated InsP formation. When bradykinin was added to cells prelabeled with [3H]choline, the phospholipase A2 products lysophosphatidylcholine and glycerophosphocholine were generated. In cells pretreated with dexamethasone, lysophosphatidylcholine and glycerophosphocholine formation induced by bradykinin were inhibited. Treatment of cells with phorbol ester enhanced bradykinin-induced formation of these metabolites. The data suggest that bradykinin receptors are coupled by GTP-binding proteins to both phospholipase C and phospholipase A2 and that phospholipase A2 is the enzyme that catalyzes release of arachidonate for prostaglandin synthesis.
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PMID:Dissociation of bradykinin-induced prostaglandin formation from phosphatidylinositol turnover in Swiss 3T3 fibroblasts: evidence for G protein regulation of phospholipase A2. 288 13

The effects of guanine nucleotides, thrombin, and platelet cytosol (100,000 X g supernatant) on the hydrolysis of polyphosphoinositides by phospholipase C was examined in isolated platelet membranes labeled with [3H]inositol. Guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) (10 microM) caused a 2-fold stimulation of polyphosphoinositide hydrolysis, compared to background. GTP gamma S (10 microM) plus thrombin (1 unit/ml) stimulated the release of inositol triphosphate, inositol diphosphate, and inositol phosphate 500, 300, and 250%, respectively, compared to GTP gamma S alone. Cytosol prepared from unlabeled platelets slightly increased the release of inositol phosphates from [3H]inositol-labeled membranes. Addition of cytosol plus GTP gamma S (10 microM), however, resulted in a 300% enhancement of the release of inositol phosphates compared to membranes incubated with thrombin and GTP gamma S. The stimulatory effects of cytosol and GTP gamma S on polyphosphoinositide hydrolysis were also observed when membranes were replaced by sonicated lipid vesicles prepared from a total platelet lipid extract. These data suggest that PIP2 hydrolysis in platelets is catalyzed by a soluble phospholipase C which is regulated by a GTP-binding regulatory protein.
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PMID:Regulation of membrane-associated and cytosolic phospholipase C activities in human platelets by guanosine triphosphate. 301 54

It has recently been shown in this laboratory that permeabilization of human platelets with 15-25 micrograms/ml saponin allows ADP-ribosylation by pertussis toxin of the alpha i-subunit of Gi (Ni), a guanine nucleotide-binding regulatory protein. The same assay conditions have been used to determine phospholipase C in permeabilized platelets. Guanosine 5'-O-thiotriphosphate- (GTP[gamma S]-) activated phospholipase C in permeabilized platelets whose inositol phospholipids were prelabeled with [3H]inositol. Phospholipase C activity was measured by [3H]polyphosphoinositide decreases and formation of [3H]inositol bisphosphate and [3H]inositol trisphosphate. Prostacyclin, cyclic AMP or pretreatment of permeabilized platelets with pertussis toxin did not alter this effect under conditions in which the alpha i-subunit was effectively ADP-ribosylated by pertussis toxin. This information indicated that ADP-ribosylation of Gi-protein was not directly related to activation or inhibition of platelet phospholipase C by GTP [gamma S]. Thrombin also activated phospholipase C in permeabilized platelets and, surprisingly, this action was enhanced by pertussis toxin pretreatment. This indicates that ADP-ribosylation of Gi-protein facilitates the action of thrombin on phospholipase C.
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PMID:Effect of pertussis toxin on the phosphodiesteratic cleavage of the polyphosphoinositides by guanosine 5'-O-thiotriphosphate and thrombin in permeabilized human platelets. 302 Dec 35


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