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)

[14C]Choline was incorporated into microsomal membranes in vivo, and from CDP-[14C]choline in vitro, and the site of incorporation determined by hydrolysis of the outer leaflet of the membrane bilayer using phospholipase C from Clostridium welchii. Labelled phosphatidylcholine was found to be concentrated in the outer leaflet of the membrane bilayer with a specific activity approximately three times that of the inner leaflet. During incorporation of CDP-choline and treatment with phospholipase C the vesicles retained labelled-protein contents indicating that they remained intact. When the microsomes were opened with taurocholate after incorporation of [14C]choline in vivo, the labelled phosphatidylcholine behaved as a single pool. Selective hydrolysis of labelled phosphatidylcholine in intact vesicles is not, therefore, a consequence of specificity of phospholipase C. These results indicate that the phosphatidylcholine of the outer leaflet of the microsomal membrane bilayer is preferentially labelled by the choline-phosphotransferase pathway and that this pool of phospholipid does not equilibrate with that of the inner leaflet.
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PMID:Asymmetry of the site of choline incorporation into phosphatidylcholine of rat liver microsomes. 11 94

1. The phosphonium analogues of choline, phosphorylcholine, CDPcholine and phosphatidylcholine were synthesized chemically and characterized by 1H-NMR and 31P-NMR; in 1,2-distearoyl-DL-glycero-3-phosphorylphosphocholine, the 31P-NMR chemical shift of phosphonium relative to phosphate was--28.2 ppm. 2. A comparison was made of the rates of reaction of choline kinase, cholinephosphate cytidyltransferase, cholinephosphotransferase and phospholipase C on natural and phosphonium substrates. Enzyme reaction rates were similar for all but the cytidyltransferase, which exhibited a 3-fold preference for the normal substrate. 3. Weanling rats were maintained for 6 weeks on a diet in which choline was fully replaced by phospho[1,2-14C2]choline mixed with a trace of [Me-3H] choline. Incorporation of phosphocholine into liver lipids was detectable by 31P-NMR even in crude tissue homogenates. Choline-based phospholipids of liver, kidney, lung and brain were extracted, and phosphocholine incorporation calculated from 31P-NMR peak area ratios. The phosphatidylcholine analogues were separated by preparative thin-layer chromatography. Incorporation of phosphocholine ranged from 33% in lung phosphatidylcholine to 6% in kidney sphingomyelin. Variations in 14C/3H ratio between feed and phospholipid extracts indicated preferences for exogenous choline over phosphocholine varying from 1.3: 1 in brain to 3.2: 1 in liver. The results indicated that phosphocholine is a potentially useful 31P-NMR probe for the study of membrane lipids.
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PMID:The metabolism of the phosphonium analogue of choline in vitro and in vivo, and its detection in phospholipids by 31P-NMR. 93 56

The incorporation of [methyl-14C]choline into the choline-containing compounds of Ascaris suum muscle and the effects of acetylcholine and its agonists, carbachol and levamisole, on this incorporation were studied. Previous experiments reported a stimulation of phosphatidylcholine (lecithin) metabolism upon the administration of acetylcholine. Acetylcholine administered in vitro to A. suum muscle and body wall preparations resulted in a stimulation of phospholipase C activity that, in turn, produced an increased rate of hydrolysis of phosphatidylcholine to the corresponding diacylglyceride (DAG). The DAG, in turn, may act as a second messenger as it is required for the activation of an A. suum protein kinase C. Evidence presented here is in accordance with this hypothesis. The administration of cholinergics resulted in a stimulation of phosphatidylcholine turnover. Acetylcholine also stimulated isotope incorporation into glycerophosphorylcholine, presumably as a consequence of enhanced phospholipid turnover. These events appear to be associated with the ligand binding to the acetylcholine receptors of the A. suum muscle. Choline kinase activity is suggested in order to maintain the observed high ratio of phosphorylcholine to choline. Findings indicate that in the parasite's muscle phosphatidylcholine metabolism may be linked to receptor-dependent responses and subsequent signal transduction.
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PMID:Effects of cholinergic agents on the metabolism of choline in muscle from Ascaris suum. 159 77

We investigated the effect of bacterial lipopolysaccharide (LPS) on phospholipid (PL) turnover in human monocytic leukaemia U937 cells. Cells were pre-labelled with [3H]choline, [14C]ethanolamine and [3H]inositol for 24 h. By monitoring the radiolabel association with cellular PL, the data indicated that LPS (10 micrograms/ml) drastically altered the catabolism of choline-containing PL; it induced their breakdown by 50% within 20 min. The reutilization of choline or its phosphates for PL synthesis was also suggested as a result of regaining radiolabel in the next 40 min. Choline-containing PL then underwent a second degradation after 60 min; 50% decline in radiolabel was detected at 120 min. In contrast, LPS did not induce the breakdown of phosphatidylethanolamine and phosphatidylinositol through phospholipase C/phospholipase D (PLC/PLD). No significant redistribution of the radiolabel in PL was detected in any cases during chasing. The data clearly indicate that LPS stimulates phosphatidylcholine breakdown, implying that the liberation of phosphatidic acid or diacylglycerol via PLC/PLD reaction may be relevant to the initiation of LPS-induced monocytic activation.
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PMID:Bacterial lipopolysaccharide induces phosphatidylcholine breakdown in human leukaemia monocytic U937 cells. 162 16

Choline, acetylcholine and betaine used as the sole carbon, nitrogen or carbon and nitrogen source increase cholinesterase activity in addition to phosphorylcholine phosphatase and phospholipase C activities in Pseudomonas aeruginosa. The cholinesterase activity catalyses the hydrolysis of acetylthiocholine (Km approx. 0.13 mM) and propionylthiocholine (Km approx. 0.26 mM), but not butyrylthiocholine, which is a pure competitive inhibitor (Ki 0.05 mM). Increasing choline concentrations in the assay mixture decreased the affinity of cholinesterase for acetylthiocholine, but in all cases prevented inhibition raised by high substrate concentrations. Considering the properties of these enzymes, and the fact that in the corneal epithelium there exists a high acetylcholine concentration and that Pseudomonas aeruginosa produces corneal infection, it is proposed that these enzymes acting coordinately might contribute to the breakdown of the corneal epithelial membrane.
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PMID:Pseudomonas aeruginosa cholinesterase and phosphorylcholine phosphatase: two enzymes contributing to corneal infection. 165 99

Cholesterol oxidase (CO) and choline phosphohydrolase (CPH) exoenzymes were isolated from culture supernatants of Rhodococcus equi ATCC 33701 and their hemolytic and cytotoxic activities examined. The purifications involved differential ammonium sulphate precipitation, ion exchange and gel filtration chromatography. A purification of 32.8-fold and a yield of 0.3% of CO were determined by synergistic hemolysis of sheep red blood cells (SRBC) presensitized with Staphylococcus aureus beta toxin. The enzymatic activity of CO was also demonstrated by oxidation of aqueous cholesterol suspensions. The activity of CO was reversibly inhibited by concentration. A purification of 412.4-fold and a yield of 1.7% of CPH were determined by hydrolysis of p-nitrophenyphosphorylcholine. Purity of both exoenzymes was confirmed by immunoblotting. On sodium dodecyl sulphate polyacrylamide gel electrophoresis, the CO had a molecular mass (Mr) of 60 kd and the CPH a Mr of 65 kd. Choline phosphohydrolase did not hydrolyse sphingomyelin. Sphingomyelinase C (SMC) activity was however demonstrated in concentrated culture supernatants. This dissociation of SMC from CPH activity indicates that R. equi produces two distinct phospholipase C exoenzymes, a CPH and a SMC. Both CO and CPH combined, or individually, did not lyse native SRBC even with subsequent chilling of the cells at 4 degrees C ("hot-cold" treatment). Purified CO lysed beta toxin-sensitized SRBC. The CPH showed only minor hemolytic activity against such sensitized SRBC even at high concentrations. Combination of CO and CPH in lysis of beta toxin sensitized SRBC showed only minor additive rather than synergistic effects.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Purification and properties of cholesterol oxidase and choline phosphohydrolase from Rhodococcus equi. 179 Apr 88

Choline, betaine and N,N-dimethylglycine as the sole carbon and nitrogen source induced a periplasmic acid phosphatase activity in Pseudomonas aeruginosa. This enzyme produced the highest rates of hydrolysis in phosphorylcholine and phosphorylethanolamine among the various phosphoric esters tested. At saturating concentrations of Mg2+, the Km values were 0.2 and 0.7 mM for phosphorylcholine and phosphorylethanolamine respectively. At high concentrations both compounds were inhibitors of the enzyme activity. The Ksi values for phosphorylcholine and phosphorylethanolamine were 1.0 and 3.0 mM respectively. The higher catalytic efficiency was that of phosphorylcholine. Considering these results it is possible to suggest that the Pseudomonas aeruginosa acid phosphatase is a phosphorylcholine phosphatase. The existence of this activity which is induced jointly with phospholipase C by different choline metabolites, in a high phosphate medium, suggests that the attack of Pseudomonas aeruginosa on the cell host may also be produced under conditions of high phosphate concentrations, when the alkaline phosphatase is absent.
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PMID:Identification of the Pseudomonas aeruginosa acid phosphatase as a phosphorylcholine phosphatase activity. 211 92

Increasing interest in receptor-regulated phospholipase C and phospholipase D hydrolysis of cellular phosphatidylcholine motivates the development of a sensitive and simple assay for the water-soluble hydrolytic products of these reactions, phosphocholine and choline respectively. Choline was partially purified from the methanol/water upper phase of a Bligh & Dyer extract by ion-pair extraction using sodium tetraphenylboron, and the mass of choline was determined by a radioenzymic assay using choline kinase and [32P]ATP. After removal of choline from the upper phase, the mass of residual phosphocholine was determined by converting it into choline by using alkaline phosphatase, followed by radioactive phosphorylation. In addition to excellent sensitivity (5 pmol for choline and 10 pmol for phosphocholine), these assays demonstrated little mutual interference (phosphocholine----choline = 0%; choline----phosphocholine = 5%), were extremely reproducible (average S.E.M. of 3.5% for choline and 2.9% for phosphocholine), and were simple to perform with instrumentation typically available in most laboratories. In addition, the ability to apply the extraction technique to the upper phase of Bligh & Dyer extracts permitted simple analysis not only of choline and phosphocholine, but also of phosphatidylcholine and lipid products of phospholipase C and phospholipase D activity (1,2-diacylglycerol and phosphatidic acid respectively) from the same cell or tissue sample.
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PMID:Isolation and enzymic assay of choline and phosphocholine present in cell extracts with picomole sensitivity. 211 61

We have used the 1321N1 astrocytoma cell as a model system for understanding the molecular events involved in signal transduction through phospholipid metabolism. This clonal cell line expresses muscarinic cholinergic receptors (mAChR) that interact with a GTP-binding protein to regulate phospholipase C, rapidly increasing Ins 1,4,5-P3 and mobilizing intracellular Ca2+. Diacylglycerol (DAG) is also increased following mAChR stimulation but the increase in DAG is not significant until several minutes after addition of the mAChR agonist carbachol. To determine the role of Ca2+ and DAG in the activation of protein kinase C (PKC), we assessed PKC redistribution in the intact cell by measuring membrane-associated [3H]phorbol dibutyrate ([3H]PDB) binding. mAChR activation leads to a two-fold increase in [3H]PDB binding which is rapid, transient and temporally correlated with the increase in cytosolic [Ca2+]. When the rise in cytosolic [Ca2+] is buffered with Quin-2 or BAPTA the increase in [3H]PDB binding is inhibited. Studies using subtype-specific antibodies to PKC reveal only the alpha-subtype and confirm that mAChR stimulation causes redistribution of PKC immunoreactivity to a particulate cell fraction only when Ca2+ is increased. Our data suggest that the relatively slow increase in DAG is not the trigger for PKC redistribution and may be secondary to the activation of PKC. Thus, when 1321N1 cells are stimulated with phorbol 12-myristate 13-acetate (PMA) to activate PKC there is a rise in the cellular DAG content. In addition, in cells treated with PMA to down-regulate PKC, carbachol no longer significantly increases DAG mass. These data suggest that PKC is a mediator in the generation of DAG. Analysis of the fatty acid composition of the DAG formed in response to mAChR stimulation suggests that it is mostly derived from phosphatidylcholine (PC) rather than from inositol phospholipids. We examined the effect of mAChR stimulation on PC metabolism in 1321N1 cells. Cells were labelled with [3H]choline which was incorporated into PC and released into the medium when the cells were stimulated with carbachol or with PMA. [3H]Choline release increased throughout a 20-min stimulation. PKC down-regulation abolished both PMA and carbachol-stimulated [3H]choline release. These data support the hypothesis that mAChR stimulation leads to phospholipase D-mediated PC hydrolysis through activation of PKC. Activation of phospholipase D (PLD) was demonstrated by the finding that phosphatidic acid increased in response to PMA or carbachol prior to the increase in PA. In addition, phosphatidylethanol was formed in response to PMA and carbachol in cells exposed to ethanol.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Muscarinic receptor regulation of protein kinase C distribution and phosphatidylcholine hydrolysis. 213 May 11

The stable GTP analog, guanosine 5'-(3-O-thiotriphosphate), GTP gamma S, stimulated both inositol trisphosphate (InsP3) and choline generation by NIH 3T3 cell membranes. Choline generation was stimulated by GTP gamma S over the dose range for activation of GTP-binding proteins. Membranes from control and c-Ha-ras- or c-Ha-ras(61 leu)-transformed cells did not differ in the extent to which GTP gamma S stimulated InsP3 or choline formation despite 5-10 fold over expression of Ras in the transformed cells. Unlike GTP gamma S, GTP did not stimulate phospholipid hydrolysis, even in membranes from cells expressing Ras61leu, a mutant protein having reduced GTPase activity. Thus there is G protein regulation of both phosphatidylcholine-specific phospholipase D and polyphosphoinositide-specific phospholipase C in NIH 3T3 cell membranes. However, the lack of difference in GTP gamma S-stimulated phospholipid metabolism between control and ras-transformed cell membranes suggests that Ras does not function as the G protein(s) that directly regulate either phospholipase.
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PMID:GTP-binding protein-stimulated phospholipase C and phospholipase D activities in ras-transformed NIH 3T3 fibroblasts. 214 69


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