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)

Acidification of the endosomal pathway is important for ligand and receptor sorting, toxin activation, and protein degradation by lysosomal acid hydrolases. Fluorescent probes and imaging methods were developed to measure pH to better than 0.2 U accuracy in individual endocytic vesicles in Swiss 3T3 fibroblasts. Endosomes were pulse labeled with transferrin (Tf), alpha 2-macroglobulin (alpha 2M), or dextran, each conjugated with tetramethylrhodamine and carboxyfluorescein (for pH 5-8) or dichlorocarboxyfluorescein (for pH 4-6); pH in individual labeled vesicles was measured by ratio imaging using a cooled CCD camera and novel image analysis software. Tf-labeled endosomes acidified to pH 6.2 +/- 0.1 with a t1/2 of 4 min at 37 degrees C, and remained small and near the cell periphery. Dextran- and alpha 2M-labeled endosomes acidified to pH 4.7 +/- 0.2, becoming larger and moving toward the nucleus over 30 min; approximately 15% of alpha 2M-labeled endosomes were strongly acidic (pH less than 5.5) at only 1 min after labeling. Replacement of external Cl by NO3 or isethionate strongly and reversibly inhibited acidification. Addition of ouabain (1 mM) at the time of labeling strongly enhanced acidification in the first 5 min; Tf-labeled endosomes acidified to pH 5.3 without a change in morphology. Activation of phospholipase C by vasopressin (50 nM) enhanced acidification of early endosomes; activation of protein kinase C by PMA (100 nM) enhanced acidification strongly, whereas elevation of intracellular Ca by A23187 (1 microM) had no effect on acidification. Activation of protein kinase A by CPT-cAMP (0.5 mM) or forskolin (50 microM) inhibited acidification. Lysosomal pH was not affected by ouabain or the protein kinase activators. These results establish a methodology for quantitative measurement of pH in individual endocytic vesicles, and demonstrate that acidification of endosomes labeled with Tf and alpha 2M (receptor-mediated endocytosis) and dextran (fluid-phase endocytosis) is sensitive to intracellular anion composition, Na/K pump inhibition, and multiple intracellular second messengers.
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PMID:Second messengers regulate endosomal acidification in Swiss 3T3 fibroblasts. 138 79

Plasma disaturated phosphatidylcholine (DSPC) concentration has been implicated as a risk factor for atherosclerosis. However, suitable methods for the estimation of these compounds in plasma are not available. In this paper, a method for the estimation of DSPC using argentation thin-layer chromatography and high-performance liquid chromatography is described. It is quantitative for the measurement of individual and total DSPC species and is not dependent on fatty acid chain length. The method employs hydrolysis of total plasma phosphatidyl choline by phospholipase C, followed by benzoylation of the diacylglycerols. The benzoates are then fractionated on silver nitrate-impregnated silica gel thin-layer chromatography plates, and the disaturated species separated and quantitated by high-performance liquid chromatography. The method is sensitive and reproducible and allows many samples to be done at once. With this method, the amounts of DSPC were found to be significantly higher in a group of normolipidemic diabetic subjects, compared to age-matched controls.
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PMID:A high-performance liquid chromatographic procedure for the determination of disaturated phosphatidylcholine in human plasma. 207 37

For the enumeration of vegetative cells and spores of Bacillus cereus in foods, a mannitol-egg yolk-phenol red-agar has been developed which exploits the failure of B. cereus to dissimilate mannitol, and the ability of most strains to produce phospholipase C. When a high degree of selectivity was required, polymyxin B sulfate in a concentration of 10 ppm appeared to be the most effective selective additive. Useful characteristics for the identification of presumptive isolates of B. cereus were found to be: morphology, dissimilation of glucose mostly to acetyl methyl carbinol under anaerobic conditions, hydrolysis of starch and gelatin, reduction of nitrate, and growth on 0.25% chloral hydrate agar.
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PMID:Enumeration of Bacillus cereus in foods. 429 56

Sphingomyelins from human blood plasma have been converted into ceramides by enzymatic hydrolysis with phospholipase C. After acetylation the ceramides were fractionated by thin-layer chromatography on silica gel containing silver nitrate. Four main fractions obtained by this method were subsequently converted to di-O-trimethylsilyl ether derivatives and separated by gas-liquid chromatography on 1% OV-1. 2-11 components could be distinguished in each of the four fractions. The major fractions emerging from the gas chromatograph were analyzed by mass spectrometry and their main molecular species were identified. Two of the gas chromatographic fractions contained essentially pure molecular species, namely N-tetracosenoyl sphingosine and N-tetracosenoylsphinga-4, 14-dienine.
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PMID:Separation and identification of ceramides derived from human plasma sphingomyelins. 576 17

The rate of phospholipid hydrolysis in erythrocyte ghosts by Bacillus cereus phospholipase C was markedly decreased by the presence of NaCl at concentrations between 25 and 200 mM. The inhibition seemed to be due to Cl- and was unaffected by the type of cation present. The larger univalent anions such as HCO3-, Br-, Cl-, NO3-, CNO- and I- seemed most effective, whereas the bivalent anion SO42- was relatively ineffective at 0.1 M, as were acetate and formate. Tris buffers at 0.1 M caused marked inhibition. With bovine brain myelin, phospholipid hydrolysis by phospholipase C was also much more strongly inhibited by I- and Cl- than by SO42- or acetate. NaCl inhibited the hydrolysis by the enzyme of the soluble substrate dihexanoylglycerophosphocholine, thereby suggesting that the inhibiton did not arise simply from substrate effects.
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PMID:Inhibition of Bacillus cereus phospholipase C by univalent anions. 681 Aug 75

1. The synthesis of nitric oxide (NO) by immune-stimulated murine phagocytic cells (J774) and the modulation of this synthesis by tricyclodecan-9-yl-xanthogenate (D609), a specific inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), was investigated. D609 dose-dependently suppressed production of NO, as measured by the release of nitrite and nitrate, in response to lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) in intact cultured cells with an IC50 of approximately 20 micrograms ml-1. D609 at 40 micrograms ml-1 completely abrogated immune-stimulated nitrite production. 2. The inhibitory effects of D609 on nitrite production were time-dependent and restricted to the first 18 h post-stimulation. D609 did not inhibit nitrite production in the cytosol of immune-stimulated phagocytes. 3. These findings indicate that the xanthogenate, D609, is a potent inhibitor of the induction of NO-synthase activity in immune-stimulated phagocytes. Furthermore, since D609 has been demonstrated to inhibit PC-PLC specifically, our findings suggest that the activation of this enzyme by LPS and IFN-gamma is a proximal step in the signal transduction of inducible NO-synthase in phagocytic cells.
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PMID:Induction of nitric oxide synthase activity in phagocytic cells inhibited by tricyclodecan-9-yl-xanthogenate (D609). 753 78

Listeria monocytogenes secretes a phospholipase C (PLC) which has 39% amino acid sequence identity with the broad-specificity PLC from Bacillus cereus. Recent work indicates that the L. monocytogenes enzyme plays a role during infections of mammalian cells (J.-A. Vazquez-Boland, C. Kocks, S. Dramsi, H. Ohayon, C. Geoffroy, J. Mengaud, and P. Cossart, Infect. Immun. 60:219-230, 1992). The homogeneous enzyme has a specific activity of 230 mumol/min/mg when phosphatidylcholine (PC) is dispersed in sodium deoxycholate. With phospholipid-Triton X-100 mixed micelles, the enzyme had a broad pH optimum between 5.5 and 8.0, and the rates of lipid hydrolysis were in the following order: PC > phosphatidylethanolamine (PE) > phosphatidylserine > sphingomyelin >> phosphatidylinositol (PI). Activity on PC was stimulated 35% by 0.5 M NaCl and 60% by 0.05 mM ZnSO4. When Escherichia coli phospholipids were dispersed in Triton X-100, PE and phosphatidylglycerol, but not cardiolipin, were hydrolyzed. The enzyme was active on all phospholipids of vesiculated human erythrocytes including PI, which was rapidly hydrolyzed at pH 7.0. PI was also hydrolyzed in PI-PC-cholesterol liposomes by the nonspecific PLC from L. monocytogenes and by the homologous enzyme from B. cereus. The water-soluble hydrolysis product was identified as inositol-1-phosphate. For the hydrolysis of human erythrocyte ghost phospholipids, a broad pH optimum was also observed. 32P-labelled Clostridium butyricum protoplasts, which are rich in ether lipids, were treated with PLC. The enzyme hydrolyzed the plasmalogen form of PE, its glycerol acetal, and cardiolipin, in addition to PE. I-, Cl- and F- stimulated activity on either PC- Triton X-100 mixed micelles or human erythrocyte ghosts, unlike the enzyme from B. cereus which is strongly inhibited by halides. Tris-HCl, phosphate, and calcium nitrate had similar inhibitory effects on the enzyme on the enzymes from L. monocytogenes and B. cereus.
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PMID:Nonspecific phospholipase C of Listeria monocytogenes: activity on phospholipids in Triton X-100-mixed micelles and in biological membranes. 833 Oct 63

In this study, the mechanism involved in the antiplatelet activity of rutaecarpine in human platelet suspensions was investigated. In platelet suspensions (4.5 x 10(8)/ml), rutaecarpine (100 and 200 microM) did not influence the binding of FITC-triflavin to platelet glycoprotein IIb/IIIa complex. Additionally, rutaecarpine (200 microM) did not significantly change the fluorescence of platelet membrane labeled with diphenylhexatriene (DPH). On the other hand, rutaecarpine (50 and 100 microM) dose-dependently inhibited the increase in intracellular free Ca2+ of Fura 2-AM loaded platelets stimulated by collagen. Moreover, rutaecarpine (100 and 200 microM) did not significantly affect the thromboxane synthetase activity of aspirin-treated platelet microsomes. Furthermore, retaecarpine (100 and 200 microM) significantly inhibited [3H]arachidonic acid released in collagen-activated platelets but not in unactivated-platelets. Nitric oxide (NO) production in human platelets was measured by a chemiluminesence detection method in this study. Rutaecarpine (100 and 200 microM) did not significantly affect nitrate production in collagen (10 microg/ml)-induced human platelet aggregation. On the other hand, various concentrations of rutaecarpine (50, 100, and 200 microM) dose-dependently inhibited [3H]inositol monophosphate formation stimulated by collagen (10 microg/ml) in [3H]myoinositol-loaded platelets at different incubation times (1, 2, 3, and 5 minutes). It is concluded that the antiplatelet activity of rutaecarpine may possibly be due to the inhibition of phospholipase C activity, leading to reduce phosphoinositide breakdown, followed by the inhibition of thromboxane A2 formation, and then inhibition of [Ca2+]i mobilization of platelet aggregation stimulated by agonists.
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PMID:The antiplatelet activity of rutaecarpine, an alkaloid isolated from Evodia rutaecarpa, is mediated through inhibition of phospholipase C. 979 12

In this study, Escherichia coli lipopolysaccharide (LPS) dose-dependently (100-300 microg/ml) and time-dependently (10-60 min) inhibited platelet aggregation in human platelets stimulated by agonists. LPS also dose-dependently inhibited the phosphoinositide breakdown and the intracellular Ca+2 mobilization in human platelets stimulated by collagen. LPS (300 microg/ml) also significantly inhibited the thromboxane A2 formation stimulated by collagen in human platelets. Moreover, LPS (100-300 microg/ml) dose-dependently decreased the fluorescence of platelet membranes tagged with diphenylhexatrience. In addition, LPS (200 and 300 microg/ml) significantly increased the formation of cyclic GMP but not cyclic AMP in platelets. LPS (200 microg/ml) also significantly increased the production of nitrate within a 30 min incubation period. Rapid phosphorylation of a platelet protein of Mr 47,000, a marker of protein kinase C activation, was triggered by phorbol-12-13-dibutyrate (PDBu, 50 nM). This phosphorylation was markedly inhibited by LPS (200 microg/ml) within a 30 min incubation period. These results indicate that the antiplatelet activity of LPS may be involved in two important pathways. (1) LPS may induce conformational changes in the platelet membrane, leading to change in the activity of phospholipase C. (2) LPS also activated the formation of nitric oxide (NO)/cyclic GMP in human platelets, resulting in inhibition of platelet aggregation. Therefore, LPS-mediated alteration of platelet function may contribute to bleeding diathesis in septicaemic and endotoxaemic patients.
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PMID:Mechanisms involved in the antiplatelet activity of Escherichia coli lipopolysaccharide in human platelets. 979 85

Tumor necrosis factor (TNF)-alpha, a pluripotent cytokine implicated in the pathogenesis of airway inflammation, has been shown to provoke hypersecretion of mucin by airway epithelial cells in vitro. In this study, we investigated potential signaling pathways mediating TNF-alpha-induced mucin secretion using guinea pig tracheal epithelial (GPTE) cells in air-liquid interface culture. Exogenously applied TNF-alpha (human recombinant) stimulated mucin secretion in a concentration-dependent manner, with maximal effects at 10 to 15 ng/ml (286 to 429 U/ml). The pathway of stimulated secretion appeared to involve generation of intracellular nitric oxide (NO), activation of soluble guanylate cyclase (GC-S), production of cyclic guanosine monophosphate (cGMP), and activation of cGMP-dependent protein kinase (PKG). TNF-alpha increased production of nitrite and nitrate by GPTE cells; both mucin secretion and cGMP production were attenuated by NG-monomethyl-L-arginine (1 mM), a competitive inhibitor of nitric oxide synthase (NOS), or by the GC-S inhibitor LY83583 (50 microM); and mucin secretion in response to TNF-alpha or to the cGMP analogue dibutyryl cGMP (100 and 500 microM) was attenuated by the specific PKG inhibitor KT5823 (1 microM). Increased mucin secretion and increased cGMP production in response to TNF-alpha both appeared to be mediated by a phospholipase C that hydrolyzes phosphatidylcholine (PC-PLC), and by protein kinase C (PKC), since both responses were attenuated by either D609 (10 and 20 microg/ml), a specific PC-PLC inhibitor, or by each of three PKC inhibitors: Calphostin C (0.3 and 0.5 microM), bisindoylmaleimide (GF 109203X, Go 6850; 20 nM), or Ro31-8220 (10 microM). Collectively, the results suggest that TNF-alpha stimulates secretion of mucin by GPTE cells via a mechanism(s) dependent on PC-PLC and PKC, and involving activation of NOS, generation of NO, production of cGMP, and activation of PKG.
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PMID:Tumor necrosis factor-alpha stimulates mucin secretion and cyclic GMP production by guinea pig tracheal epithelial cells in vitro. 1003 Aug 39


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