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)

There is much evidence that G-proteins transduce the signal from receptors for Ca(2+)-mobilizing agonists to the phospholipase C that catalyzes the hydrolysis of phosphoinositides. However, the specific G-proteins involved have not been identified. We have recently purified a 42 kDa protein from liver that activates phosphoinositide phospholipase C and cross-reacts with antisera to a peptide common to G-protein alpha-subunits. It is proposed that this protein is the alpha-subunit of the G-protein that regulates the phospholipase in this tissue. Ca(2+)-mobilizing agonists and certain growth factors also promote the hydrolysis of phosphatidylcholine through the activation of phospholipases C and D in many cell types. This yields a larger amount of diacylglycerol for a longer time than does the hydrolysis of inositol phospholipids. Consequently phosphatidylcholine breakdown is probably a major factor in long-term regulation of protein kinase C. The functions of phosphatidic acid produced by phospholipase D are speculative, but there is evidence that it is a major source of diacylglycerol in many cell types. The regulation of phosphatidylcholine phospholipases is multiple and involves direct activation by G-proteins, and regulation by Ca2+, protein kinase C and perhaps growth factor receptor tyrosine kinases.
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PMID:Cell signalling through phospholipid breakdown. 165 98

Tumor necrosis factor (TNF) is a proinflammatory polypeptide that is able to induce a great diversity of cellular responses via modulating the expression of a number of different genes. One major pathway by which TNF receptors communicate signals from the membrane to the cell nucleus involves protein kinase C (PKC). In the present study, we have addressed the molecular mechanism of TNF-induced PKC activation. To this, membrane lipids of the human histiocytic cell line U937 were labeled by incubation with various radioactive precursors, and TNF-induced changes in phospholipid, neutral lipid, and water-soluble metabolites were analyzed by thin layer chromatography. TNF treatment of U937 cells resulted in a rapid and transient increase of 1'2'diacylglycerol (DAG), a well-known activator of PKC. The increase in DAG was detectable as early as 15 s after TNF treatment and peaked at 60 s. DAG increments were most pronounced (approximately 360% of basal levels) when cells were preincubated with [14C]lysophosphatidylcholine, which was predominantly incorporated into the phosphatidylcholine (PC) pool of the plasma-membranes. Further extensive examination of changes in metabolically labeled phospholipids indicated that TNF-stimulated hydrolysis of PC is accompanied by the generation of phosphorylcholine and DAG. These results suggest the operation of a PC-specific phospholipase C. Since no changes in phosphatidic acid (PA) and choline were observed and the production of DAG by TNF could not be blocked by either propranolol or ethanol, a combined activation of phospholipase D and PA-phosphohydrolase in DAG production appears unlikely. TNF-stimulated DAG production as well as PKC activation could be blocked by the phospholipase inhibitor p-bromophenacylbromide (BPB). Since BPB did not inactivate PKC directly, these findings underscore that TNF activates PKC via formation of DAG. TNF stimulation of DAG production could be inhibited by preincubation of cells with a monoclonal anti-TNF receptor (p55-60) antibody, indicating that activation of a PC-specific phospholipase C is a TNF receptor-mediated event.
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PMID:Tumor necrosis factor induces rapid production of 1'2'diacylglycerol by a phosphatidylcholine-specific phospholipase C. 165 88

We explored the nature and time course of the multiple signal transduction pathways for V1-vascular vasopressin (AVP) receptors of A7r5 aortic smooth muscle cells in culture by using radioligand binding techniques, intracellular calcium monitoring, and polyphosphoinositide and phospholipid analyses. V1-vascular AVP receptors of A7r5 cells were characterized by the agonist radioligand [3H]AVP and the antagonist radioligand [3H]d(CH2)5Tyr(Me)AVP. Affinity and capacity of agonist but not antagonist binding were modulated by MgCl2 and aluminum fluoride, suggesting that the receptors are coupled to a guanine nucleotide regulatory protein. In fura-2-loaded A7r5 cells, AVP induced within seconds a dose-dependent increase of free intracellular Ca++ ([Ca++]i) consisting of a rapid transient spike and a sustained increase lasting for 3-5 min. The baseline [Ca++]i was 136 +/- 18 nM, the maximum [Ca++]i response to AVP was 1,582 +/- 297 nM, and AVP ED50 was 1.87 +/- 0.15 nM. Diverse experiments performed with EGTA, 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethylester, Mn++, ionomycin, terbutylbenzo hydroquinone, and nicardipine suggested that the initial spike resulted from both intracellular Ca++ release from the endoplasmic reticulum and extracellular Ca++ influx, whereas the sustained phase depended on dihydropyridine-insensitive extracellular Ca++ influx. Experiments done with indomethacin and arachidonic acid indicated that AVP-induced extracellular Ca++ influx was in part dependent on phospholipase A2 activation. In [3H]myoinositol and [3H]arachidonate-labeled A7r5 cells, AVP stimulated inositol 1,4,5 trisphosphate and 1,2 diacylglycerol production via activation of phospholipase C. Also, AVP stimulated a transphosphatidylation reaction through activation of phospholipase D in A7r5 cells labeled with [3H]1-O-alkyl lysoglycerophosphocholine. Thus, the stimulation of V1-vascular AVP receptors of A7r5 cells triggers several signaling pathways. The immediate and transient [Ca++]i rise due to mobilization of intracellular and extracellular Ca++ is associated with the activation of phospholipases A2 and C, and the sustained activation of phospholipase D.
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PMID:Multiple signaling pathways of V1-vascular vasopressin receptors of A7r5 cells. 165 17

We established a cell-free system in which epinephrine and other lipolytic agents stimulated lipolysis of endogenous lipid droplets from fat cells by hormone-sensitive lipase. The endogenous lipid droplets were prepared by hypotonic treatment of fat cells and their successive washing with buffer containing 0.025% Triton X-100. In the cell-free system, propranolol inhibited lipolysis induced by various lipolytic agents such as norepinephrine, theophylline and cyclic AMP (cAMP), whereas phenoxybenzamine did not inhibit lipolysis. The binding of these lipolytic agents to endogenous lipid droplets was inhibited by propranolol, but not by phenoxybenzamine. The "propranolol-sensitive" binding of these lipolytic agents to the droplets may be involved in lipolysis. Treatment of the droplets with phospholipase C, but not phospholipase D, inhibited the propranolol-sensitive binding of these lipolytic agents to the droplets. These results suggest that the phosphate group of phospholipid in the droplets may be the site of propranolol-sensitive of binding of theophylline, and cAMP in addition to norepinephrine.
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PMID:Propranolol-sensitive binding of lipolytic agents to lipid droplets from adipocytes. 165 19

Chemoattractant receptors on leukocytes initiate migratory and cytotoxic activities via GTP-binding proteins. Recent cloning of cDNA encoding the formylpeptide receptor indicates it to be a member of the class of seven membrane spanning domain receptors which couple to G proteins. Leukocyte activation by chemoattractants requires sequential metabolic pathways involving phospholipase C then phospholipase D. Interestingly, the formylpeptide receptor physically associates with both a heterotrimeric G protein and a low molecular mass GTP-binding protein. These multiple GTP-binding proteins may regulate the function of chemoattractant receptors.
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PMID:Molecular mechanisms of leukocyte activation by chemoattractants. 166 88

A phosphatidylinositol-specific phospholipase C (PI-PLC) has been isolated from bovine brain (purification factor of 5.6 x 10(4)). By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it had a Mr of 57,000. Neither amino nor neutral sugars were detected in the purified enzyme. The pH optimum was 7.0-7.5, and the activity decreased only slightly at pH 8.0. When phosphatidylinositol was used as a substrate, the optimum Ca2+ requirement was 4 mM, and Km was 260 microM. When phosphatidylinositol 4,5-bisphosphate was used, the optimum Ca2+ requirement was 10(-7) M, and the Km was reduced to 90 microM. Lipid specificity studies showed that equal amounts of inositol phosphate and diacylglycerol were released from phosphatidylinositol but 4 times as much inositol 1,4,5-trisphosphate was released from phosphatidylinositol 4,5-bisphosphate. Other lipids, phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin, were not substrates. Failure to detect phosphatidic acid confirmed the absence of a phospholipase D activity in the purified enzyme. Myelin basic protein (MBP) stimulated the PI-PLC activity between 2- and 3-fold. Histone had a small effect only, whereas bovine serum albumin and cytochrome C had no effect. Phosphorylation of MBP reduced the stimulatory effect. Protein-protein interactions between MBP and PI-PLC have been demonstrated both immunologically and by sucrose density gradients. A stoichiometry of 1:1 has been suggested by the latter method. A number of peptides have been prepared by chemical, enzymatic, and synthetic methods. Peptides containing the MBP sequences consisting of residues 24-33 and 114-122 stimulated the PI-PLC but were less effective than the intact protein.
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PMID:A 57-kDa phosphatidylinositol-specific phospholipase C from bovine brain. 170 49

Decay-accelerating factor (DAF) is anchored in cell membranes by a glycosyl-plasmanylinositol (GPI) moiety that is transferred to it en bloc in the rough endoplasmic reticulum. To analyze the biochemical reactions involved in preassembly of this structure, a human hematopoietic cell-free system was employed. Incubation of cell extracts with UDP-[3H]GlcNAc and butanol partitioning of reaction mixtures yielded two products similar in TLC mobility to intermediates described in Trypanosoma brucei. Both species were sensitive to Bacillus thuringiensis phosphatidylinositol-specific phospholipase C, indicative of association of [3H]GlcNAc label with a plasmanylinositol-containing acceptor. In contrast to trypanosome intermediates, which contain phosphatidylinositol (1,2-diacylglycerophosphoinositol), however, alkali treatment and phospholipase A2 digestion generated butanol-phase products characteristic of glycosylated plasmanylinositol (1-alkyl-2-acylglycerophosphoinositol). Kinetic and pulse-chase experiments indicated that the slower-migrating species was a product of the faster and that it, but not the faster, was sensitive to both GPI-specific phospholipase D and nitrous acid deamination, consistent with conversion of GlcNAc- to GlcN-plasmanylinositol. Accordingly, acetic anhydride acetylation retransformed the slower species back to the faster. Further incubation with cell extracts converted the slower species into more polar products. Lysates of normal and of affected blood leukocytes from two paroxysmal nocturnal hemoglobinuria (PNH) patients supported assembly of the two intermediates within 1 min. Thus, the initial enzymes mediating human GPI-anchor assembly are GlcNAc-plasmanylinositol transferase and GlcNAc-plasmanylinositol deacetylase, their substrates contain plasmanylinositols, and the products of their activities are normal in affected PNH cells.
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PMID:Assembly and deacetylation of N-acetylglucosaminyl-plasmanylinositol in normal and affected paroxysmal nocturnal hemoglobinuria cells. 170 86

The role of lipid-bound second messengers in the regulation of neurotransmitter secretion is an important but poorly understood subject. Both bovine adrenal chromaffin cells and rat phoeochromocytoma (PC12) cells, two widely studied models of neuronal function, respond to bradykinin by generating phosphatidic acid (PA). This putative second messenger may be produced by two receptor-linked pathways: sequential action of phospholipase C (PLC) and diacylglycerol kinase (DAG kinase), or directly by phospholipase D (PLD). Here we show that bradykinin stimulation of chromaffin cells prelabelled (24 h) with 32Pi leads to production of [32P]PA which is not affected by 50 mM butanol. However, bradykinin stimulation of PC12 cells leads to [32P]PA formation, all of which is converted to phosphatidylbutanol in the presence of butanol. When chromaffin cells prelabelled with [3H]choline were stimulated with bradykinin there was no enhancement of formation of water soluble products of phosphatidylcholine hydrolysis. When chromaffin cells were permeabilised with pneumolysin and incubated in the presence of [gamma-32P]ATP, the formation of [32P]PA was still stimulated by bradykinin. These results show that, although both neuronal models synthesize PA in response to bradykinin, they do so by quite different routes: PLC/DAG kinase for chromaffin cells and PLD for PC12 cells. The observation that neither bradykinin nor tetradecanoyl phorbol acetate stimulate PLD in chromaffin cells suggests that these cells lack PLD activity. The conservation of PA formation, albeit by different routes, may indicate an essential role of PA in the regulation of cellular events by bradykinin.
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PMID:Lack of phospholipase D activity in chromaffin cells: bradykinin-stimulated phosphatidic acid formation involves phospholipase C in chromaffin cells but phospholipase D in PC12 cells. 171 14

Our recent studies have demonstrated the presence in neonatal islet cells and intact adult islets of a phosphatidylcholine-directed phospholipase D (PLD) which is activated after phorbol ester stimulation. The present study describes PLD activation in the presence of a carbohydrate insulin secretagogue. At the highest concentration tested (20 mM) the triose, glyceraldehyde, induced formation of phosphatidic acid in cells prelabeled with [14C]arachidonic acid or [3H]myristic acid (164 +/- 7 and 210 +/- 9% of basal phosphatidic acid values, respectively). Experimental confirmation of a concentration-dependent specific activation of PLD was provided by the formation of a transphosphatidylation product, phosphatidylethanol, after stimulation with glyceraldehyde in the presence of added ethanol (1.5%). Additionally, there was an early (within 5 min) rise in [14C]arachidonate-labeled diacylglycerol (139 +/- 7% of basal) accompanied by an increase in intracellular diacylglycerol mass (51 +/- 2 pmol/mg protein) and an increase in membrane-associated protein kinase C activity (183 +/- 5% of basal) which preceded the activation of PLD, as indicated by the time course of glyceraldehyde-stimulated phosphatidylethanol formation in the presence of ethanol. Pretreatment of islet cells with 2 microM 12-O-tetradecanoylphorbol-13-acetate for 18 h, to down-regulate protein kinase C, was without effect on diacylglycerol and phosphatidic acid production after 5 min but inhibited completely the production of phosphatidylethanol at 30 min. The phosphohydrolase inhibitor propranolol (100 microM) potentiated the accumulation of phosphatidic acid and phosphatidylethanol incubation following incubation with glyceraldehyde. These findings demonstrate for the first time that a physiological nutrient activates a phospholipase directed against endogenous phosphatidylcholine in intact islet cells; furthermore, they indicate a role for PLD in a delayed formation of phosphatidic acid in the islet cell. The finding of an early rise in glyceraldehyde-stimulated diacylglycerol (which may be formed de novo or by the action of phospholipase C), suggests that PLD is recruited by the activation of protein kinase C by this nutrient.
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PMID:Activation of phospholipase D by glyceraldehyde in isolated islet cells follows protein kinase C activation. 172 27

Complement receptor (CR)-mediated phagocytosis is associated with an increased accumulation of diglyceride (sn-1,2-diacylglycerol and/or 1-O-alkyl-2-acyl-glycerol) in human neutrophils. The C3bi-mediated increase in diglyceride (5-20 min) was only partially impaired when phosphoinositide-specific phospholipase C (PLC) activity was abolished by reduction of cytosolic free Ca2+. At an early time point (1 min), however, diglyceride production was barely detectable in control cells, whereas production was considerable in cells with a reduced cytosolic free Ca2+ concentration. C3bi stimulation of 32P-labeled neutrophils caused a rapid and significant breakdown of [32P]phosphatidylcholine (PC) which was not affected by inhibition of Ca(2+)-dependent phosphoinositide-specific PLC. Thus, PC hydrolysis could be involved in C3bi-induced diglyceride formation. Stimulation of cells labeled with [3H]1-O-alkyl-lyso-PC ([3H]alkyl-lyso-PC), resulted in an increased formation of [3H]1-O-alkyl-phosphatidic acid ([3H]alkyl-PA) and a later and slower formation of [3H]1-O-alkyl-diglyceride ([3H]alkyl-diglyceride); this suggests activation of phospholipase D (PLD). When these labeled cells were stimulated in the presence of 0.5% ethanol a marked accumulation of [3H]1-O-alkyl-phosphatidylethanol ([3H]alkyl-PEt) was observed in both controls and calcium-reduced cells, further strengthening the suggested involvement of PLD activity. In parallel with the sustained increase in diglyceride formation, CR-mediated phagocytosis was also associated with phosphorylation of a cellular protein kinase C substrate (MARCKS). Therefore it seems reasonable to suggest a causal relationship between C3bi-induced PLD activation, which results in diglyceride formation, and activation of protein kinase C. In electropermeabilized cells which were incapable of ingesting particles, C3bi particles were still able to activate PLD and induce formation of diglyceride. This signaling event must therefore be triggered by binding of particles to the cell and not by the engulfment process. Most importantly, introduction of the protein kinase C inhibitor peptides, PKC(19-36) and PKC(19-31), into these permeabilized cells resulted in a clear reduction of the C3bi-induced production of diglyceride, indicating that CR-mediated activation of protein kinase C directly triggers a positive feedback mechanism for additional diglyceride formation. Taken together, these data further clarify the mechanisms of CR-mediated diglyceride formation and give added support to the concept that protein kinase C plays an important role in the phagocytic process.
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PMID:Complement receptor-mediated phagocytosis is associated with accumulation of phosphatidylcholine-derived diglyceride in human neutrophils. Involvement of phospholipase D and direct evidence for a positive feedback signal of protein kinase. 173 62


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