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 molecular species of 1,2-diacyl-sn-glycerol (DAG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2) from brains of adult rats (weighing 150 g) were determined. The DAG, isolated from brain lipid extracts by TLC, was benzoylated, and the molecular species of the purified benzoylated derivatives were separated from each other by reverse-phase HPLC. The total amount and the concentration of each species were quantified by using 1,2-distearoyl-sn-glycerol (18:0-18:0) as an internal standard. About 30 different molecular species containing different fatty acids at the sn-1 and sn-2 positions of DAG were identified in rat brains (1 min postmortem), and the predominant ones were 18:0-20:4 (35%), 16:0-18:1 (15%), 16:0-16:0 (9%), and 16:0-20:4 (8%). The molecular species of PC, PE, PS, and PI were determined by hydrolyzing the lipids with phospholipase C to DAG, which was then benzoylated and subjected to reverse-phase HPLC. PIP and PIP2 were first dephosphorylated to PI with alkaline phosphatase before hydrolysis by phospholipase C. The molecular species composition of phosphoinositides showed predominantly the 18:0-20:4 species (50% in PI and approximately 65% in PIP and PIP2). PS contained mainly the 18:0-22:6 (42%) and 18:0-18:1 (24%) species. PE was mainly composed of the 18:0-20:4 (22%), 18:0-22:6 (18%), 16:0-18:1 (15%), and 18:0-18:1 (15%) species. In PC the main molecular species were 16:0-18:1 (36%), 16:0-16:0 (19%), and 18:0-18:1 (14%). Studies on postmortem brains (30 s to 30 min) showed a rapid increase in the total amount (from 40-50 nmol/g in 0 min to 210-290 nmol/g in 30 min) and in all the molecular species of DAG. Comparatively larger increases (seven- to 10-fold) were found for the 18:0-20:4 and 16:0-20:4 species. Comparison of DAG species with the molecular species of different glycerolipids indicated that the rapid postmortem increase in content of DAG was mainly due to the breakdown of phosphoinositides. However, a slow but continuous breakdown of PC to DAG was also observed.
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PMID:Molecular species of diacylglycerols and phosphoglycerides and the postmortem changes in the molecular species of diacylglycerols in rat brains. 184 96

We have previously shown that two ectoenzymes, acetylcholinesterase (AChE) and alkaline phosphatase, are released from the surface and from particulate fractions of the parasite Schistosoma mansoni, by a phosphatidylinositol-specific phospholipase C (PtdIns-PLC) of bacterial origin. Exposure to PtdIns-PLC not only removes large amounts of AChE from the surface of intact, viable Schistosoma in culture, but is accompanied by a concomitant increase in overall levels of AChE in the parasite. The same phenomenon is observed with PtdIns-PLC from two different bacterial sources; Staphylococcus aureus and Bacillus thuringiensis. The increase in AChE levels may be ascribed to de novo synthesis since exposure to PtdIns-PLC, in the presence of the protein-synthesis inhibitor cycloheximide, totally blocked the increase in AChE activity. Furthermore, PtdIns-PLC induced an increased incorporation of [35S]methionine into the AChE immunoprecipitated by a specific anti-AChE serum. This increase is selective for AChE, since total protein synthesis remained almost unchanged after PtdIns-PLC addition, and little or no effect was observed on the enzymatic activity of alkaline phosphatase, which is also glycophosphatidylinositol anchored. Since cleavage of the phosphatidylinositol anchor by PtdIns-PLC should liberate diacylglycerol, which may act as second messenger, we investigated the effect of exogenous diacylglycerols on the synthesis of AChE in S. mansoni. Three different diacylglycerols were tested as possible inducers of AChE activity in the parasite. Both 1-oleoyl-2-acetyl-sn-glycerol and 1,2-dimyristoyl-sn-glycerol were able to increase AChE activity by 35-40% at concentrations of 25 micrograms/ml. A higher concentration of 1,2-dioctanoyl-sn-glycerol (70 micrograms/ml) was needed to produce an equivalent effect. Moreover, addition of phorbol-12-myristate-13-acetate, together with the calcium ionophore A23187, produced a similar increase in AChE activity. Finally, polymixin B, a specific inhibitor of protein kinase C, partially blocked the increase in AChE activity induced by PtdIns-PLC. Our results suggest the involvement of glycophosphatidyl membrane-anchor breakdown products as putative second messengers in the parasite S. mansoni.
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PMID:Phosphatidylinositol-specific phospholipase C induces biosynthesis of acetylcholinesterase via diacylglycerol in Schistosoma mansoni. 184 73

Plasmids encoding mercury resistance carried by Pseudomonas aeruginosa PAO1161 and PA103 were found to be involved in regulating the secretion of protease, phospholipase C, and alkaline phosphatase. Previously, mutations in Pseudomonas strains that caused pleiotropic effects on the production of extracellular enzymes were mapped to the bacterial chromosome. We show that pleiotropic changes in extracellular enzyme production can also be regulated by plasmids. In this study, the effects on secretion of exoenzymes by two mercury resistance plasmids, FP2 from PAO1161 and pRLW103 from PA103, were assayed in P. aeruginosa PAO1 and PAO18. The introduction of either plasmid into PAO1 resulted in a significant decrease in exoprotease production. Additionally, pRLW103 significantly increased the production of alkaline phosphatase by both strains. Phospholipase C was produced only in strain PAO18 containing the pRLW103 plasmid. FP2 had no effect on alkaline phosphatase or phospholipase C production in either strain and was found to decrease exoprotease secretion only in strain PAO1. The results indicate the P. aeruginosa mercury resistance plasmids vary in their ability to modify exoenzyme expression, and this ability is influenced by the host strain.
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PMID:Effects of FP2 and a mercury resistance plasmid from Pseudomonas aeruginosa PA103 on exoenzyme production. 190 22

The molecular nature and possible presence of a glycan-phosphatidylinositol anchor (GPI-anchor) in CA125 molecules was investigated. Serial lectin affinity chromatography and N- or O-glycanase treatment to reduce antigenicity showed that CA125 contained certain N- and O-glycosylated sugar chains in the molecule, like a glycoprotein. CA125 released from ovarian cancer tissues increased time-dependently following phosphatidylinositol-specific phospholipase C (PI-PLC) treatment, concomitant with the release of tissue-unspecific alkaline phosphatase. Western blotting of CA125 treated by PI-PLC showed a single band of 90 kD instead of the 162- and 76-kD bands of the native antigen. Further, ovarian cancer tissues subjected to PI-PLC treatment lost the immunohistochemical localization of CA125 with OC125 antibody. Consequently, it is strongly suggested that CA125 is a glycoprotein that has both N- and O-linked sugar chains and a membranous GPI-anchoring moiety, and further, that its 90-kD form is the antigen without the GPI-anchor.
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PMID:Molecular nature and possible presence of a membranous glycan-phosphatidylinositol anchor of CA125 antigen. 196 50

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

Alkaline phosphatase (ALP) activity has been demonstrated in periodontal ligament (PDL). On the basis of electron microscopic study, distribution of the enzyme in PDL tissue has also been indicated not only as a cell associated activity but also as an extracellular matrix associated activity. This study is concerned with the purification and characterization of the enzyme obtained from bovine PDL tissue. Purification of ALP extracted from the tissue included solubilization with 10 mM Tris-HCl buffer, pH 7.4, containing 0.2 mM MgCl2 and 0.1% Nonidet P-40 and fractionation by sequential chromatography utilizing DEAE-sephacel, Sepharose CL-6B and concanavalin A Sepharose 4B. Purity was established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This was followed by staining for ALP activity first with 2 mM beta-naphthyl acid phosphate and 1 mM Fast Blue BB Salt and then the protein with Coomassie Brilliant Blue. SDS-PAGE of the crude enzyme preparations gave a broad band with apparent molecular weight of 110,000-130,000 dalton. ALP activities were separated into two major peaks using Sepharose CL-6B chromatography. The void volume peak showed a purified form of 110,000 dalton ALP (110K ALP) while the second peak contained 120,000-130,000 dalton ALP (120-130K ALP) and other proteins. Sequentially, 120-130K ALP was purified by chromatography on concanavalin A Sepharose 4B. A polyclonal antibody was raised against purified bovine PDL 110K ALP in a rabbit. Immunodiffusion analysis showed that a polyclonal antibody against 110K ALP recognized 120-130K ALP. Analytical affinity chromatography on concanavalin A Sepharose 4B indicated that 110K ALP and 120-130K ALP had distinct affinity to the column which may depend upon the sugar chain structure. Digestion of 110K ALP with phosphatidylinositol-specific phospholipase C affected electrophoretic mobility but 120-130K ALP had no effect. It is suggested that 110K ALP is attached to a cell membrane anchored by a phosphatidylinositol glycan. In conclusion, bovine PDL contains two types of alkaline phosphatase i.e. 110K ALP and 120-130K ALP. Both ALPs are immunologically related although they have different sugar chain moieties. Furthermore, 110K ALP has a membrane anchoring domain. These results suggest that 110K ALP would be localized on the surface of the cell membrane and 120-130K ALP may associated with the extracellular matrix.
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PMID:[Purification and characterization of alkaline phosphatase obtained from bovine periodontal ligament]. 213 40

Phosphatidylinositol 4-phosphate (PIP) kinase (E.C. 2.7.1.68) has been purified about 1200-fold from rat liver plasma membranes, taking advantage of affinity chromatography on quercetin-Sepharose as a novel step. The purified PIP kinase showed no contamination by the following enzyme activities: phosphatidylinositol (PI) kinase (EC 2.7.1.67), protein kinase C (EC 2.7.1.-), diacylglycerol kinase (EC 2.7.1.-), phospholipase C (EC 3.1.4.11), protein-tyrosine kinase (EC 2.7.1.112), alkaline phosphatase (EC 3.1.3.1), triphosphoinositide phosphomonoesterase (EC 3.1.3.36), adenylate kinase (EC 2.7.4.3) and cAMP-dependent protein kinase (EC 2.7.1.37). The liver membrane enzyme requires high Mg2+ concentrations with a KM value of 10 mM. Ca2+ or Mn2+ could replace Mg2+ to a certain, though small, extent. Apparent KM values with respect to PIP and ATP were 10 and 65 microM, respectively. GTP was slightly utilized by the kinase as phosphate donor while CTP was not. Quercetin inhibited the enzyme with Ki = 34 microM. Extending our previous observations (Urumow, T. and Wieland, O.H. (1986) FEBS Lett. 207, 253-257 and Urumow, T. and Wieland, O.H. (1988) Biochim. Biophys. Acta 972, 232-238) [gamma S]pppG still stimulated the PIP kinase in extracts of solubilized liver membranes. 20-40% (NH4)2SO4 precipitation of the membrane extracts yielded a fraction that contained the bulk of enzyme activity but did not respond to stimulation by [gamma S]pppG any longer. This was restored by recombination with a protein fraction collected at 40-70% (NH4)2SO4 saturation, presumably containing a GTP binding protein and/or some other factor separated from the PIP kinase. In the reconstituted system [gamma S]pppG stimulated PIP kinase in a concentration dependent manner with maximal activation at 5 microM. This effect was not mimicked by [gamma S]pppA and was blocked by [beta S]ppG. These results strongly support our view that in liver membranes PIP kinase is regulated by a G-protein.
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PMID:Purification and partial characterization of phosphatidylinositol-4-phosphate kinase from rat liver plasma membranes. Further evidence for a stimulatory G-protein. 215 97

The alkaline phosphatase (AP) synthesized by human tumor cells closely resembles human placental AP (PLAP). Little is known about the molecular events that lead to the expression of a placenta-like AP in tumor cells. The complementary DNA encoding the AP expressed by a choriocarcinoma cell line, BeWo, was isolated and characterized. The complementary DNA is the product of the germ cell AP (Nagao isozyme) gene and not of the term PLAP gene. Like placental AP, the tumor AP can be released from the cell membrane by a phosphaditylinositol-specific phospholipase C and has a phosphaditylinositol-glycan (PI-glycan) moiety at the COOH terminus. Immunoprecipitation of phosphaditylinositol-specific phospholipase C-treated AP and analysis by polyacrylamide gel electrophoresis or isoelectric focusing demonstrates that at least 95% of the AP contains PI-glycan. Two-dimensional gel electrophoresis reveals two precursors of the mature AP. One of these does not bind an antibody against the Trypanosoma variable surface glycoprotein cross-reacting determinant and probably does not contain PI-glycan. This precursor had a shorter half-life than the more prominent PI-glycan-containing precursor in pulse-chase experiments, suggesting a precursor-product relationship between the two proteins. These data demonstrate that BeWo AP is the product of a gene normally expressed in testis, thymus, and germ cells, but not in placenta. Thus, the expression of BeWo AP results from the repression of the PLAP gene and derepression of the germ cell AP gene and, as such, the expression is ectopic. The BeWo AP (Nagao isozyme) is modified with PI-glycan that is added soon after translation, not cotranslationally.
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PMID:Expression of a Nagao-type, phosphatidylinositol-glycan anchored alkaline phosphatase in human choriocarcinomas. 216 49

A number of studies have shown membrane phospholipid metabolism to have an important role in biological mineralization. We considered the effects of exogenously applied phosphatidic acid (PA), a minor component of membrane phospholipids, on an osteoblast-like cell line, MOB 3-4. Exogenous PA (10-40 micrograms/ml) raised the level of cytoplasmic free Ca2+ [( Ca2+]i), independent of the level of extracellular Ca2+, in a dose-dependent fashion, and this Ca2+ response to PA gradually fell on serial application of PA. In a dose-dependent manner, exogenous PA also increased inositol 1,4,5-trisphosphate (IP3) accumulation and cytoplasmic pH, but decreased basal cAMP level. This cytoplasmic alkalinization was inhibited by pretreatment with nonspecific protein kinase C (PKC) inhibitors, such as sphingosine or H-7. A long-term incubation with PA increased alkaline phosphatase (ALP) activity and cell proliferation. Exogenous PA thus appeared to increase IP3 accumulation by activating phospholipase C, raise [Ca2+] by releasing Ca2+ from intracellular stores, induce cytoplasmic alkalinization via a PKC-dependent mechanism, and simultaneously decrease basal cAMP level. We suggest that these initial responses may be responsible for the increase in ALP activity and the proliferation of PA-treated MOB 3-4 cells.
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PMID:Initial responses of a clonal osteoblast-like cell line, MOB 3-4, to phosphatidic acid in vitro. 216 76


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