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)

Plasma or serum [ 0.1-1.0 ml] was digested with phospholipase C and total lipid extracts were prepared and silylated in the presence of tridecanoylglycerol as internal standard. The neutral lipid and free fatty acid profiles were determined by means of an automated GLC system equipped with an unheated on-column inlet, time actuated liquid injector, programmed heating, cooling and equilibration cycles, and an electronic peak area integrator. The separations were accomplished on a 50 cm x 2 mm i.d. steel column packed with 3% OV-1 on100-120 mesh Gas Chrom Q using nitrogen as a carrier gas in the temperature range 175-350 degrees C. The tube number, peak retention time and peak area were recorded on a punched paper tape, which was subsequently read into a computer via a time-share terminal. The composition of the sample was calculated in relation to the internal standard using a modification of a commercially available computer program and the results were expressed as mg or mole % and characteristic molar ratios of lipid classes. In addition to estimates for total cholesterol and triglyceride, the method provides a detailed account of individual or small groups of molecular species of various lipid classes, which is a major advantage over other automated methods of plasma lipid analyses.
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PMID:Determination of plasma lipid profiles by automated gas chromatography and computerized data analysis. 115 32

Affinity partitioning has been employed in the purification of membranes rich in cholinergic receptor from Torpedo californica electric organs. The procedure involves a modification of poly(ethylene oxide)-dextran aqueous phase partitioning systems where a ligand selective for the receptor is conjugated to the poly(ethylene oxide). Specific partitioning of the receptor-containing membranes into the poly(ethylene oxide)-rich phase occurs when bis-alpha,omega-trimethylamino poly(ethylene oxide) or bis-rho-tri-methylammonium phenylamino poly(ethylene oxide) was added to the phase system in low mole ratio. bis-alpha,omega-Methylamino poly(ethylene oxide), which should impart equivalent interfacial electromotive potential to the system but bind poorly to the receptor sites, was much less effective in producing phase distribution changes. The ligand-polymer-dependent phase distribution shifts were blocked by bisquaternary methonium ligands at concentrations consistent with their relative affinities for the cholinergic receptor. Titration or receptor sites with cobra alpha-toxin decreased the phase distribution changes in a linear fashion up to the point of stoichiometry. These observations are consistent with the phase distribution changes being consequent to ligand-polymer association with the pharmacologically important site on the receptor. The affinity partitioning procedure, when employed following an initial purification of the membranes by differential and density gradient centrifugation, yields membrane preparations with a high degree of morphological uniformity and a specific activity between 2.9 and 4.6 nmol of bound cobra alpha-toxin/mg of protein.
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PMID:Affinity partitioning of membranes. Cholinergic receptor-containing membranes from Torpedo californica. 124 60

We studied the binding of phosphoinositide-specific phospholipase C-delta 1 (PLC-delta) to vesicles containing the negatively charged phospholipids phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylserine (PS). PLC-delta did not bind significantly to large unilamellar vesicles formed from the zwitterionic lipid phosphatidylcholine (PC) but bound strongly to vesicles formed from mixtures of PC and PIP2. The apparent association constant for the putative 1:1 complex formed between PLC-delta and PIP2 was Ka congruent to 10(5) M-1. The binding strength increased further (Ka congruent to 10(6) M-1) when the vesicles also contained 30% PS. High-affinity binding of PLC-delta to PIP2 did not require Ca2+. PLC-delta bound only weakly to vesicles formed from mixtures of PC and either PS or phosphatidylinositol (PI); binding increased as the mole fraction of acidic lipid in the vesicles increased. We also studied the membrane binding of a small basic peptide that corresponds to a conserved region of PLC. Like PLC-delta, the peptide bound weakly to vesicles containing monovalent negatively charged lipids; unlike PLC-delta, it did not bind strongly to vesicles containing PIP2. Our data suggest that a significant fraction of the PLC-delta in a cell could be bound to PIP2 on the cytoplasmic surface of the plasma membrane.
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PMID:Phosphoinositide-specific phospholipase C-delta 1 binds with high affinity to phospholipid vesicles containing phosphatidylinositol 4,5-bisphosphate. 133 29

We added phospholipase C-delta 1 (PLC-delta) to the aqueous subphase beneath monolayers formed from mixtures of phosphatidylinositol 4,5-bisphosphate (2% PIP2), phosphatidylserine (33% PS), and phosphatidylcholine (65% PC) and then measured the initial rate of hydrolysis of PIP2 after addition of 10 microM free calcium. Increasing the surface pressure of the monolayer, pi, from 20 to 40 mN/m decreased the rate of hydrolysis 200-fold. The rate of hydrolysis depends exponentially on the surface pressure: rate alpha exp(-pi Ap/kT) where k is the Boltzmann constant, T is the temperature, and Ap congruent to 1 nm2. Similar results were obtained with different (1 and 100 microM) free [Ca2+] and with different mole fractions of PIP2. The results are consistent with a model in which PLC-delta binds to PIP2 with high affinity (Ka = 10(6) M-1) in the absence of calcium ions [Rebecchi, M.J., Peterson, A., & McLaughlin, S. (1993) Biochemistry (preceding paper in this issue)], and a portion of PLC-delta of area Ap inserts into the monolayer doing work = pi Ap prior to hydrolysis of PIP2. Removing the monovalent acidic lipid PS from the monolayer decreases the activity of PLC-delta 4-fold, this effect of PS on activity is similar to the effect of monovalent acidic lipids on the binding of PLC-delta to PIP2 in bilayer vesicles.
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PMID:Phosphoinositide-specific phospholipase C-delta 1: effect of monolayer surface pressure and electrostatic surface potentials on activity. 133 30

We have previously suggested the existence of two distinct states for cholesterol in cell membranes as revealed by high- and low-affinity binding sites for theta-toxin of Clostridium perfringens. In liposomes, phospholipid and cholesterol compositions, but not membrane protein composition, have been shown to be major determinants for the topology of membrane cholesterol. The effects of lipidic factors on cholesterol topology were investigated in detail by analyzing toxin binding to large unilamellar liposomes composed of cholesterol and phospholipids (neutral phospholipids/phosphatidylglycerol = 82:18, mol/mol). The numbers of high- and low-affinity toxin-binding sites depend strictly on the cholesterol mole percentage in liposomes. High-affinity toxin-binding sites appear only in liposomes with high cholesterol contents. Liposomes whose cholesterol/phospholipid ratio is 0.4 or less have no high-affinity sites regardless of their phospholipid compositions, while low-affinity sites appear in liposomes with lower cholesterol contents. The threshold values for the cholesterol mole percentage above which high-affinity toxin-binding sites appear were examined. The values decrease in accordance with the increase in the mole fraction of 18-carbon hydrocarbon chains among the total 14-18 carbon-hydrocarbon chains of the liposomal phospholipids. Furthermore, both the partial replacement of phosphatidylcholine with phosphatidylethanolamine and the digestion of phospholipids with phospholipase C also affect the threshold values. Thus the cholesterol mole percentage, in combination with phospholipid chain length and other factors, determines the topology of membrane cholesterol providing distinctively different affinity sites for theta-toxin.
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PMID:Effect of lipidic factors on membrane cholesterol topology--mode of binding of theta-toxin to cholesterol in liposomes. 150 83

Cetyltrimethylammonium and n-octadecyldimethylsulfonium bromides inhibit the Clostridium perfringens phospholipase C-catalyzed hydrolysis of 1-S-phosphocholine-2-O-hexadecanoyl-1-mercapto-2-ethanol (1) at pH 7.5, 37 degrees C, mu = 0.15 with KCl. Mixed micelles containing 1 and either inhibitor are substrates for the enzyme and the fraction of activity remaining is a monotonic, but non-linear function of the mole fraction of inhibitor. Simple saturation kinetics are observed as the concentration of 1 is increased in mixed micelles containing a constant mole fraction of inhibitor. Inhibition constants for cetyltrimethylammonium and n-octadecyldimethylsulfonium bromides are 0.66 +/- 0.04 and 0.25 +/- 0.02 mM, respectively. The data suggest that the significant inhibition previously observed for soluble alkyldisulfonium salts (K50 for dodecamethylene-bis(dimethylsulfonium) bromide, 27 microM) is dependent upon bifunctional cationic interactions rather than hydrophobic binding.
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PMID:Inhibition of the Clostridium perfringens phospholipase C hydrolysis of a thiophosphate analog of lysophosphatidylcholine by micelle-bound ammonium and sulfonium cations. 180 98

A highly sensitive method was developed for quantitative analysis of phospholipid molecular species. Diradylglycerols prepared from phospholipids with phospholipase C were converted to the anthroyl-diradylglycerol derivatives, which could be separated into molecular species and sensitively quantified by reverse-phase HPLC using a fluorescence detector. All the molecular species of the derivatives had the same peak area per mole, and the peak areas were proportional to the amounts of the derivatives. Quantification could be carried out at the femtomole level.
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PMID:A highly sensitive method for quantitative analysis of phospholipid molecular species by high-performance liquid chromatography. 188 Jan 30

The scrapie (PrPSc) and cellular (PrPC) prion proteins are encoded by the same gene, and their different properties are thought to arise from posttranslational modifications. We have found a phosphatidylinositol glycolipid on both PrPC and PrP 27-30 (derived from PrPSc by limited proteolysis at the amino terminus). Ethanolamine, myo-inositol, phosphate, and stearic acid were identified as glycolipid components of gel-purified PrP 27-30. PrP 27-30 contains 2.8 moles of ethanolamine per mole. Incubation of PrP 27-30 with a bacterial phosphatidylinositol-specific phospholipase C (PIPLC) releases covalently bound stearic acid, and allows PrP 27-30 to react with antiserum specific for the PIPLC-digested glycolipid linked to the carboxyl terminus of the trypanosomal variant surface glycoprotein. PIPLC catalyzes the release of PrPC from cultured mammalian cells into the medium. These observations indicate that PrPC is anchored to the cell surface by the glycolipid.
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PMID:Scrapie prion protein contains a phosphatidylinositol glycolipid. 244 40

Large unilamellar vesicles composed of phosphatidylcholine/phosphatidylethanolamine/cholesterol (50:25:25 mole ratio) were treated with phospholipase C. The early stages of phospholipid cleavage are accompanied by mixing of bilayer lipids (monitored by dequenching of octadecylrhodamine fluorescence) and leakage-free mixing of vesicle contents [measured by using 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) and p-xylylenebis(pyridinium bromide) (DPX)]. These results are interpreted in terms of vesicle fusion induced by the catalytic activity of phospholipase C. The use of sonicated unilamellar vesicles decreases the lag time, but does not modify the amplitude, of the fusion process. The presence of both phosphatidylethanolamine and cholesterol appears to be essential for measurable fusion effects to occur with low levels of phospholipid hydrolysis. Optimal fusion rates are observed with about 10-20 enzyme molecules per large unilamellar vesicle. This system of catalytically induced liposome fusion may be of relevance for the interpretation of physiological membrane fusion processes.
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PMID:Liposome fusion catalytically induced by phospholipase C. 281 74

Pseudomonas aeruginosa produces at least two distinct hemolysins: heat-labile hemolysin, which is known as phospholipase C, and heat-stable hemolysin, which is considered to be a glycolipid but its true nature is not found yet. Cellophane agar plate method was applied to hasten and enhance the hemolysin production by Pseudomonas aeruginosa, and the hemolysin obtained by this method was examined biochemically. This heat-stable hemolysin consisted of two major acidic glycolipids. One was considered to be the same glycolipid as that obtained by the same method of Jarvis and Johnson's from the peptone-glycerol culture filtrate of the same organism, which is composed of two moles of rhamnose and two moles of beta-hydroxy decanoic acid, and the other one being a glycolipid composed of one mole of rhamnose and two moles of beta-hydroxy decanoic acid. By this method, the rhamnolipid with one mole of rhamnose was produced more than the rhamnolipid with two moles of rhamnose, and they were produced in the same ratio from the first to fifth culture day. From the examinations of hemolytic activities of the hydrolyzed products of these glycolipids, the hemolysis producing moiety of the hemolysin was considered to be the dimer of beta-hydroxy decanoic acid contained in the glycolipid molecule. Hemolytic activities of these glycolipids are regarded as their detergent like effects. Since these activities are interfered by small amount of serum protein, their pathogenic role would not be important in vitro. However, their interaction with other toxic substances produced by this organism would be subject to be solved.
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PMID:[Heat-stable hemolysin of Pseudomonas aeruginosa]. 311 62


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