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)

Using the technique of HPLC with Partisil SAX columns, we have found that stimulation of amoebae of Dictyostelium discoideum with the chemoattractant cyclic AMP induces the rapid accumulation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), with a peak at 5 s. A smaller HPLC peak (designated P3) that elutes just after the Ins(1,4,5)P3 peak accumulates more slowly to a maximum at 20 s. In control studies, the changes in Ins(1,4,5)P3 were shown not to be due to varying recovery from the cell extracts and a comparison of reverse-phase and Partisil SAX HPLC columns showed similar values for determinations by either method. The involvement of a G-protein in this chemotactic system was confirmed by the finding that accumulation of Ins(1,4,5)P3 was elicited by the addition of GTP gamma S (5'-[gamma-thio]triphosphate) to saponin-permeabilized amoebae. A study of the changes in the lipid-soluble phosphatidyl inositol phosphates demonstrated that cyclic AMP also stimulated a rapid loss of radioactivity from 32P-labelled phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), which corresponded in its timing to the rise in Ins(1,4,5)P3, indicating that a phosphoinositidase C (phospholipase C) is present that can be stimulated by occupation of the cell surface cyclic AMP receptors.
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PMID:Inositol tris- and polyphosphate formation during chemotaxis of Dictyostelium. 255 21

The influence of nucleotides and pyrophosphate on phospholipase C from rabbit thymocytes was investigated by using two different methods for the determination of phospholipase C activity. In a first approach the release of radiolabeled inositol phosphates from [3H]inositol-labeled membranes was examined. By a second type of experiment the cleavage of exogenously added radiolabeled phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) was measured. Using internally labeled membranes only guanosine 5'-O-(thiotriphosphate) exhibited a stimulatory effect on the phospholipase C suggesting the involvement of a G-protein. When exogenous [3H]PtdIns-4,5-P2 was used as substrate, cleavage of PtdIns-4,5-P2 was stimulated by all nucleotides investigated; in addition pyrophosphate showed a stimulatory effect. From these data we conclude that the increased cleavage of exogenous PtdIns-4,5-P2 induced by GTP analogues is not conclusive in terms of the involvement of a G-protein. Rather than induced by a G-protein this activation may be caused by an increased substrate accessibility. Our experiments with endogenous substrate clearly established the regulatory role of G-proteins for membrane-bound phospholipase C.
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PMID:Effects of nucleotides on the activity of phospholipase C in rabbit thymus lymphocytes. Differences in assays using endogenous [3H]inositol-prelabeled membranes or exogenous [3H]phosphatidylinositol 4,5-bisphosphate as substrate. 264 8

The effect of a number of growth factors on phosphatidylcholine (PtdCho) turnover in Swiss-3T3 cells was studied. Phorbol 12-myristate 13-acetate (PMA), bombesin, platelet-derived growth factor (PDGF) and vasopressin rapidly stimulated PtdCho hydrolysis, diacylglycerol (DAG) production, and PtdCho synthesis. Insulin and prostaglandin F2 alpha (PGF2 alpha) stimulated PtdCho synthesis, but not its breakdown, whereas epidermal growth factor (EGF) and bradykinin were without effect. Stimulation of PtdCho hydrolysis by the above ligands resulted in increased production of phosphocholine and DAG (due to phospholipase C activity) and significant amounts of choline, suggesting activation of a phospholipase D as well. CDP-choline and glycerophosphocholine levels were unchanged. Down-regulation of protein kinase C with PMA (400 nM, 40 h) abolished the stimulation of PtdCho hydrolysis and PtdCho synthesis by PMA, bombesin, PDGF and vasopressin, but not the stimulation of PtdCho synthesis by insulin and PGF2 alpha. PtdCho hydrolysis therefore occurs predominantly by activation of protein kinase C (either by PMA or PtdIns hydrolysis) leading to elevation of DAG levels derived from non-PtdIns(4,5)P2 sources. PtdCho synthesis occurs by both a protein kinase C-dependent pathway (stimulated by PMA, PDGF, bombesin and vasopressin) and a protein kinase C-independent pathway (stimulated by insulin and PGF2 alpha). DAG production from PtdCho hydrolysis is not the primary signal to activate protein kinase C, but may contribute to long-term activation of this kinase.
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PMID:Stimulation of phosphatidylcholine breakdown and diacylglycerol production by growth factors in Swiss-3T3 cells. 269 Aug 29

The effect of the GTP analogue guanosine 5'-[gamma-thio]triphosphate (GTP[S]) on the polyphosphoinositide phospholipase C (PLC) of rat liver was examined by using exogenous [3H]phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. GTP[S] stimulated the membrane-bound PLC up to 20-fold, with a half-maximal effect at approx. 100 nM. Stimulation was also observed with guanosine 5'-[beta gamma-imido]triphosphate, but not with adenosine 5'-[gamma-thio]triphosphate, and was inhibited by guanosine 5'-[beta-thio]diphosphate. Membrane-bound PLC was entirely Ca2+-dependent, and GTP[S] produced both a decrease in the Ca2+ requirement and an increase in activity at saturating [Ca2+]. The stimulatory action of GTP[S] required millimolar Mg2+. [8-arginine]Vasopressin (100 nM) stimulated the PLC activity approx. 2-fold in the presence of 10 nM-GTP[S], but had no effect in the absence of GTP[S] or at 1 microM-GTP[S]. The hydrolysis of PtdIns(4,5)P2 by membrane-bound PLC was increased when the substrate was mixed with phosphatidylethanolamine, phosphatidylcholine or various combinations of these with phosphatidylserine. With PtdIns(4,5)P2, alone or mixed with phosphatidylcholine, GTP[S] evoked little or no stimulation of the PLC activity. However, maximal stimulation by GTP[S] was observed in the presence of a 2-fold molar excess of phosphatidylserine or various combinations of phosphatidylethanolamine and phosphatidylserine. Hydrolysis of [3H]phosphatidylinositol 4-phosphate by membrane-bound PLC was also increased by GTP[S]. However, [3H]phosphatidylinositol was a poor substrate, and its hydrolysis was barely affected by GTP[S]. Cytosolic PtdIns(4,5)P2-PLC exhibited a Ca2+-dependence similar to that of the membrane-bound activity, but was unaffected by GTP[S]. It is concluded that rat liver plasma membranes possess a Ca2+-dependent polyphosphoinositide PLC that is activated by hormones and GTP analogues, depending on the Mg2+ concentration and phospholipid environment. It is proposed that GTP analogues and hormones, acting through a guanine nucleotide-binding protein, activate the enzyme mainly by lowering its Ca2+ requirement.
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PMID:Guanine-nucleotide and hormone regulation of polyphosphoinositide phospholipase C activity of rat liver plasma membranes. Bivalent-cation and phospholipid requirements. 282 42

The photoreceptor, like many other cells, undergoes receptor-mediated breakdown of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. The lack of phosphatidylinositol (PdtIns) breakdown during receptor stimulation suggests the existence of a phospholipase C specific for polyphosphoinositides. Phospholipase C activity in frog rod outer segments was assayed with several substrates. The activity was selective for the polyphosphoinositides, phosphatidylinositol 4-phosphate [PtdIns(4)P] and PtdIns(4,5)P2. PtdIns was hydrolysed at 2% of the rate of hydrolysis of PtdIns(4,5)P2. No activity was detected when phosphatidylcholine, phosphatidylserine, or phosphatidylethanolamine were used as substrates. The enzymatic activity was optimal at neutral pH. These findings suggest, but do not prove, that this activity might contain the light-regulated phospholipase C of the photoreceptor.
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PMID:Phospholipase C activity and substrate specificity in frog photoreceptors. 283 Jan 27

Epidermal growth factor (EGF) stimulated the rapid accumulation of inositol trisphosphate in WB cells, a continuous line of rat hepatic epithelial cells. Since we previously had shown that EGF stimulates EGF receptor synthesis in these cells, we tested whether hormones that stimulate PtdIns(4,5)P2 hydrolysis would increase EGF receptor protein synthesis and mRNA levels. Epinephrine, angiotensin II, and [Arg8]vasopressin activate phospholipase C in WB cells as evidenced by the accumulation of the inositol phosphates, inositol monophosphate, inositol bisphosphate, and inositol trisphosphate. A 3-4-h treatment with each hormone also increased the rate of EGF receptor protein synthesis by 3-6-fold as assessed by immunoprecipitation of EGF receptor from [35S]methionine-labeled cells. Northern blot analyses of WB cell EGF receptor mRNA levels revealed that agents linked to the phosphoinositide signaling system increased receptor mRNA content within 1-2 h. A maximal increase of 3-7-fold was observed after a 3-h exposure to EGF and hormones. The phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), which activates protein kinase C also stimulated EGF receptor synthesis. Pretreatment of WB cells for 18 h with high concentrations of TPA "down-regulated" protein kinase C and blocked TPA-directed EGF receptor mRNA synthesis. In contrast, the effect of EGF on EGF receptor mRNA levels was not significantly decreased by TPA pretreatment. Epinephrine-induced increases in EGF receptor mRNA were reduced from 4- to 2-fold. Similarly, 18 h TPA pretreatment abolished the effect of TPA on EGF receptor protein synthesis but did not affect EGF-dependent EGF receptor protein synthesis. The 18-h TPA pretreatment diminished by 30-50% the induction of receptor protein synthesis by epinephrine or angiotensin II. We conclude that in WB cells EGF receptor synthesis can be regulated by EGF and other hormones that stimulate PtdIns(4,5)P2 hydrolysis. In these cells, EGF receptor synthesis appears to be regulated by several mechanism: one pathway is dependent upon EGF receptor activation and can operate independently of protein kinase C activation; another pathway is correlated with PtdIns(4,5)P2 hydrolysis and is dependent, at least in part, upon protein kinase C activation.
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PMID:Epidermal growth factor (EGF) and hormones stimulate phosphoinositide hydrolysis and increase EGF receptor protein synthesis and mRNA levels in rat liver epithelial cells. Evidence for protein kinase C-dependent and -independent pathways. 284 41

Sickle-cell-anaemia erythrocytes (SS cells) are known to have a high Ca2+ content (particularly the dense cell fraction) and to take up Ca2+ on deoxygenation. It has been reported that this high Ca2+ was responsible for the activation of the Ca2+-dependent K+ loss, and of the Ca2+-sensitive polyphosphoinositide phospholipase C (PIC) in dense SS cells. We found that, either in the total population of SS cells or in the light or dense fractions, the content of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] was not changed, whereas that of phosphatidylinositol 4-phosphate was increased and that of phosphatidic acid (PtdOH) was decreased compared with normal (AA) erythrocytes. Deoxygenation-induced Ca2+ entry into SS cells did not change the concentration or, in 32P-prelabelled cells, the radioactivity of polyphosphoinositides and PtdOH. It also failed to induce the formation of inositol 1,4,5-trisphosphate, the product of PtdIns(4,5)P2 hydrolysis by PIC, which was measured by an original method using ion-pair reverse-phase h.p.l.c. Thus there was no evidence of an endogenous Ca2+ effect on the PIC activity in SS cells, in agreement with the demonstration that the excess Ca2+ in SS cells is compartmentalized into internal vesicles and unavailable as free Ca2+. The 32P incorporation in polyphosphoinositides and PtdOH was markedly higher in SS than in AA cells, but this increase was the same in both dense and light SS cells. The increase in the turnover of these phospholipids in SS cells is consistent either with an activation of the lipid kinases and phosphatases or with perturbation in the metabolic compartmentation of these lipids.
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PMID:Endogenous calcium in sickle cells does not activate polyphosphoinositide phospholipase C. 284 44

The present study examined the possible role of increased phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) breakdown in the regulation of actin assembly in human neutrophils. Tetracaine, a local anesthetic, was used since it has recently been proposed to inhibit the phosphorylation of phosphatidylinositol 4-phosphate to form PtdIns(4,5)P2. Surprisingly, it was found that incubation with tetracaine alone increased the breakdown of PtdIns(4,5)P2, measured as total inositol trisphosphate formation. This occurred without any rise above basal in the cellular content of filamentous actin. However, in the presence of formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe), tetracaine potentiated the chemotactic-induced increase of both inositol trisphosphate formation and actin polymerization. To further explore the relationship between increased PtdIns(4,5)P2 breakdown and actin polymerization, the activity of phospholipase C was depressed by lowering the cytosolic free calcium ion level or by incubating the cells with ionomycin. In these cells, fMet-Leu-Phe stimulation still raised the cellular content of filamentous actin to a level similar to levels in nontreated cells, despite the absence of PtdIns(4,5)P2 hydrolysis. Consequently, increased breakdown of PtdIns(4,5)P2 alone is not enough to initiate actin polymerization, nor is the polymerization of actin dependent on an increased PtdIns(4,5)P2 breakdown. However, we cannot exclude the possibility that increased turnover of phosphoinositides might act as a modulator of actin assembly.
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PMID:Increased breakdown of phosphatidylinositol 4,5-bisphosphate is not an initiating factor for actin assembly in human neutrophils. 284 63

1. In the intact guinea-pig myometrium, carbachol and oxytocin stimulated a specific receptor-mediated phospholipase C activation, catalysing the breakdown of PtdIns(4,5)P2 with the sequential generation of InsP3, InsP2 and InsP. Stimulation of muscarinic receptors also triggered an inhibition of cyclic AMP accumulation caused by prostacyclin. 2. NaF plus AlCl3 mimicked the effects of carbachol and oxytocin by inducing, in a dose-dependent manner, the generation of all three inositol phosphates as well as uterine contractions. AlCl3 enhanced the fluoride effect, supporting the concept that A1F4- was the active species. Under similar conditions, fluoroaluminates activated the guanine nucleotide regulatory protein Gi, reproducing the inhibitory effect of carbachol on cyclic AMP concentrations. 3. Both carbachol- and oxytocin-mediated increases in inositol phosphates, as well as contractions, were insensitive to pertussis toxin, under conditions where the expression of Gi was totally prevented. Cholera toxin, which activates Gs and enhances cyclic AMP accumulation, failed to affect basal or oxytocin-evoked inositol phosphate generation, but induced a slight, though consistent, attenuation of the muscarinic inositol phosphate response, which was similarly evoked by forskolin. 4. The data provide evidence that, in the myometrium, (a) a G protein mediates the generation of inositol phosphates and the Ca2+-dependent contractile event, (b) the relevant G protein that most probably couples muscarinic and oxytocin receptors to phospholipase C is different from Gi and Gs, the proteins that couple receptors to adenylate cyclase, and (c) cyclic AMP does not seem to control the phosphoinositide cycle, but rather exerts a negative regulation at the muscarinic-receptor level.
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PMID:Fluoroaluminates mimic muscarinic- and oxytocin-receptor-mediated generation of inositol phosphates and contraction in the intact guinea-pig myometrium. Role for a pertussis/cholera-toxin-insensitive G protein. 284 25

The molecular events involved in the cellular actions of insulin remain unexplained. Some of the acute actions of the hormone may be due to the intracellular generation of a chemical substance which modulates certain enzyme activities. Such an enzyme-modulating substance has been identified as an inositol phosphate-glycan, produced by the insulin-sensitive hydrolysis of a glycosyl-phosphatidylinositol (glycosyl-PtdIns) precursor. This precursor glycolipid is structurally similar to the glycosyl-phosphoinositide membrane protein anchor. The exposure of fat, liver or muscle cells to insulin results in the hydrolysis of glycosyl-PtdIns, giving rise to the inositol phosphate glycan and diacylglycerol. This hydrolysis reaction is catalysed by a glycosyl-PtdIns-specific phospholipase C. This enzyme has been characterized and purified from a plasma membrane fraction of liver. This reaction also results in the acute release of certain glycosyl-PtdIns-anchored proteins from the cell surface. Elucidation of the functional role of glycosyl-phosphoinositides in the generation of second messengers or the release of proteins may provide further insights into the pleiotropic nature of insulin action.
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PMID:The function of glycosyl phosphoinositides in hormone action. 290 43


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